Embryology - User contributions [en-gb]

Lecture - Week 1 and 2 Development

2015-11-07T03:38:03Z

Z3292373:

{{Header}}

[[Week 1]] | [[Week 2]] | [[Zygote]] | [[Morula]] | [[Blastocyst]] | [[Implantation]]

==Introduction==
[[File:Week1_summary.jpg|thumb|450px|Week 1 and 2 Development (see [[Week 1 Movie]])]]
This lecture will discuss the first two weeks of human embryogenesis and describe the cleavage stages, blastocyst formation and hatching, and the generation of the bilaminar embryo. There will also be an introduction to the uterine changes at implantation, that will be covered in detail in the placentation lecture.

[[Media:2015ANAT2341 Lecture 3 - Week 1 and 2 Development.pdf|Lecture - Print PDF]]
==Objectives==

* Understand the events during week 1 of development (Zygote, Blastomeres, Morula, Blastocyst)
* Understand the events during week 2 of development (Trophoblast, Syncytiotrophoblast, Cytotrophoblast, Embryoblast, Implantation)
* Brief understanding of early placentation
* Brief understanding of maternal changes

==Lecture Resources==
{| class="wikitable mw-collapsible mw-collapsed"
! Movies
|-
| valign="bottom"|{{Human blastocyst movie 1}}
| valign="bottom"|{{Human blastocyst movie 2}}
| valign="bottom"|{{Human blastocyst movie 3}}
|-
| valign="bottom"|{{Mouse zygote movie}}
| valign="bottom"|{{Mouse zygote movie 1}}
| valign="bottom"|{{Mouse zygote movie 2}}
| valign="bottom"|{{Mouse Blastocyst movie}}
|-
| valign="bottom"|{{Morula model movie}}
| valign="bottom"|{{Blastocyst model movie}}
|-
! Week 2
|-
| valign="bottom"|{{Week 2 implant movie}}
| valign="bottom"|{{Week 2 bilaminar movie}}
| valign="bottom"|{{Embryo 1.6mm movie 1‎}}
|}

{| class="wikitable mw-collapsible mw-collapsed"
! References
|-
| {{Embryo logocitation}}
|
* [[Media:2015ANAT2341 Lecture 3 - Week 1 and 2 Development.pdf|2015 Lecture PDF]]
* [[Menstrual Cycle|Menstrual Cycle]] | [[Week 1]] | [[Week 2]] | [[Implantation]]
* Lecture Archive: [[2009_Lecture_3|2009]] | [[2010_Lecture_3|2010]] | [http://embryology.med.unsw.edu.au/embryology/index.php?title=Lecture_-_Week_1_and_2_Development&oldid=61429 2011] | [http://embryology.med.unsw.edu.au/embryology/index.php?title=Lecture_-_Week_1_and_2_Development&oldid=97577 2012] | [[Media:ANAT2341_Lecture_2_-_Beverdam_-_Week_1_and_2.pdf|2013]] | [[Media:ANAT2341 Lecture 3 - 2014 Week 1 and 2 Development.pdf|2014]]
|-
| {{MPT2011cover_citation}}
| The following chapter links only work with a UNSW connection.
* [http://www.unsw.eblib.com.wwwproxy0.library.unsw.edu.au/patron/Read.aspx?p=1430154&pg=35 First Week of Human Development]
* [http://www.unsw.eblib.com.wwwproxy0.library.unsw.edu.au/patron/Read.aspx?p=1430154&pg=63 Second Week of Human Development]
|-
| {{SBBFP2015cover_citation}}
| The following chapter links only work with a UNSW connection.
* [https://login.wwwproxy0.library.unsw.edu.au/login?url=http://www.unsw.eblib.com/patron/FullRecord.aspx?p=2074524 Chapter 1 - Gametogenesis, Fertilization, and First Week]
|}

{| class="wikitable mw-collapsible mw-collapsed"
! ECHO360 Recording
|-
| [[File:ECHO360_icon.gif|right|link=https://lectures.unsw.edu.au/ess/portal/section/5157_00900]]
[https://lectures.unsw.edu.au/ess/portal/section/8aefd634-898b-4cdd-bbd5-c6052c39b759 2015 Lecture 3]

Links only work with currently enrolled UNSW students.
|}

==Fertilization==
[[File:ova20he.jpg|thumb|alt=Antral Follicle and Oocyte|Antral Follicle and Oocyte]]
[[File:Intracytoplasmic_sperm_insemination.jpg|thumb|alt=Intracytoplasmic sperm insemination|Intracytoplasmic sperm insemination ([[Assisted_Reproductive_Technology|ART]])]]
{|
|
* Fertilization usually occurs in first 1/3 of oviduct.
* Fertilization can also occur outside oviduct, associated with In Vitro Fertilization (IVF, GIFT, ZIFT...) and ectopic pregnancy.
* The majority of fertilized eggs do not go on to form an embryo.
|
{{Fertilization movie 2}}
|}
===Fertilization - Spermatozoa ===
* '''Sperm Binding''' - zona pellucida protein ZP3 acts as receptor for sperm
* '''Acrosome Reaction''' - exyocytosis of acrosome contents (Calcium mediated) [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.3741 MBoC - Figure 20-31. The acrosome reaction that occurs when a mammalian sperm fertilizes an egg]
** enzymes to digest the zona pellucida, exposes sperm surface proteins to bind ZP2
* '''Membrane Fusion''' - between spermatozoa and oocyte, allows spermatozoa nuclei passage into oocyte cytoplasm

[[File:Model of gamete recognition zona pellucida.jpg|500px]]
===Fertilization- Oocyte===
* '''Membrane Depolarization''' - caused by sperm membrane fusion, primary block to polyspermy
* '''Cortical Reaction''' - IP3 pathway elevates intracellular Calcium, exocytosis of cortical granules [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.3743 MBoC - Figure 20-32. How the cortical reaction in a mouse egg is thought to prevent additional sperm from entering the egg]
** enzyme alters ZP3 so it will no longer bind sperm plasma membrane
* '''Meiosis 2''' - completion of 2nd meiotic division
** forms second polar body (a third polar body may be formed by meiotic division of the first polar body)

== Zygote Formation==
* zygote ([[Carnegie stage 1]]) is the first diploid cell formed following fertilisation.
* male and female pronuclei, 2 nuclei approach each other and nuclear membranes break down.
* DNA replicates, first mitotic division
* sperm contributes centriole which organizes mitotic spindle
{|
| [[File:Stage1_size_with_ruler.jpg|200px]]
| [[File:Human_zygote_two_pronuclei_22.jpg|300px]]
| [[File:Parental_genome_mix_01_icon.jpg|200px]]
|}

{| class="wikitable mw-collapsible mw-collapsed"
! Pronuclear Fusion and Parental Genomes Movies
|-
| <html5media height="340" width="320">File:Pronuclear_fusion_001.mp4</html5media>

[[Media:Pronuclear_fusion_001.mp4|'''Click Here''' to play on mobile device]]

[[Development_Animation_-_Pronuclear_Fusion|Movie - Pronuclear Fusion]]
| <html5media height="340" width="320">File:Parental_genome_mix_02.mp4</html5media>

[[Media:Parental_genome_mix_02.mp4|'''Click Here''' to play on mobile device]]

[[Movie_-_Parental_genomes|Movie - Parental Genomes]]
|}

:'''''Conceptus''' - the term refers to all material derived from this fertilised zygote, includes both the embryo and the non-embryonic tissues (placenta, fetal membranes).''

:'''Links:''' [[Fertilization]] | [[Carnegie stage 1]]

==Cleavage of Zygote==
{| class="wikitable mw-collapsible mw-collapsed"
! Zygote Division Movie
|-
| <html5media height="400" width="400">File:Mouse_zygote_division.mp4</html5media>

[[Media:Mouse_zygote_division.mp4|'''Click Here''' to play on mobile device]] | [[Mouse_Zygote_1_Movie|Mouse Zygote Movie page]]

|}
{|
|
* cleavage of zygote forms 2 blastomeres and is also cleavage with no cytoplasm synthesis.
** special "embryonic" cell cycle '''S''' phases and '''M''' phases alternate without any intervening '''G1''' or '''G2''' phases (MSMSMSMS, adult MG1SG2) therefore individual cell volume decreases.
* cell division is initially synchronous, then asynchronously
* cell division becomes slower (centre cells, larger) and faster in peripheral cells
* zona pellucid still intact (division occurs within the ZP)
| [[Image:Cell_cycle1.jpg|400px]]
|}

[[File:Human blastocyst day 1-6.jpg]]

Human Zygote to Blastocyst Development (day 1 to 6)

:'''Links:''' [[Carnegie stage 2]] |

== Morula ==

{| border='0px'
|-
| [[File:Human embryo day 2.jpg|Stage 2 Day 2]]
| [[File:Human embryo day 3.jpg|Stage 2 Day 3]]
|-
| Human Embryo (day 2)
| Human Embryo (day 3)
|-
|}
* about '''day 4''' is a solid ball of 16-20 cells with peripheral cells flattened against zona pellucida
* compaction occurs forming a cavity and leading to the next blastocyst stage

:'''Links:''' [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=hmg.figgrp.928 Figure 8.19. Changes in DNA methylation during mammalian development]

==Blastocyst==
[[File:Human embryo day 5 label.jpg|thumb|300px|Blastocyst (day 5)]]
* about '''day 5''' have 2 identifiable cell types and a fluid-filled cavity (blastoceol)
** outer cell layer - '''trophoblast''', peripheral flattened cells, forms the placenta and placental membranes
** inner cell mass - '''embryoblast''', mass of rounder cells located on one wall of the blastocoel, forms entire embryo

{| class="wikitable mw-collapsible mw-collapsed"
! Human Blastocyst Movies
|-
| <html5media height="470" width="500">File:Human_blastocyst_day_3-6.mp4</html5media>

[[Media:Human_blastocyst_day_3-6.mp4|'''Click Here''' to play on mobile device]]
|}

===Blastula Cell Communication===
Two forms of cellular junctions
* '''gap junctions''', allow electrically couple cells of epithelium surrounding a fluid-filled cavity
*''' tight junctions''', close to outer surface create a seal, isolates interior of embryo from external medium
{|
| [[File:Gap junction 01.jpg|400px]]
| [[File:Tight_junction_01.jpg|400px]]
|-
| Gap junctions
| Tight junctions
|}
===Blastocyst Hatching===
{| class="wikitable mw-collapsible mw-collapsed"
! Human Blastocyst Hatching Movie
|-
| <html5media height="470" width="500">File:Human_blastocyst_hatching_day_5-6.mp4</html5media>

[[Media:Human_blastocyst_hatching_day_5-6.mp4|'''Click Here''' to play on mobile device]] | [[Blastocyst_Hatching_Movie|Movie page]]
|}

[[File:Human_carnegie_stage_3_label.jpg|400px|Hatching Blastocyst]]

'''Blastocyst Hatching''' - zona pellucida lost, ZP has sperm entry site, and entire ZP broken down by uterine secretions and possibly blastula secretions.
'''Uterine Glands''' - secretions required for blastocyst motility and nutrition

[[File:Human_blastocyst_day_1-6.jpg|600px]]

:'''Links:''' [[Carnegie stage 3]] | [http://www.ncbi.nlm.nih.gov/books/NBK26863/figure/A3927 Figure 21-69. The blastula]

== Week 2 - Implantation ==
{| class="wikitable mw-collapsible mw-collapsed"
! Implantation Movie
|-
| width=520px|<html5media height="580" width="500">File:Week2_001.mp4</html5media>

[[Media:Week2_001.mp4|'''Click Here''' to play on mobile device]] | [[Implantation_Movie|Movie page]]
|}
The second week of human development is concerned with the process of implantation and the differentiation of the blastocyst into early embryonic and placental forming structures.
{|
|
* implantation commences about '''day 6 to 7'''
* '''Adplantation''' - begins with initial adhesion to the uterine epithelium
** blastocyst then slows in motility, "rolls" on surface, aligns with the inner cell mass closest to the epithelium and stops
* '''Implantation''' - migration of the blastocyst into the uterine epithelium, process complete by about '''day 9'''
** interaction between trophoblast cells and endometrial epithelium (apoptosis and decidualization)
* '''coagulation plug''' - left where the blastocyst has entered the uterine wall '''day 12'''

'''Normal Implantation Sites''' - in uterine wall superior, posterior, lateral
|
| valign="bottom"|{{Week 2 implant movie}}
|}

[[File:Stage5 bf11L.jpg|alt=Human embryo Carnegie stage 5|400px]]

===Endometrial Receptivity===
{|
|
* In humans, receptivity occurs 6 days after the post-ovulatory progesterone surge and lasts about 2 to 4 days.
** Similar "receptivity window" in other species (rat day 5 and mouse day 4.5).
* Many studies have looked into identifying markers for this receptivity period both to optimise and to block this process.
| [[File:Implantation_LIF.jpg|400px]]
|}
===Abnormal Implantation===
{| class="wikitable mw-collapsible mw-collapsed"
! Ectopic Ultrasound Movie
| <html5media height="550" width="660">File:Ectopic_01.mp4</html5media>

[[Media:Ectopic_01.mp4|'''Click Here''' to play on mobile device]]
|}

[[Image:Tubal pregnancy.gif|thumb|Ectopic tubal pregnancy]]

Abnormal implantation sites or Ectopic Pregnancy occurs if implantation is in uterine tube or outside the uterus.
* sites - external surface of uterus, ovary, bowel, gastrointestinal tract, mesentry, peritoneal wall
* If not spontaneous then, embryo has to be removed surgically
'''Tubal pregnancy''' - 94% of ectopic pregnancies
* if uterine epithelium is damaged (scarring, pelvic inflammatory disease)
* if zona pellucida is lost too early, allows premature tubal implantation
* embryo may develop through early stages, can erode through the uterine horn and reattach within the peritoneal cavity

{|
| [[File:Tubal_pregnancy_historic.jpg|200px]]
| [[File:Abnormal_implantation_sites.jpg|200px]]
|}

:'''Links:''' [[Implantation]] | [[Abnormal_Development_-_Ectopic_Implantation|Ectopic Pregnancy]] | [[Ultrasound_-_Ectopic_01|Movie - Ectopic pregnancy ultrasound]]

== Uterus ==
{|
|
[[File:Menstrual cycle.png|400px|Menstrual cycle]]

* Endometrium - 3 layers in secretory phase of menstrual cycle: compact, spongy, basal
* Myometrium - muscular layer outside endometrium, contracts in parturition
* Perimetrium - tunica serosa of the uterus continuous with the peritoneal wall
| [[File:Uterus proliferative phase.jpg]]
|}

<gallery>
Uterus proliferative phase.jpg|Uterus proliferative phase
Uterine gland proliferative phase.jpg|Uterine gland proliferative phase
Uterus secretory phase.jpg|Uterus secretory phase
Uterine gland secretory phase.jpg|Uterine gland secretory phase
</gallery>
===Endometrial Layers===
* Compact - implantation occurs in this layer, dense stromal cells, uterine gland necks, capillaries of spiral arteries
* Spongy - swollen stromal cells, uterine gland bodies, spiral arteries
* Basal - not lost during menstruation or childbirth, own blood supply

===Decidual Reaction===
* transformation of endometrial stromal cells
* occurs initially at site of implantation and includes both cellular and matrix changes
* reaction spreads throughout entire uterus, not at cervix
* deposition of fibrinoid and glycogen and epithelial plaque formation (at anchoring villi)
* presence of decidual cells are indicative of pregnancy
[[File:Bailey494.jpg|thumb|300px]]
===Other Uterine Changes===
* '''Cervix''' - at mouth of uterus, secretes mucus (CMP), forms a plug/barrier, mechanical and antibacterial
* '''Vascular''' - increased number of blood vessels

===Decidua===
The endometrium becomes the decidua and forms 3 distinct anatomical regions (at approx 3 weeks)
* Decidua Basalis at implantation site
* Decidua Capsularis enclosing the conceptus
* Decidua Parietalis the remainder of uterus
** Decidua Capsularis and Parietalis fuse eventually fuse and uterine cavity is lost by 12 weeks

=== Uterus Abnormalities ===
'''Endometriosis''' endometrial tissue located in other regions of the uterus or other tissues. This misplaced tissue develops into growths or lesions which respond to the menstrual cycle hormonal changes in the same way that the tissue of the uterine lining does; each month the tissue builds up, breaks down, and sheds.

== Conceptus==
===Bilaminar Embryoblast ===
{|
|
* about '''day 8 to 9'''
* The outer trophoblast and inner embryoblast layers now both differentiate to form two distinct cellular layers.
* The trophoblast layer forms the '''syncitotrophoblast''' and '''cytotrophoblast''' layers.
* The embryoblast (inner cell mass) forms the '''epiblast''' and '''hypoblast''' layers.
** '''Epiblast''' - will form the 3 germ layers.
** '''Hypoblast''' - transient layer replaced by endoderm.
* This early stage of embryo development is referred to as the '''bilaminar embryo'''.
| <html5media height="300" width="250">File:Chorion 001.mp4</html5media>

[[Development_Animation_-_Chorionic_Cavity|Movie - Week 2 Bilaminar Embryo]]
|}

===Bilaminar Trophoblast===
Two trophoblast layers Cytotrophoblast and Syncitiotrophoblast.

'''Cytotrophoblasts''' - form a continuous cellular layer that covers the developing placental villi.

'''Syncitiotrophoblasts'''
* '''secrete proteolytic enzymes''', enzymes break down extracellular matrix around cells
* Allow passage of blastocyst into endometrial wall, totally surround the blastocyst
* generate spaces that fill with maternal blood- lacunae
* '''secrete Human Chorionic Gonadotropin''' (hCG), hormone, maintains decidua and Corpus Luteum, basis of pregnancy diagnostic test, present in urine is diagnostic of pregnancy
** levels peak at 8 to 10 weeks of pregnancy, then decline and are lower for rest of pregnancy
** 1-2 months: 5,000-200,000 mIU/ml; Non-pregnant females: <5.0 mIU/ml; Postmenopausal females: <9.5 mIU/ml)
** Later in development placenta will secrete hCG

{|
| [[File:Human ovary - corpus luteum 21.jpg|250px]]
| [[File:Trophoblast_cell_hCG.jpg|250px]]
| [[File:Pregnancy test.gif|250px]]
|-
| Human ovary corpus luteum
| Trophoblast hCG
| Pregnancy Test Kit
|}

[[Image:Ovary_corpus_luteum.jpg|300px|Ovary corpus luteum]]

== Twinning ==

Twinning can be due to two separate fertilization events (dizygotic twins) or as an abnormality of a single fertilization (monozygotic twins) event during the early weeks of development.

===Dizygotic Twinning===
Dizygotic twins (fraternal, non-identical) arise from separate [[F#fertilization|fertilization]] events involving two separate [[O#oocyte|oocyte]] (egg, ova) and [[S#spermatozoa|spermatozoa]] (sperm).
* In '''dizygotic twinning''' the genetic material is different and implantation and placentation is also different.

===Monozygotic Twinning===
* In '''monozygotic twinning''' the genetic material is initially identical and degree of twinning will depend upon the timing (early to late) from separate fetal membranes and placenta to conjoined twins.
** morula stage (diamniotic dichorionic), early blastocyst (diamniotic monochorionic), late blastocyst to bilaminar (monoamniotic monochorionic), bilaminar to trilaminar embryo (conjoined)
* Monozygotic twins are a unique research resource for comparing environmental effects on development and health.
* Congenital abnormality statistics for twins is generally increased in various conditions.

Monoygotic twins (identical) produced from a single [[F#fertilization|fertilization]] event (one fertilised egg and a single spermatazoa, form a single zygote), these twins therefore share the same genetic makeup. Occurs in approximately 3-5 per 1000 pregnancies, more commonly with aged mothers. The later the twinning event, the less common are initially separate placental membranes and finally resulting in conjoined twins.

{| class="prettytable" 100%
| width=10% | '''Week'''
|
| colspan="7" | Week 1
| colspan="7" | Week 2

|-bgcolor="lightsteelblue"
| '''Day'''
| width=5% | 0
| width=5% | 1
| width=5% | 2
| width=5% | 3
| width=5% | 4
| width=5% | 5
| width=5% | 6
| width=5% | 7
| width=5% | 8
| width=5% | 9
| width=5% | 10
| width=5% | 11
| width=5% | 12
| width=5% | 13
| width=5% | 14

|-
| '''Cell Number'''
| 1
| 1
| 2
| 16
| 32
| 128
|
|
|
| bilaminar
|
|
|
|
|

|-
| '''Event'''
| Ovulation
| fertilization
| First cell division
| Morula
| Early blastocyst
| Late blastocyst

Hatching
| Implantation starts
|
|
| [[Molecular_Development_-_X_Inactivation|X inactivation]]
|
|
|
|
|

|-
|
| [[File:Follicle 001 icon.jpg|50px]]
| [[File:Early_zygote.jpg|50px]]
| [[File:Human embryo day 2.jpg|50px]]
| [[File:Human embryo day 3.jpg|50px]]
| [[File:Human embryo day 5.jpg|50px]]
| [[File:CSt3.jpg|50px]]
| [[File:Week2_001 icon.jpg|50px]]
|
|
|
|
|
|
|
|
|-bgcolor="lightsteelblue"
| '''Monoygotic'''

'''Twin Type'''
|
| colspan="3" | Diamniotic

Dichorionic
| colspan="3" | Diamniotic

Monochorionic
| colspan="3" | Monoamniotic

Monochorionic
| colspan="5" | Conjoined

|-
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|

|}

Table based upon recent Twinning Review.<ref><pubmed>12957099</pubmed></ref>

<references/>

:'''Links:''' [[Abnormal_Development_-_Twinning| Twinning]] | [http://www.twins.org.au/index.php?page=31 Australian Twin Registry]

Now watch the [[Week 1 Movie|Week 1 overview]].
{| class="wikitable mw-collapsible mw-collapsed"
! Week 1 Movie
|-
| <html5media height="260" width="660">File:Week1_001.mp4</html5media>

[[Media:Week1_001.mp4|'''Click Here''' to play on mobile device]]
|}

{{2015ANAT2341}}

[[Category:Science-Undergraduate]]

[[Category:Week 1]] [[Category:Week 2]]

User:Z3292373

2015-11-05T07:04:52Z

Z3292373:

'''The Completer Finisher'''
is a perfectionist and will often go the extra mile to make sure everything is "just right," and the things he or she delivers can be trusted to have been double-checked and then checked again. The Completer Finisher has a strong inward sense of the need for accuracy, and sets his or her own high standards rather than working on the encouragement of others. They may frustrate their teammates by worrying excessively about minor details and by refusing to delegate tasks that they do not trust anyone else to perform.

I had no idea that Oocytes and spermatozoa were not matured at the time of release, for no reason at all i assumed that it was bucket science and the two just needed mixing and "let them do their thing". Which is ridicules now that i think about it.

=Laboratory Work=

==Lab 1==
A summery of two recent research articles on fertility or fertilization.
=== Article One:Degradation of Paternal Mitochondria by Fertilization-Triggered Autophagy in ''C.elegans'' Embryos <ref><pubmed>21998252</pubmed></ref> ===
Miyuki Sato,Ken Sato

Using ''C.elegans'' as a model organism this paper investigates the possible mechanisms in which paternal mitochondria might be removed from the Oocyte after fertilisation.
Initially mitochondria from the spermatozoa were track through embriogenisis via marking with MitoTracker red (MT) and Heat Shock Green Fluorescence Protein (HS-GFP). This initial tracking showed paternal mitochondria entering the cell at fertilisation and being randomly inherited by blastomeres through to the 4 cell stage then clearing by the 16 cell stage.

To establish a link between this pattern and autophagy, autophagisomes with GFP marked homologue of a protein in their membrane (LGG-1) were fertilised. These autophagisomes were built up around the pronuclear paternal DNA (''C.eleigans'' spermatazoa have their mitochondria distributed around the head of the gamete and posses no tail) and then dispersed around the cytoplasm as the blastocyst developed. Like the paternal mitochondria the autophagisomes then cleared by the 16 cell stage as well as appearing sparadicaly in places related with regular development.

Further fertilisation with mutant sperm line ''spe-9'', that cannot produce the proteins for normal fertilisation, did not produce the initial induction of autophagy. Whilst insertion of sperm line ''spe-11'', that cannot fertilise but permit embryogenesis but show the same patterning of auto phagisomes around the paternal pronuclei. This suggested that induction of autophagy is based on the entry of paternal proteins.
By arresting the Oocyte in metaphase 1 by ''emb-27(RNAi)'' it was also shown that the induction happens independent of progress into anaphase 1.

Confirmation of the role of autophagy in the removal of paternal mitochondria was done by the use of mutants with compromised autophagy regulators. Gamete mutants ''lgg-1(tm3489)'' that could not produce the autophagicyte membrane could proceed through fertilisation at a reduced capacity however could not progress past the L1 larval stage. In these matings the paternal mitochondria persisted past the 16 cell stage.
In contrast if mutant Oocytes were mated with wild type spermatozoa then paternal mitochondria would be present until the Lima Bean stage and then cleared.
Spermatozoa that contained the same ''lgg-1'' mutation however had no change to the regular clearing of paternal mitochondria when mated with wildtype oocytes.
This suggested that the maternal autophagocytes were the main contributors to paternal mitochondrial clearing. However paternal ''lgg-1'' expression could, at a reduced capacity, compensate for compromised maternal autophagy.

=== Atricle Two: Developmental potential of zona pellucida–free oocytes obtained following mild in vitro fertilization <ref><pubmed>25256934</pubmed></ref> ===
Satoshi Ueno et al.

In this research, from a Japanese fertility clinic normal oocyte and zona free (ZF) oocytes where taken from the same patient in the same collection cycle. The ZF oocytes did not have an intrinsic absence but breakages of the zona palucida through which the oocyct was protruding. The oocyte was retrieved from the extrusion. These were then fertilised via intracytoplasmic sperm injection, cultured and followed through cleavage and blastocyst formation.

A comparison of the blastocyst formation from successfully fertilised ZF and normal oocytes showed no statistical difference in viability. The same was found of thoughs ZF derived blasticysts that were carried to full term.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:28, 4 September 2015 (AEST) These are accurate suppers of these 2 papers (5/5)

==Lab 2==
Introduction to addition of images to website.

[[File:Cells_of_the_innate_and_adaptive_immune_system_present_in_the_uterus_at_the_time_of_implantation.jpg|600px]]

Cells of the innate and adaptive immune system present in the uterus at the time of implantation <ref><pubmed>26136750</pubmed></ref>| [http://journal.frontiersin.org/article/10.3389/fimmu.2015.00321/abstract '''frontiers''' in Immunology]

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:30, 4 September 2015 (AEST) Image uploaded with correct reference, copyright and student template. Please in future use a briefer image title for example, File:Cells of the innate and adaptive immune system present in the uterus at the time of implantation.jpg, could have been simply File:Cells of the innate and adaptive immune system at implantation.jpg. (5/5)

==Lab 3==

Brief description of 3 research articles on your subject.

===Article 1:===
PMID 25629662 '''Mitocondrial donation--how many women could benefit?'''<ref><pubmed> 25629662 </pubmed></ref>

This is a statistical analysis of the prevalence of women of child bearing age that have pathogenic mutation to their mitochondria that could benefit from mitochondrial donation in the UK. And the affects of the mitochondrial mutation on fertility as compared to background natural birth rate. They found no difference in fertility rates and 4% of women at risk of passing on symptomatic mitochondrial disease.

===Article 2:===
PMID 20393463 '''Pronuclear tranfer in human embryos to prevent transmition of mitochondrial DNA disease'''<ref><pubmed> 20393463 </pubmed></ref>

As the name suggest this paper looks at pronuclear transfer as way to remove donor mitochondria measured by mt-DNA. And it effectiveness in doing so. And the processes that occur in the oocyte when this method is used.

===Article 3:===
PMID 18674747 '''Pathogenic mitochondrial DNA mutations are common in the general population.'''<ref><pubmed> 18674747 </pubmed></ref>
Another on the prevalence of mitochondrial mutations in the populous. This time via mtDNA sequencing from umbilical samples from live births, looking for ten specific mt-DNA mutations. It found a frequency rate of 0.54% for these mutations. Although they had limited data on the prevalence of these mutations maternally.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:32, 4 September 2015 (AEST) These are relevant references to your group project. (5/5)
==Lab 4==

Three question quiz on Placenta Development
===Take the Quiz===

<quiz display=simple>

{Which of these is '''NOT''' a part of Tertiary Chorionic Villi formation?
|type="()"}
- Mesenchyme differentiating into blood vessels and cells.
- Fusing with placental vessels.
+ Extra-embryonic mesoderm grows into the villi.
- All of the above
|| The ingress of extra-embryonic mesoderm is part of '''Secondary''' [[Placenta - Villi Development#Chorionic Villi|Chorionic Villi]] development.

{Which of these structures make up part of the maternal surface of the placenta?
|type="()"}
- Amniotic membrane.
+ Cotyledons
-Chorionic plate.
-Syncytiotrophoblasts

|| [[C#cotyledon|Cotyledons]] from the greek for "Cup,bowl" named after the proto leaf structure in germinating plants gives the leaf-like or cobblestone like appearance, on the [[Lecture - Placenta Development#Placenta at Birth|maternal surface]] of the placenta.

{What is the name given to the abnormal development of the placenta described by the attachment of the placenta deep into the uterine wall and also penetrating into the uterine muscle but not into the uterine serosa ?
|type="()"}
+ Placenta Increta.
- Placenta Accreta
- Placenta Percreta
- Placenta Previa

|| [[P#placenta accreta|Placenta Accreta]] occurs when the placenta adheres to the myometrium without inter-lying decidua basalis.In [[Lecture - Placenta Development#Placental Abnormalities|Placenta Percreta]] the placental villi penetrate all the way through to the Serosa. [[Lecture - Placenta Development#Placental Abnormalities|Placenta Previa]] occurs when the placenta adheres over the ostium (or external orifice) of the uterus blocking the birth canal.

</quiz>

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:33, 4 September 2015 (AEST) Only 2 questions. I will come back later. [[ANAT2341 Student 2015 Quiz Questions]]

{{Template:Quizzes}}

==Lab 5==

===Brief Overview of '''Hirschsprung's disease'''===

Hirschsprung's disease (HSCR) also know as ''congenital aganglionic megacolon'' or ''intestinal aganglionosis'' is a disorder of the gastrointestinal tract characterised by a lack of neurons in the intestinal tract (IT). Most commonly affecting regions of the colon and more distal sections of the hindgut although can be prevalent from the stomach to the rectum. This causes the inability of the enteric nervous system (ENS) to control secretions and blood-flow in the affected area as well as maintain peristalsis leading to sustained contraction of the smooth muscle and hence obstruction and distension of the bowel <ref><pubmed>17514199</pubmed></ref>. Clinically this is displayed by the absence of a meconium stool in the first 48 hours after birth and confirmed by radiological examination with a barium enema <ref><pubmed>6691093</pubmed></ref>. Treatment requires removal of the defective region via surgery and has many possible complications <ref><pubmed>9722005</pubmed></ref>.t

The ENS is derived from the Neural Crest. Vagal neural crest cells (NCC) contributing to the fore-,mid- and hind-gut<ref><pubmed>8565847</pubmed></ref>, Sacral NCC's contributing to the distal hindgut<ref><pubmed>9753687</pubmed></ref>. Malformations of it's development such as HSCR are termed neurocristopathies. In the case of HSCR, most commonly it a restriction in the migration and proliferation of the neural crest cells in early development (weeks 4-7) impeding their colonisation of the gut. The cause of this impediment is varied. Around half of cases can be linked to the GDNF/RET (glial cell line derived neurotrophic factor/receptor tyrosine kinase)gene families that regulate to progression of the NCC cells through the mesoderm <ref><pubmed>12399307</pubmed></ref>. Mutations in any number of these genes can lead to delay or inability of the NCCs in their progress rostro-cordauly.

Another cuase comes from disruption of Endothelin pathways that although also control migration, maintain the enteric NCC progenitors cells in their proliferative state <ref><pubmed>16624853</pubmed></ref>. Mutations in this pathway have shown to stop differentiation of the NCC cells at the distal bowel <ref><pubmed>16339294</pubmed></ref>, meaning although fully colonised the ENS cells have reduced excitatory fibres and abnormal neurotransmitter release.

Outside of genetic mutations retinal (vitamin A) deficiency has been link to HSCR <ref><pubmed>12702665</pubmed></ref>. Although vitamin A deficiency has been linked to numerous congenital defects of which HSCR is a small part. The complexity of systems that contribute the enteric nervous system mean that HSCR, although fairly understood as a disease, has many possible causes not yet linked.

==Lab 6==
Complete work on group project.

==Lab 7==
A brief description of the findings of a recent research paper on Odontogenisis.

=== Enamel hypomineralisation due to endocrine disruptors.===
jedeon K et al.<ref><pubmed>25158179</pubmed></ref>.

Endocrine disruptors and their possible health effects have been gaining a lot of traction in the media in the past decade. Most touted of these being the estrogen-like Bisphenol A (BPA) found in many plastic products. This study compared the affects of BPA and two other endocrine disruptors (EDs) genistein (G) and vinclozolin (V) on tooth development by use of mouse models and cell culture.

Their results showed that phenotypicaly rats exposed to BPA devolop the greatest degree of hypomineralisation, 75% of rats treated. Compared to lowest reading of 35% for though exposed to GV excluding the control group.
Further more in HAT-7 rat ameloblast-like cell culture and analysis with RT-qPCR it was shown that BPA was up-regulating enamelin mRNA levels and down-regulating klk4 mRNA. The reson that mixtures of EDs were not as disruptive as BPA alone was discovered to be link to V which had a down-regulation effect on enamelin mRNA promoters only.
It was suggested that there was most likely other target genes that the EDs affect that gave rise to there results.

==Lab 9==
2.Great stuff guys,
Good layout, nice flow of topics and comprehensive. Language is easy to follow. Well research and supported. There are couple of parts that need some references WIKI original recommends that if you make a statement of fact or something that can be disputed you should add a reference ie. last statement of epidemiology . But, work in progress, i understand.
You could hyperlink some of the more unfamiliar words to the UNSW embryology glossary and other pages to get the wiki "click through" effect.
I hate to recommend it because i really like how clean and "wiki" like your page is but we have to add images so perhaps a map of the genes and mutations, show the promoters and such?
The symptoms section has some repetition to its structure i think you should condense it all into the table then write a lead in paragraph to the table. Lead in could have a bit about when the symptoms usually come on in life?
I would recommend moving diagnosis to above treatment and after pathology to help with flow. This would also semi-separate the page into theory and clinical.
For the pathology image if you make a one by one table and put the image into it, it should sit in alignment on the page. Its just my browser but on a smaller screen it cuts out to the left. Not a big deal. try to have the images on a line to them selves or at the end of paragraphs rather then word wrapping the text. Makes it look neater no matter how big you have the window.
That's all i can think of. Other wise looks like it going to be one of the best of the class. Very professional.

3.This is really good and basically finished,
Images are relevant and informative, then drawn image is especially great. Flow and headings are spot on, nice table. Realy professional.
Not a huge amount to say on this one just a bit of housework to do before you publish.
Make sure any factual statements you have are refrenced
Id move the first image of the FRS so your not leading in with it. Drop the Definition subheading and just make in your introduction section. Makes it more wiki like. For a style guide have a look at the wiki page https://en.wikipedia.org/wiki/Ovarian_hyperstimulation_syndrome. Taking the underline from under the subheading will make it read nicer, but then I have a person hate of underlines.
You could add an epidemiology section to move to world map to and there’s lots information you could put in such a section.
Your image under pathogenesis is word wrapped to the left. I would change that to in line or word wrap to the right. Given the detail of the image I would suggest in line. Likewise with your rat specimen image bring it down under the heading. If you put it in its own box you could add some details about what we’re looking at in the image.
Topics like Environmental factors could use some more detail. Although I know were all still working on our pages.
Make sure to hyperlink unusual words or other topics mentioned to the sites glossary or pages for that lost clicking through wiki effect.
Apart from that another top notch page, great detail, really interesting and direct. Fantastic work guys.

4.
Great work guys, a lot of information in there.
Well referenced and easily readable. Nicely broken up with some informative images. Tables are great and make it easy to find information on a big page. And the first embedded video I’ve seen which is nice.
Looks like it all the information is there already just a couple of heading to finish off so well done.
Stylistically the only things ill pick on are,
The numbered list in treatments, doesn’t fit with the formatting of the rest of the page. Just turn them into subheading or tables like the others.
Some of the tables are standalone ie. Male infertility disorder that should really have some intro text to them. Also make sure they have a description underneath each one describing what’s in table. And reference of where the information’s coming from.
You could add a section on epidemiology/prevalence of types of infertility. But the intro could be enough if you flesh something out in there.
Don’t forget to hyperlink some of the important words to the sites glossary and other pages so people can find extra reading or background. And I think you’ll have a really informative page.
Fantastic work guys.

5.
Hey guys been working hard I see,
Lots of information, well referenced (chemotherapy section pending) and really interesting stuff.
The layouts a bit hard to follow, I’d suggest dropping things down a heading level so that, example, infertility was the heading, with page beak. And targeted drugs surgery etc. wear a third level head and just bold.
At times the page seems to go off on a tangent such as how chemo is administered. Try to tie it in with what relation it has on onco-fertility or consider leaving it out and just linking to further information on the subject.
For the above to points have a look at a wiki page that also deals with a dense subject like the world war 2 page https://en.wikipedia.org/wiki/World_War_II look at what they’ve done to group the information and break out blocks of text.
The list of drugs at the end would go much nicer into a table after leading in with the text. I recommend you rotate you tables too so that they read left to right rather than top to bottom ie switch rows and columns and then put the data into bullet list in each table area rather than dashing out the excess space.
Most of the images are great and interesting (oocytes and tissues), Some don’t add much to the page (DNA breaks, Lady get intravenous). Have a look through them and think about whether the image provides any extra information, assists understanding of topic or is eye-catching, attention grabbing, if it doesn’t do one of the three grab another image.
Consider adding some hyperlinks to the sites glossary and other pages of site key words.
It’s all there and the information’s really good. If you work out the formatting so the text is easily digestible you’ll have a great page.

6.
Fantastic job guys, this ones hard to pick on.
Layout is great, flows, logical topics, easy to read, nicely broken up with informative or interesting images. Just smashing. References appear to all be solid.
So what can I suggest? You could try making up a table for the advantages and diss advantages so it’s easy to compare each tech. The PCR Cycle copies table is just a list of exponential growth and can go.
Hyperlink some of your key words to the glossary or other pages so people can get background or further reading on topics involved.
Change the heading to spell out Assisted Repro… etc. so that its stated before you abbreviate. Collect up the info to make your intro and so far you have the best project of the group. Pretty flawless guys.

==Lab 10==
===Hyaloid Vascular System===

Link to permalink image: [https://embryology.med.unsw.edu.au/embryology/Slides/Embryo_Stages/Stage22/08-eye/Stage22-08-eye.html?zoom=5&lat=-3273&lon=4370&layers=B | Hyaloid Vascular System]

The hyaloid vascular system (HVS) is a network of blood vessels, supplied by the hyaloid artery and extending from the optic disk to the superior part of the retina. These nourish the eye during development . This structure is transient disappearing before birth, but is commonly present in premature infants<ref><pubmed> 23772130 </pubmed></ref>. Persistence post nataly leads to severe blindness. It is a common target of studies into the signaling pathways of vascuralisation and regression.

'''Embryology link''' [[Vision - Retina Development #Week 8]]

==Lab 11==
Still no sign of the CATI form, so here's my pledge to do it when it comes out.
And here it is, almost forgot
[[File:Proof of eval of ANAT2341.jpg|600px]]

==Lab Attendance==
--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:45, 7 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:31, 14 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:43, 21 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:36, 28 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:20, 4 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:08, 18 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 14:00, 25 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:55, 9 October 2015 (AEDT)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:45, 16 October 2015 (AEDT)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:21, 23 October 2015 (AEDT)

=Notes=
[[Test student 2015]]

{{Uploading Images in 5 Easy Steps table}}

[http://www.biomedcentral.com]

{{StudentPage2015}}

=References=

File:Proof of eval of ANAT2341.jpg

2015-11-05T07:00:14Z

Z3292373: just a proof that a student did the coarse evaluation. feel free to delete after 2015

just a proof that a student did the coarse evaluation. feel free to delete after 2015

User:Z3292373

2015-10-29T05:21:59Z

Z3292373:

'''The Completer Finisher'''
is a perfectionist and will often go the extra mile to make sure everything is "just right," and the things he or she delivers can be trusted to have been double-checked and then checked again. The Completer Finisher has a strong inward sense of the need for accuracy, and sets his or her own high standards rather than working on the encouragement of others. They may frustrate their teammates by worrying excessively about minor details and by refusing to delegate tasks that they do not trust anyone else to perform.

I had no idea that Oocytes and spermatozoa were not matured at the time of release, for no reason at all i assumed that it was bucket science and the two just needed mixing and "let them do their thing". Which is ridicules now that i think about it.

=Laboratory Work=

==Lab 1==
A summery of two recent research articles on fertility or fertilization.
=== Article One:Degradation of Paternal Mitochondria by Fertilization-Triggered Autophagy in ''C.elegans'' Embryos <ref><pubmed>21998252</pubmed></ref> ===
Miyuki Sato,Ken Sato

Using ''C.elegans'' as a model organism this paper investigates the possible mechanisms in which paternal mitochondria might be removed from the Oocyte after fertilisation.
Initially mitochondria from the spermatozoa were track through embriogenisis via marking with MitoTracker red (MT) and Heat Shock Green Fluorescence Protein (HS-GFP). This initial tracking showed paternal mitochondria entering the cell at fertilisation and being randomly inherited by blastomeres through to the 4 cell stage then clearing by the 16 cell stage.

To establish a link between this pattern and autophagy, autophagisomes with GFP marked homologue of a protein in their membrane (LGG-1) were fertilised. These autophagisomes were built up around the pronuclear paternal DNA (''C.eleigans'' spermatazoa have their mitochondria distributed around the head of the gamete and posses no tail) and then dispersed around the cytoplasm as the blastocyst developed. Like the paternal mitochondria the autophagisomes then cleared by the 16 cell stage as well as appearing sparadicaly in places related with regular development.

Further fertilisation with mutant sperm line ''spe-9'', that cannot produce the proteins for normal fertilisation, did not produce the initial induction of autophagy. Whilst insertion of sperm line ''spe-11'', that cannot fertilise but permit embryogenesis but show the same patterning of auto phagisomes around the paternal pronuclei. This suggested that induction of autophagy is based on the entry of paternal proteins.
By arresting the Oocyte in metaphase 1 by ''emb-27(RNAi)'' it was also shown that the induction happens independent of progress into anaphase 1.

Confirmation of the role of autophagy in the removal of paternal mitochondria was done by the use of mutants with compromised autophagy regulators. Gamete mutants ''lgg-1(tm3489)'' that could not produce the autophagicyte membrane could proceed through fertilisation at a reduced capacity however could not progress past the L1 larval stage. In these matings the paternal mitochondria persisted past the 16 cell stage.
In contrast if mutant Oocytes were mated with wild type spermatozoa then paternal mitochondria would be present until the Lima Bean stage and then cleared.
Spermatozoa that contained the same ''lgg-1'' mutation however had no change to the regular clearing of paternal mitochondria when mated with wildtype oocytes.
This suggested that the maternal autophagocytes were the main contributors to paternal mitochondrial clearing. However paternal ''lgg-1'' expression could, at a reduced capacity, compensate for compromised maternal autophagy.

=== Atricle Two: Developmental potential of zona pellucida–free oocytes obtained following mild in vitro fertilization <ref><pubmed>25256934</pubmed></ref> ===
Satoshi Ueno et al.

In this research, from a Japanese fertility clinic normal oocyte and zona free (ZF) oocytes where taken from the same patient in the same collection cycle. The ZF oocytes did not have an intrinsic absence but breakages of the zona palucida through which the oocyct was protruding. The oocyte was retrieved from the extrusion. These were then fertilised via intracytoplasmic sperm injection, cultured and followed through cleavage and blastocyst formation.

A comparison of the blastocyst formation from successfully fertilised ZF and normal oocytes showed no statistical difference in viability. The same was found of thoughs ZF derived blasticysts that were carried to full term.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:28, 4 September 2015 (AEST) These are accurate suppers of these 2 papers (5/5)

==Lab 2==
Introduction to addition of images to website.

[[File:Cells_of_the_innate_and_adaptive_immune_system_present_in_the_uterus_at_the_time_of_implantation.jpg|600px]]

Cells of the innate and adaptive immune system present in the uterus at the time of implantation <ref><pubmed>26136750</pubmed></ref>| [http://journal.frontiersin.org/article/10.3389/fimmu.2015.00321/abstract '''frontiers''' in Immunology]

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:30, 4 September 2015 (AEST) Image uploaded with correct reference, copyright and student template. Please in future use a briefer image title for example, File:Cells of the innate and adaptive immune system present in the uterus at the time of implantation.jpg, could have been simply File:Cells of the innate and adaptive immune system at implantation.jpg. (5/5)

==Lab 3==

Brief description of 3 research articles on your subject.

===Article 1:===
PMID 25629662 '''Mitocondrial donation--how many women could benefit?'''<ref><pubmed> 25629662 </pubmed></ref>

This is a statistical analysis of the prevalence of women of child bearing age that have pathogenic mutation to their mitochondria that could benefit from mitochondrial donation in the UK. And the affects of the mitochondrial mutation on fertility as compared to background natural birth rate. They found no difference in fertility rates and 4% of women at risk of passing on symptomatic mitochondrial disease.

===Article 2:===
PMID 20393463 '''Pronuclear tranfer in human embryos to prevent transmition of mitochondrial DNA disease'''<ref><pubmed> 20393463 </pubmed></ref>

As the name suggest this paper looks at pronuclear transfer as way to remove donor mitochondria measured by mt-DNA. And it effectiveness in doing so. And the processes that occur in the oocyte when this method is used.

===Article 3:===
PMID 18674747 '''Pathogenic mitochondrial DNA mutations are common in the general population.'''<ref><pubmed> 18674747 </pubmed></ref>
Another on the prevalence of mitochondrial mutations in the populous. This time via mtDNA sequencing from umbilical samples from live births, looking for ten specific mt-DNA mutations. It found a frequency rate of 0.54% for these mutations. Although they had limited data on the prevalence of these mutations maternally.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:32, 4 September 2015 (AEST) These are relevant references to your group project. (5/5)
==Lab 4==

Three question quiz on Placenta Development
===Take the Quiz===

<quiz display=simple>

{Which of these is '''NOT''' a part of Tertiary Chorionic Villi formation?
|type="()"}
- Mesenchyme differentiating into blood vessels and cells.
- Fusing with placental vessels.
+ Extra-embryonic mesoderm grows into the villi.
- All of the above
|| The ingress of extra-embryonic mesoderm is part of '''Secondary''' [[Placenta - Villi Development#Chorionic Villi|Chorionic Villi]] development.

{Which of these structures make up part of the maternal surface of the placenta?
|type="()"}
- Amniotic membrane.
+ Cotyledons
-Chorionic plate.
-Syncytiotrophoblasts

|| [[C#cotyledon|Cotyledons]] from the greek for "Cup,bowl" named after the proto leaf structure in germinating plants gives the leaf-like or cobblestone like appearance, on the [[Lecture - Placenta Development#Placenta at Birth|maternal surface]] of the placenta.

{What is the name given to the abnormal development of the placenta described by the attachment of the placenta deep into the uterine wall and also penetrating into the uterine muscle but not into the uterine serosa ?
|type="()"}
+ Placenta Increta.
- Placenta Accreta
- Placenta Percreta
- Placenta Previa

|| [[P#placenta accreta|Placenta Accreta]] occurs when the placenta adheres to the myometrium without inter-lying decidua basalis.In [[Lecture - Placenta Development#Placental Abnormalities|Placenta Percreta]] the placental villi penetrate all the way through to the Serosa. [[Lecture - Placenta Development#Placental Abnormalities|Placenta Previa]] occurs when the placenta adheres over the ostium (or external orifice) of the uterus blocking the birth canal.

</quiz>

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:33, 4 September 2015 (AEST) Only 2 questions. I will come back later. [[ANAT2341 Student 2015 Quiz Questions]]

{{Template:Quizzes}}

==Lab 5==

===Brief Overview of '''Hirschsprung's disease'''===

Hirschsprung's disease (HSCR) also know as ''congenital aganglionic megacolon'' or ''intestinal aganglionosis'' is a disorder of the gastrointestinal tract characterised by a lack of neurons in the intestinal tract (IT). Most commonly affecting regions of the colon and more distal sections of the hindgut although can be prevalent from the stomach to the rectum. This causes the inability of the enteric nervous system (ENS) to control secretions and blood-flow in the affected area as well as maintain peristalsis leading to sustained contraction of the smooth muscle and hence obstruction and distension of the bowel <ref><pubmed>17514199</pubmed></ref>. Clinically this is displayed by the absence of a meconium stool in the first 48 hours after birth and confirmed by radiological examination with a barium enema <ref><pubmed>6691093</pubmed></ref>. Treatment requires removal of the defective region via surgery and has many possible complications <ref><pubmed>9722005</pubmed></ref>.t

The ENS is derived from the Neural Crest. Vagal neural crest cells (NCC) contributing to the fore-,mid- and hind-gut<ref><pubmed>8565847</pubmed></ref>, Sacral NCC's contributing to the distal hindgut<ref><pubmed>9753687</pubmed></ref>. Malformations of it's development such as HSCR are termed neurocristopathies. In the case of HSCR, most commonly it a restriction in the migration and proliferation of the neural crest cells in early development (weeks 4-7) impeding their colonisation of the gut. The cause of this impediment is varied. Around half of cases can be linked to the GDNF/RET (glial cell line derived neurotrophic factor/receptor tyrosine kinase)gene families that regulate to progression of the NCC cells through the mesoderm <ref><pubmed>12399307</pubmed></ref>. Mutations in any number of these genes can lead to delay or inability of the NCCs in their progress rostro-cordauly.

Another cuase comes from disruption of Endothelin pathways that although also control migration, maintain the enteric NCC progenitors cells in their proliferative state <ref><pubmed>16624853</pubmed></ref>. Mutations in this pathway have shown to stop differentiation of the NCC cells at the distal bowel <ref><pubmed>16339294</pubmed></ref>, meaning although fully colonised the ENS cells have reduced excitatory fibres and abnormal neurotransmitter release.

Outside of genetic mutations retinal (vitamin A) deficiency has been link to HSCR <ref><pubmed>12702665</pubmed></ref>. Although vitamin A deficiency has been linked to numerous congenital defects of which HSCR is a small part. The complexity of systems that contribute the enteric nervous system mean that HSCR, although fairly understood as a disease, has many possible causes not yet linked.

==Lab 6==
Complete work on group project.

==Lab 7==
A brief description of the findings of a recent research paper on Odontogenisis.

=== Enamel hypomineralisation due to endocrine disruptors.===
jedeon K et al.<ref><pubmed>25158179</pubmed></ref>.

Endocrine disruptors and their possible health effects have been gaining a lot of traction in the media in the past decade. Most touted of these being the estrogen-like Bisphenol A (BPA) found in many plastic products. This study compared the affects of BPA and two other endocrine disruptors (EDs) genistein (G) and vinclozolin (V) on tooth development by use of mouse models and cell culture.

Their results showed that phenotypicaly rats exposed to BPA devolop the greatest degree of hypomineralisation, 75% of rats treated. Compared to lowest reading of 35% for though exposed to GV excluding the control group.
Further more in HAT-7 rat ameloblast-like cell culture and analysis with RT-qPCR it was shown that BPA was up-regulating enamelin mRNA levels and down-regulating klk4 mRNA. The reson that mixtures of EDs were not as disruptive as BPA alone was discovered to be link to V which had a down-regulation effect on enamelin mRNA promoters only.
It was suggested that there was most likely other target genes that the EDs affect that gave rise to there results.

==Lab 9==
2.Great stuff guys,
Good layout, nice flow of topics and comprehensive. Language is easy to follow. Well research and supported. There are couple of parts that need some references WIKI original recommends that if you make a statement of fact or something that can be disputed you should add a reference ie. last statement of epidemiology . But, work in progress, i understand.
You could hyperlink some of the more unfamiliar words to the UNSW embryology glossary and other pages to get the wiki "click through" effect.
I hate to recommend it because i really like how clean and "wiki" like your page is but we have to add images so perhaps a map of the genes and mutations, show the promoters and such?
The symptoms section has some repetition to its structure i think you should condense it all into the table then write a lead in paragraph to the table. Lead in could have a bit about when the symptoms usually come on in life?
I would recommend moving diagnosis to above treatment and after pathology to help with flow. This would also semi-separate the page into theory and clinical.
For the pathology image if you make a one by one table and put the image into it, it should sit in alignment on the page. Its just my browser but on a smaller screen it cuts out to the left. Not a big deal. try to have the images on a line to them selves or at the end of paragraphs rather then word wrapping the text. Makes it look neater no matter how big you have the window.
That's all i can think of. Other wise looks like it going to be one of the best of the class. Very professional.

3.This is really good and basically finished,
Images are relevant and informative, then drawn image is especially great. Flow and headings are spot on, nice table. Realy professional.
Not a huge amount to say on this one just a bit of housework to do before you publish.
Make sure any factual statements you have are refrenced
Id move the first image of the FRS so your not leading in with it. Drop the Definition subheading and just make in your introduction section. Makes it more wiki like. For a style guide have a look at the wiki page https://en.wikipedia.org/wiki/Ovarian_hyperstimulation_syndrome. Taking the underline from under the subheading will make it read nicer, but then I have a person hate of underlines.
You could add an epidemiology section to move to world map to and there’s lots information you could put in such a section.
Your image under pathogenesis is word wrapped to the left. I would change that to in line or word wrap to the right. Given the detail of the image I would suggest in line. Likewise with your rat specimen image bring it down under the heading. If you put it in its own box you could add some details about what we’re looking at in the image.
Topics like Environmental factors could use some more detail. Although I know were all still working on our pages.
Make sure to hyperlink unusual words or other topics mentioned to the sites glossary or pages for that lost clicking through wiki effect.
Apart from that another top notch page, great detail, really interesting and direct. Fantastic work guys.

4.
Great work guys, a lot of information in there.
Well referenced and easily readable. Nicely broken up with some informative images. Tables are great and make it easy to find information on a big page. And the first embedded video I’ve seen which is nice.
Looks like it all the information is there already just a couple of heading to finish off so well done.
Stylistically the only things ill pick on are,
The numbered list in treatments, doesn’t fit with the formatting of the rest of the page. Just turn them into subheading or tables like the others.
Some of the tables are standalone ie. Male infertility disorder that should really have some intro text to them. Also make sure they have a description underneath each one describing what’s in table. And reference of where the information’s coming from.
You could add a section on epidemiology/prevalence of types of infertility. But the intro could be enough if you flesh something out in there.
Don’t forget to hyperlink some of the important words to the sites glossary and other pages so people can find extra reading or background. And I think you’ll have a really informative page.
Fantastic work guys.

5.
Hey guys been working hard I see,
Lots of information, well referenced (chemotherapy section pending) and really interesting stuff.
The layouts a bit hard to follow, I’d suggest dropping things down a heading level so that, example, infertility was the heading, with page beak. And targeted drugs surgery etc. wear a third level head and just bold.
At times the page seems to go off on a tangent such as how chemo is administered. Try to tie it in with what relation it has on onco-fertility or consider leaving it out and just linking to further information on the subject.
For the above to points have a look at a wiki page that also deals with a dense subject like the world war 2 page https://en.wikipedia.org/wiki/World_War_II look at what they’ve done to group the information and break out blocks of text.
The list of drugs at the end would go much nicer into a table after leading in with the text. I recommend you rotate you tables too so that they read left to right rather than top to bottom ie switch rows and columns and then put the data into bullet list in each table area rather than dashing out the excess space.
Most of the images are great and interesting (oocytes and tissues), Some don’t add much to the page (DNA breaks, Lady get intravenous). Have a look through them and think about whether the image provides any extra information, assists understanding of topic or is eye-catching, attention grabbing, if it doesn’t do one of the three grab another image.
Consider adding some hyperlinks to the sites glossary and other pages of site key words.
It’s all there and the information’s really good. If you work out the formatting so the text is easily digestible you’ll have a great page.

6.
Fantastic job guys, this ones hard to pick on.
Layout is great, flows, logical topics, easy to read, nicely broken up with informative or interesting images. Just smashing. References appear to all be solid.
So what can I suggest? You could try making up a table for the advantages and diss advantages so it’s easy to compare each tech. The PCR Cycle copies table is just a list of exponential growth and can go.
Hyperlink some of your key words to the glossary or other pages so people can get background or further reading on topics involved.
Change the heading to spell out Assisted Repro… etc. so that its stated before you abbreviate. Collect up the info to make your intro and so far you have the best project of the group. Pretty flawless guys.

==Lab 10==
===Hyaloid Vascular System===

Link to permalink image: [https://embryology.med.unsw.edu.au/embryology/Slides/Embryo_Stages/Stage22/08-eye/Stage22-08-eye.html?zoom=5&lat=-3273&lon=4370&layers=B | Hyaloid Vascular System]

The hyaloid vascular system (HVS) is a network of blood vessels, supplied by the hyaloid artery and extending from the optic disk to the superior part of the retina. These nourish the eye during development . This structure is transient disappearing before birth, but is commonly present in premature infants<ref><pubmed> 23772130 </pubmed></ref>. Persistence post nataly leads to severe blindness. It is a common target of studies into the signaling pathways of vascuralisation and regression.

'''Embryology link''' [[Vision - Retina Development #Week 8]]

==Lab 11==
Still no sign of the CATI form, so here's my pledge to do it when it comes out.

==Lab Attendance==
--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:45, 7 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:31, 14 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:43, 21 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:36, 28 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:20, 4 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:08, 18 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 14:00, 25 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:55, 9 October 2015 (AEDT)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:45, 16 October 2015 (AEDT)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:21, 23 October 2015 (AEDT)

=Notes=
[[Test student 2015]]

{{Uploading Images in 5 Easy Steps table}}

[http://www.biomedcentral.com]

{{StudentPage2015}}

=References=

2015 Group Project 1

2015-10-23T16:14:58Z

Z3292373: /* Glossary */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leigh Syndrome.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical progression=
Three-person in-vitro fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. The main approaches to achieve this goal involve the replacement of the mitochondrial genome between gametes or embryos<ref name=pmid24382342><pubmed> 24382342</pubmed></ref><ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. However, this approach is considered to be inadequate to prevent the inheritance of diseased mitochondria because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes<ref name=pmid24382342/>.
*Newly emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. However, none of these techniques have been proven in generating healthy human offspring due to the technical difficulty, as well as the ethical issues being recognized worldwide<ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate)<ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies<ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in-vitro fertilization (IVF) technique that introduces a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocytes or zygotes from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the development of the embryo<ref><pubmed> 15140871 </pubmed></ref>. Following ovulation, the survival of the zygote depends almost exclusively on maternal messenger RNA and proteins that accumulate during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8 cell stage in humans, where the new zygote genome is activated and replaces the maternal cytoplasm to become predominant in regulating the zygote development<ref><pubmed> 3352746 </pubmed></ref>. The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome<ref><pubmed> 10429238 </pubmed></ref>.

Researchers are still investigating the molecular and cellular mechanisms by which ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of an oocyte or repair of an embryo<ref name=pmid24382342/><ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>.

In method one, the cytoplasm is withdrawn from a donor’s oocyte and then injected into a patient’s oocyte together with the sperm cells that will then fertilize the oocyte. In method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified cell structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|Embryo repair by cytoplasmic transfer <ref name='3egirl'/>]]
|}
===Cytoplasmic transfer cases in humans<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>===
{|

|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risks of cytoplasmic transfer – Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one mitochondrial DNA (mtDNA) type within the cytoplasm of an individual<ref><pubmed>20735895</pubmed></ref><ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals. However, human mtDNA sequencing has now shown that each person has some low-frequency karyotypes of mtDNA, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cells. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy<ref name=PMID23271951><pubmed>23271951</pubmed></ref>.
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing<ref name=PMID23271951/>.]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low-frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA<ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedures''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns that arises regarding cytoplasmic transfer in IVF procedure<ref><pubmed>16939888</pubmed></ref>. Severe diseases can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system<ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations<ref><pubmed>24709341</pubmed></ref>.

==Spindle-chromosome transfer==

Spindle-chromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background<ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families<ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to create both cattle and mice after subsequent fertilization, and has resulted in live monkeys (Macaca mulatta) after sperm injection<ref name=PMID25573721/>. Spindle transfer between human oocytes has also resulted in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy<ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to assess the safety of this procedure. Tachibana et al. (2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - Removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to threadlike spindle fibers (the spindle–chromosomal complex).
*b - Transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - Fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - Developing blastocyst was implanted in a surrogate mother.
*e - Mother gave birth to a healthy baby.

Some of the resulting embryos were successful and resulted in healthy offspring with low mtDNA carryover. However, it is still too early to determine whether spindle-chromosome transfer is a safe procedure because defects may develop later in life, or in subsequent generations. Thus long-term studies are required to assess the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer<ref name=pmid19710649/>.]]
|-
|}

===Current research===
Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. "''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''". Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human pre-implantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities<ref name=PMID25472922><pubmed> 25472922</pubmed></ref><ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==
Pronuclear transfer is similar to maternal spindle transfer but is performed as a repair of an embryo. The mother’s egg is first fertilized and then the nuclear DNA is transferred to a fertilized donor egg containing healthy mitochondria, from which the donor's original nuclear DNA has been removed<ref name=PMID25573721/>.

*Pronuclear transfer procedures were first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA<ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003, scientists at Sun Yat-Sen University in China were the first to attempt this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses<ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010, researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days. This marked the procedure as a success in preventing mitochondrial disease<ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer<ref name=PMID25573721/>.]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote<ref name ='humanmodel2003'/>.
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human embryo model===

A research group at Newcastle University performed pronuclear transfer on human embryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilized human zygotes generated following in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres.

The safety effects of this procedure (zygote mtDNA carryover) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities.

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research<ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice models. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth<ref><pubmed>16275929</pubmed></ref>. Human embryo model studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos<ref name=pmid20393463/>. One disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar body transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amounts of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|An early human zygote<ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>.]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases<ref name=PMID25472922/>.

The advantages of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* Each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significantly increases the efficiency of using a donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer<ref name=PMID25472922/>.]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer<ref name=PMID25472922/>.]]
|}

===Mice model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicates that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
Other research has coupled polar body transfer with pronuclei transfer or spindle-choromosome transfer on a mice model, which increased the yield of reconstructed embryos with low mtDNA carryover<ref name=PMID25573721/>.

==Other approaches==

===Germinal vesicle nuclear transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte<ref><pubmed>19924284</pubmed></ref>.]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocyte arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization<ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal vesicle transfer (GVT)''' is the transfer of a GV from an unfertilized oocyte into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to metaphase II through in-vitro maturation (IVM) and is then fertilized through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before being transferred to a patient<ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for women whose oocytes fail to fertilize, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques, a major concern of GVT is that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the mitochondrial gene transfer techniques are ethical. The Nuffield Council on Bioethics in the United Kingdom examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders . . . we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”<ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of mitochondrial gene transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that, unlike the use of donor eggs or embryos, children born with mitochondrial gene transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with mitochondrial gene transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the children's point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identities they are aware that they share a mitochondrial genome with a donor. Second, children conceived through mitochondrial gene transfer may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents<ref><pubmed>26239841</pubmed></ref>.

=Legal status=
==Permitted==

The United Kingdom is the only country that legally allows the inheritable genetic modification of humans. On 24 February 2015, the House of Lords approved regulations. Earlier in the month, the House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against<ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>.

==Under discussion==

In the United States, the legality of mitochondrial manipulation techniques is still under discussion. On 25 and 26 February 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On 27 January 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

[*] These countries have the same law, "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention.

=Further reading=

*The ethics of creating children with three genetic parents. PMID 23608245

*Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases. PMID 24382342

*Mitochondrial function in the human oocyte and embryo and their role in developmental competence. PMID 20933103

*Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord. PMID 26020522

*The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility. PMID 25421171

*Risks inherent to mitochondrial replacement. PMID 25807984

=Glossary=
'''Cortical necrosis''' Breakdown of the kidney tissue.

'''Heteroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements.

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA.

'''Maternal spindle transfer''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue.

'''Ooplasmic transfer''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear transfer''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T16:13:29Z

Z3292373: /* Further reading */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leigh Syndrome.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical progression=
Three-person in-vitro fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. The main approaches to achieve this goal involve the replacement of the mitochondrial genome between gametes or embryos<ref name=pmid24382342><pubmed> 24382342</pubmed></ref><ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. However, this approach is considered to be inadequate to prevent the inheritance of diseased mitochondria because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes<ref name=pmid24382342/>.
*Newly emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. However, none of these techniques have been proven in generating healthy human offspring due to the technical difficulty, as well as the ethical issues being recognized worldwide<ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate)<ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies<ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in-vitro fertilization (IVF) technique that introduces a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocytes or zygotes from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the development of the embryo<ref><pubmed> 15140871 </pubmed></ref>. Following ovulation, the survival of the zygote depends almost exclusively on maternal messenger RNA and proteins that accumulate during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8 cell stage in humans, where the new zygote genome is activated and replaces the maternal cytoplasm to become predominant in regulating the zygote development<ref><pubmed> 3352746 </pubmed></ref>. The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome<ref><pubmed> 10429238 </pubmed></ref>.

Researchers are still investigating the molecular and cellular mechanisms by which ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of an oocyte or repair of an embryo<ref name=pmid24382342/><ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>.

In method one, the cytoplasm is withdrawn from a donor’s oocyte and then injected into a patient’s oocyte together with the sperm cells that will then fertilize the oocyte. In method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified cell structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|Embryo repair by cytoplasmic transfer <ref name='3egirl'/>]]
|}
===Cytoplasmic transfer cases in humans<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>===
{|

|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risks of cytoplasmic transfer – Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one mitochondrial DNA (mtDNA) type within the cytoplasm of an individual<ref><pubmed>20735895</pubmed></ref><ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals. However, human mtDNA sequencing has now shown that each person has some low-frequency karyotypes of mtDNA, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cells. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy<ref name=PMID23271951><pubmed>23271951</pubmed></ref>.
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing<ref name=PMID23271951/>.]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low-frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA<ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedures''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns that arises regarding cytoplasmic transfer in IVF procedure<ref><pubmed>16939888</pubmed></ref>. Severe diseases can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system<ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations<ref><pubmed>24709341</pubmed></ref>.

==Spindle-chromosome transfer==

Spindle-chromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background<ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families<ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to create both cattle and mice after subsequent fertilization, and has resulted in live monkeys (Macaca mulatta) after sperm injection<ref name=PMID25573721/>. Spindle transfer between human oocytes has also resulted in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy<ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to assess the safety of this procedure. Tachibana et al. (2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - Removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to threadlike spindle fibers (the spindle–chromosomal complex).
*b - Transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - Fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - Developing blastocyst was implanted in a surrogate mother.
*e - Mother gave birth to a healthy baby.

Some of the resulting embryos were successful and resulted in healthy offspring with low mtDNA carryover. However, it is still too early to determine whether spindle-chromosome transfer is a safe procedure because defects may develop later in life, or in subsequent generations. Thus long-term studies are required to assess the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer<ref name=pmid19710649/>.]]
|-
|}

===Current research===
Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. "''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''". Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human pre-implantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities<ref name=PMID25472922><pubmed> 25472922</pubmed></ref><ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==
Pronuclear transfer is similar to maternal spindle transfer but is performed as a repair of an embryo. The mother’s egg is first fertilized and then the nuclear DNA is transferred to a fertilized donor egg containing healthy mitochondria, from which the donor's original nuclear DNA has been removed<ref name=PMID25573721/>.

*Pronuclear transfer procedures were first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA<ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003, scientists at Sun Yat-Sen University in China were the first to attempt this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses<ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010, researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days. This marked the procedure as a success in preventing mitochondrial disease<ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer<ref name=PMID25573721/>.]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote<ref name ='humanmodel2003'/>.
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human embryo model===

A research group at Newcastle University performed pronuclear transfer on human embryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilized human zygotes generated following in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres.

The safety effects of this procedure (zygote mtDNA carryover) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities.

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research<ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice models. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth<ref><pubmed>16275929</pubmed></ref>. Human embryo model studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos<ref name=pmid20393463/>. One disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar body transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amounts of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|An early human zygote<ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>.]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases<ref name=PMID25472922/>.

The advantages of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* Each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significantly increases the efficiency of using a donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer<ref name=PMID25472922/>.]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer<ref name=PMID25472922/>.]]
|}

===Mice model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicates that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
Other research has coupled polar body transfer with pronuclei transfer or spindle-choromosome transfer on a mice model, which increased the yield of reconstructed embryos with low mtDNA carryover<ref name=PMID25573721/>.

==Other approaches==

===Germinal vesicle nuclear transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte<ref><pubmed>19924284</pubmed></ref>.]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocyte arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization<ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal vesicle transfer (GVT)''' is the transfer of a GV from an unfertilized oocyte into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to metaphase II through in-vitro maturation (IVM) and is then fertilized through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before being transferred to a patient<ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for women whose oocytes fail to fertilize, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques, a major concern of GVT is that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the mitochondrial gene transfer techniques are ethical. The Nuffield Council on Bioethics in the United Kingdom examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders . . . we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”<ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of mitochondrial gene transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that, unlike the use of donor eggs or embryos, children born with mitochondrial gene transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with mitochondrial gene transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the children's point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identities they are aware that they share a mitochondrial genome with a donor. Second, children conceived through mitochondrial gene transfer may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents<ref><pubmed>26239841</pubmed></ref>.

=Legal status=
==Permitted==

The United Kingdom is the only country that legally allows the inheritable genetic modification of humans. On 24 February 2015, the House of Lords approved regulations. Earlier in the month, the House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against<ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>.

==Under discussion==

In the United States, the legality of mitochondrial manipulation techniques is still under discussion. On 25 and 26 February 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On 27 January 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

[*] These countries have the same law, "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention.

=Further reading=

*The ethics of creating children with three genetic parents. PMID 23608245

*Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases. PMID 24382342

*Mitochondrial function in the human oocyte and embryo and their role in developmental competence. PMID 20933103

*Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord. PMID 26020522

*The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility. PMID 25421171

*Risks inherent to mitochondrial replacement. PMID 25807984

=Glossary=
'''Cortical necrosis''' Breakdown of the kidney tissue.

'''Heteroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements.

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA.

'''Maternal Spindle Transfer''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue.

'''Ooplasmic Transfer''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T16:10:22Z

Z3292373: /* Legal status */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leigh Syndrome.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical progression=
Three-person in-vitro fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. The main approaches to achieve this goal involve the replacement of the mitochondrial genome between gametes or embryos<ref name=pmid24382342><pubmed> 24382342</pubmed></ref><ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. However, this approach is considered to be inadequate to prevent the inheritance of diseased mitochondria because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes<ref name=pmid24382342/>.
*Newly emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. However, none of these techniques have been proven in generating healthy human offspring due to the technical difficulty, as well as the ethical issues being recognized worldwide<ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate)<ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies<ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in-vitro fertilization (IVF) technique that introduces a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocytes or zygotes from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the development of the embryo<ref><pubmed> 15140871 </pubmed></ref>. Following ovulation, the survival of the zygote depends almost exclusively on maternal messenger RNA and proteins that accumulate during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8 cell stage in humans, where the new zygote genome is activated and replaces the maternal cytoplasm to become predominant in regulating the zygote development<ref><pubmed> 3352746 </pubmed></ref>. The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome<ref><pubmed> 10429238 </pubmed></ref>.

Researchers are still investigating the molecular and cellular mechanisms by which ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of an oocyte or repair of an embryo<ref name=pmid24382342/><ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>.

In method one, the cytoplasm is withdrawn from a donor’s oocyte and then injected into a patient’s oocyte together with the sperm cells that will then fertilize the oocyte. In method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified cell structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|Embryo repair by cytoplasmic transfer <ref name='3egirl'/>]]
|}
===Cytoplasmic transfer cases in humans<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>===
{|

|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risks of cytoplasmic transfer – Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one mitochondrial DNA (mtDNA) type within the cytoplasm of an individual<ref><pubmed>20735895</pubmed></ref><ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals. However, human mtDNA sequencing has now shown that each person has some low-frequency karyotypes of mtDNA, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cells. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy<ref name=PMID23271951><pubmed>23271951</pubmed></ref>.
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing<ref name=PMID23271951/>.]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low-frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA<ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedures''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns that arises regarding cytoplasmic transfer in IVF procedure<ref><pubmed>16939888</pubmed></ref>. Severe diseases can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system<ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations<ref><pubmed>24709341</pubmed></ref>.

==Spindle-chromosome transfer==

Spindle-chromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background<ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families<ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to create both cattle and mice after subsequent fertilization, and has resulted in live monkeys (Macaca mulatta) after sperm injection<ref name=PMID25573721/>. Spindle transfer between human oocytes has also resulted in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy<ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to assess the safety of this procedure. Tachibana et al. (2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - Removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to threadlike spindle fibers (the spindle–chromosomal complex).
*b - Transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - Fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - Developing blastocyst was implanted in a surrogate mother.
*e - Mother gave birth to a healthy baby.

Some of the resulting embryos were successful and resulted in healthy offspring with low mtDNA carryover. However, it is still too early to determine whether spindle-chromosome transfer is a safe procedure because defects may develop later in life, or in subsequent generations. Thus long-term studies are required to assess the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer<ref name=pmid19710649/>.]]
|-
|}

===Current research===
Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. "''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''". Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human pre-implantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities<ref name=PMID25472922><pubmed> 25472922</pubmed></ref><ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==
Pronuclear transfer is similar to maternal spindle transfer but is performed as a repair of an embryo. The mother’s egg is first fertilized and then the nuclear DNA is transferred to a fertilized donor egg containing healthy mitochondria, from which the donor's original nuclear DNA has been removed<ref name=PMID25573721/>.

*Pronuclear transfer procedures were first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA<ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003, scientists at Sun Yat-Sen University in China were the first to attempt this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses<ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010, researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days. This marked the procedure as a success in preventing mitochondrial disease<ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer<ref name=PMID25573721/>.]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote<ref name ='humanmodel2003'/>.
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human embryo model===

A research group at Newcastle University performed pronuclear transfer on human embryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilized human zygotes generated following in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres.

The safety effects of this procedure (zygote mtDNA carryover) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities.

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research<ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice models. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth<ref><pubmed>16275929</pubmed></ref>. Human embryo model studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos<ref name=pmid20393463/>. One disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar body transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amounts of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|An early human zygote<ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>.]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases<ref name=PMID25472922/>.

The advantages of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* Each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significantly increases the efficiency of using a donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer<ref name=PMID25472922/>.]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer<ref name=PMID25472922/>.]]
|}

===Mice model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicates that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
Other research has coupled polar body transfer with pronuclei transfer or spindle-choromosome transfer on a mice model, which increased the yield of reconstructed embryos with low mtDNA carryover<ref name=PMID25573721/>.

==Other approaches==

===Germinal vesicle nuclear transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte<ref><pubmed>19924284</pubmed></ref>.]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocyte arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization<ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal vesicle transfer (GVT)''' is the transfer of a GV from an unfertilized oocyte into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to metaphase II through in-vitro maturation (IVM) and is then fertilized through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before being transferred to a patient<ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for women whose oocytes fail to fertilize, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques, a major concern of GVT is that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the mitochondrial gene transfer techniques are ethical. The Nuffield Council on Bioethics in the United Kingdom examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders . . . we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”<ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of mitochondrial gene transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that, unlike the use of donor eggs or embryos, children born with mitochondrial gene transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with mitochondrial gene transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the children's point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identities they are aware that they share a mitochondrial genome with a donor. Second, children conceived through mitochondrial gene transfer may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents<ref><pubmed>26239841</pubmed></ref>.

=Legal status=
==Permitted==

The United Kingdom is the only country that legally allows the inheritable genetic modification of humans. On 24 February 2015, the House of Lords approved regulations. Earlier in the month, the House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against<ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>.

==Under discussion==

In the United States, the legality of mitochondrial manipulation techniques is still under discussion. On 25 and 26 February 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On 27 January 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

[*] These countries have the same law, "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention.

=Further reading=

*The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

*Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

*Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

*Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

*The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

*Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Breakdown of the kidney tissue.

'''Heteroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements.

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA.

'''Maternal Spindle Transfer''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue.

'''Ooplasmic Transfer''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T16:04:58Z

Z3292373: /* Legal Status */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leigh Syndrome.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical progression=
Three-person in-vitro fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. The main approaches to achieve this goal involve the replacement of the mitochondrial genome between gametes or embryos<ref name=pmid24382342><pubmed> 24382342</pubmed></ref><ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. However, this approach is considered to be inadequate to prevent the inheritance of diseased mitochondria because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes<ref name=pmid24382342/>.
*Newly emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. However, none of these techniques have been proven in generating healthy human offspring due to the technical difficulty, as well as the ethical issues being recognized worldwide<ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate)<ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies<ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in-vitro fertilization (IVF) technique that introduces a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocytes or zygotes from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the development of the embryo<ref><pubmed> 15140871 </pubmed></ref>. Following ovulation, the survival of the zygote depends almost exclusively on maternal messenger RNA and proteins that accumulate during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8 cell stage in humans, where the new zygote genome is activated and replaces the maternal cytoplasm to become predominant in regulating the zygote development<ref><pubmed> 3352746 </pubmed></ref>. The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome<ref><pubmed> 10429238 </pubmed></ref>.

Researchers are still investigating the molecular and cellular mechanisms by which ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of an oocyte or repair of an embryo<ref name=pmid24382342/><ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>.

In method one, the cytoplasm is withdrawn from a donor’s oocyte and then injected into a patient’s oocyte together with the sperm cells that will then fertilize the oocyte. In method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified cell structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|Embryo repair by cytoplasmic transfer <ref name='3egirl'/>]]
|}
===Cytoplasmic transfer cases in humans<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>===
{|

|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risks of cytoplasmic transfer – Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one mitochondrial DNA (mtDNA) type within the cytoplasm of an individual<ref><pubmed>20735895</pubmed></ref><ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals. However, human mtDNA sequencing has now shown that each person has some low-frequency karyotypes of mtDNA, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cells. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy<ref name=PMID23271951><pubmed>23271951</pubmed></ref>.
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing<ref name=PMID23271951/>.]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low-frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA<ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedures''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns that arises regarding cytoplasmic transfer in IVF procedure<ref><pubmed>16939888</pubmed></ref>. Severe diseases can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system<ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations<ref><pubmed>24709341</pubmed></ref>.

==Spindle-chromosome transfer==

Spindle-chromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background<ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families<ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to create both cattle and mice after subsequent fertilization, and has resulted in live monkeys (Macaca mulatta) after sperm injection<ref name=PMID25573721/>. Spindle transfer between human oocytes has also resulted in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy<ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to assess the safety of this procedure. Tachibana et al. (2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - Removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to threadlike spindle fibers (the spindle–chromosomal complex).
*b - Transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - Fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - Developing blastocyst was implanted in a surrogate mother.
*e - Mother gave birth to a healthy baby.

Some of the resulting embryos were successful and resulted in healthy offspring with low mtDNA carryover. However, it is still too early to determine whether spindle-chromosome transfer is a safe procedure because defects may develop later in life, or in subsequent generations. Thus long-term studies are required to assess the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer<ref name=pmid19710649/>.]]
|-
|}

===Current research===
Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. "''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''". Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human pre-implantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities<ref name=PMID25472922><pubmed> 25472922</pubmed></ref><ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==
Pronuclear transfer is similar to maternal spindle transfer but is performed as a repair of an embryo. The mother’s egg is first fertilized and then the nuclear DNA is transferred to a fertilized donor egg containing healthy mitochondria, from which the donor's original nuclear DNA has been removed<ref name=PMID25573721/>.

*Pronuclear transfer procedures were first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA<ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003, scientists at Sun Yat-Sen University in China were the first to attempt this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses<ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010, researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days. This marked the procedure as a success in preventing mitochondrial disease<ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer<ref name=PMID25573721/>.]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote<ref name ='humanmodel2003'/>.
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human embryo model===

A research group at Newcastle University performed pronuclear transfer on human embryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilized human zygotes generated following in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres.

The safety effects of this procedure (zygote mtDNA carryover) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities.

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research<ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice models. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth<ref><pubmed>16275929</pubmed></ref>. Human embryo model studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos<ref name=pmid20393463/>. One disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar body transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amounts of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|An early human zygote<ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>.]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases<ref name=PMID25472922/>.

The advantages of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* Each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significantly increases the efficiency of using a donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer<ref name=PMID25472922/>.]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer<ref name=PMID25472922/>.]]
|}

===Mice model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicates that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
Other research has coupled polar body transfer with pronuclei transfer or spindle-choromosome transfer on a mice model, which increased the yield of reconstructed embryos with low mtDNA carryover<ref name=PMID25573721/>.

==Other approaches==

===Germinal vesicle nuclear transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte<ref><pubmed>19924284</pubmed></ref>.]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocyte arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization<ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal vesicle transfer (GVT)''' is the transfer of a GV from an unfertilized oocyte into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to metaphase II through in-vitro maturation (IVM) and is then fertilized through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before being transferred to a patient<ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for women whose oocytes fail to fertilize, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques, a major concern of GVT is that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the mitochondrial gene transfer techniques are ethical. The Nuffield Council on Bioethics in the United Kingdom examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders . . . we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”<ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of mitochondrial gene transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that, unlike the use of donor eggs or embryos, children born with mitochondrial gene transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with mitochondrial gene transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the children's point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identities they are aware that they share a mitochondrial genome with a donor. Second, children conceived through mitochondrial gene transfer may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents<ref><pubmed>26239841</pubmed></ref>.

=Legal status=
==Permitted==

The United Kingdom is the only country that legally allows the inheritable genetic modification of humans. On 24 February 2015, the House of Lords approved regulations. Earlier in the month, the House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against<ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>.

==Under discussion==

In the United States, the legality of mitochondrial manipulation techniques is still under discussion. On 25 and 26 February 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On 27 January 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

*<nowiki> These countries have the same law, "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention.

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T15:59:40Z

Z3292373: /* Ethics */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leigh Syndrome.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical progression=
Three-person in-vitro fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. The main approaches to achieve this goal involve the replacement of the mitochondrial genome between gametes or embryos<ref name=pmid24382342><pubmed> 24382342</pubmed></ref><ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. However, this approach is considered to be inadequate to prevent the inheritance of diseased mitochondria because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes<ref name=pmid24382342/>.
*Newly emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. However, none of these techniques have been proven in generating healthy human offspring due to the technical difficulty, as well as the ethical issues being recognized worldwide<ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate)<ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies<ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in-vitro fertilization (IVF) technique that introduces a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocytes or zygotes from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the development of the embryo<ref><pubmed> 15140871 </pubmed></ref>. Following ovulation, the survival of the zygote depends almost exclusively on maternal messenger RNA and proteins that accumulate during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8 cell stage in humans, where the new zygote genome is activated and replaces the maternal cytoplasm to become predominant in regulating the zygote development<ref><pubmed> 3352746 </pubmed></ref>. The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome<ref><pubmed> 10429238 </pubmed></ref>.

Researchers are still investigating the molecular and cellular mechanisms by which ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of an oocyte or repair of an embryo<ref name=pmid24382342/><ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>.

In method one, the cytoplasm is withdrawn from a donor’s oocyte and then injected into a patient’s oocyte together with the sperm cells that will then fertilize the oocyte. In method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified cell structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|Embryo repair by cytoplasmic transfer <ref name='3egirl'/>]]
|}
===Cytoplasmic transfer cases in humans<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>===
{|

|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risks of cytoplasmic transfer – Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one mitochondrial DNA (mtDNA) type within the cytoplasm of an individual<ref><pubmed>20735895</pubmed></ref><ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals. However, human mtDNA sequencing has now shown that each person has some low-frequency karyotypes of mtDNA, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cells. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy<ref name=PMID23271951><pubmed>23271951</pubmed></ref>.
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing<ref name=PMID23271951/>.]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low-frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA<ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedures''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns that arises regarding cytoplasmic transfer in IVF procedure<ref><pubmed>16939888</pubmed></ref>. Severe diseases can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system<ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations<ref><pubmed>24709341</pubmed></ref>.

==Spindle-chromosome transfer==

Spindle-chromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background<ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families<ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to create both cattle and mice after subsequent fertilization, and has resulted in live monkeys (Macaca mulatta) after sperm injection<ref name=PMID25573721/>. Spindle transfer between human oocytes has also resulted in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy<ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to assess the safety of this procedure. Tachibana et al. (2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - Removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to threadlike spindle fibers (the spindle–chromosomal complex).
*b - Transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - Fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - Developing blastocyst was implanted in a surrogate mother.
*e - Mother gave birth to a healthy baby.

Some of the resulting embryos were successful and resulted in healthy offspring with low mtDNA carryover. However, it is still too early to determine whether spindle-chromosome transfer is a safe procedure because defects may develop later in life, or in subsequent generations. Thus long-term studies are required to assess the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer<ref name=pmid19710649/>.]]
|-
|}

===Current research===
Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. "''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''". Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human pre-implantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities<ref name=PMID25472922><pubmed> 25472922</pubmed></ref><ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==
Pronuclear transfer is similar to maternal spindle transfer but is performed as a repair of an embryo. The mother’s egg is first fertilized and then the nuclear DNA is transferred to a fertilized donor egg containing healthy mitochondria, from which the donor's original nuclear DNA has been removed<ref name=PMID25573721/>.

*Pronuclear transfer procedures were first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA<ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003, scientists at Sun Yat-Sen University in China were the first to attempt this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses<ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010, researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days. This marked the procedure as a success in preventing mitochondrial disease<ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer<ref name=PMID25573721/>.]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote<ref name ='humanmodel2003'/>.
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human embryo model===

A research group at Newcastle University performed pronuclear transfer on human embryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilized human zygotes generated following in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres.

The safety effects of this procedure (zygote mtDNA carryover) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities.

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research<ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice models. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth<ref><pubmed>16275929</pubmed></ref>. Human embryo model studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos<ref name=pmid20393463/>. One disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar body transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amounts of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|An early human zygote<ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>.]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases<ref name=PMID25472922/>.

The advantages of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* Each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significantly increases the efficiency of using a donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer<ref name=PMID25472922/>.]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer<ref name=PMID25472922/>.]]
|}

===Mice model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicates that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
Other research has coupled polar body transfer with pronuclei transfer or spindle-choromosome transfer on a mice model, which increased the yield of reconstructed embryos with low mtDNA carryover<ref name=PMID25573721/>.

==Other approaches==

===Germinal vesicle nuclear transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte<ref><pubmed>19924284</pubmed></ref>.]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocyte arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization<ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal vesicle transfer (GVT)''' is the transfer of a GV from an unfertilized oocyte into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to metaphase II through in-vitro maturation (IVM) and is then fertilized through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before being transferred to a patient<ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for women whose oocytes fail to fertilize, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques, a major concern of GVT is that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the mitochondrial gene transfer techniques are ethical. The Nuffield Council on Bioethics in the United Kingdom examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders . . . we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”<ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of mitochondrial gene transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that, unlike the use of donor eggs or embryos, children born with mitochondrial gene transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with mitochondrial gene transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the children's point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identities they are aware that they share a mitochondrial genome with a donor. Second, children conceived through mitochondrial gene transfer may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents<ref><pubmed>26239841</pubmed></ref>.

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T15:55:24Z

Z3292373: /* Technical Progression */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leigh Syndrome.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical progression=
Three-person in-vitro fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. The main approaches to achieve this goal involve the replacement of the mitochondrial genome between gametes or embryos<ref name=pmid24382342><pubmed> 24382342</pubmed></ref><ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. However, this approach is considered to be inadequate to prevent the inheritance of diseased mitochondria because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes<ref name=pmid24382342/>.
*Newly emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. However, none of these techniques have been proven in generating healthy human offspring due to the technical difficulty, as well as the ethical issues being recognized worldwide<ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate)<ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies<ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in-vitro fertilization (IVF) technique that introduces a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocytes or zygotes from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the development of the embryo<ref><pubmed> 15140871 </pubmed></ref>. Following ovulation, the survival of the zygote depends almost exclusively on maternal messenger RNA and proteins that accumulate during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8 cell stage in humans, where the new zygote genome is activated and replaces the maternal cytoplasm to become predominant in regulating the zygote development<ref><pubmed> 3352746 </pubmed></ref>. The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome<ref><pubmed> 10429238 </pubmed></ref>.

Researchers are still investigating the molecular and cellular mechanisms by which ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of an oocyte or repair of an embryo<ref name=pmid24382342/><ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>.

In method one, the cytoplasm is withdrawn from a donor’s oocyte and then injected into a patient’s oocyte together with the sperm cells that will then fertilize the oocyte. In method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified cell structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|Embryo repair by cytoplasmic transfer <ref name='3egirl'/>]]
|}
===Cytoplasmic transfer cases in humans<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>===
{|

|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risks of cytoplasmic transfer – Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one mitochondrial DNA (mtDNA) type within the cytoplasm of an individual<ref><pubmed>20735895</pubmed></ref><ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals. However, human mtDNA sequencing has now shown that each person has some low-frequency karyotypes of mtDNA, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cells. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy<ref name=PMID23271951><pubmed>23271951</pubmed></ref>.
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing<ref name=PMID23271951/>.]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low-frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA<ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedures''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns that arises regarding cytoplasmic transfer in IVF procedure<ref><pubmed>16939888</pubmed></ref>. Severe diseases can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system<ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations<ref><pubmed>24709341</pubmed></ref>.

==Spindle-chromosome transfer==

Spindle-chromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background<ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families<ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to create both cattle and mice after subsequent fertilization, and has resulted in live monkeys (Macaca mulatta) after sperm injection<ref name=PMID25573721/>. Spindle transfer between human oocytes has also resulted in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy<ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to assess the safety of this procedure. Tachibana et al. (2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - Removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to threadlike spindle fibers (the spindle–chromosomal complex).
*b - Transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - Fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - Developing blastocyst was implanted in a surrogate mother.
*e - Mother gave birth to a healthy baby.

Some of the resulting embryos were successful and resulted in healthy offspring with low mtDNA carryover. However, it is still too early to determine whether spindle-chromosome transfer is a safe procedure because defects may develop later in life, or in subsequent generations. Thus long-term studies are required to assess the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer<ref name=pmid19710649/>.]]
|-
|}

===Current research===
Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. "''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''". Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human pre-implantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities<ref name=PMID25472922><pubmed> 25472922</pubmed></ref><ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==
Pronuclear transfer is similar to maternal spindle transfer but is performed as a repair of an embryo. The mother’s egg is first fertilized and then the nuclear DNA is transferred to a fertilized donor egg containing healthy mitochondria, from which the donor's original nuclear DNA has been removed<ref name=PMID25573721/>.

*Pronuclear transfer procedures were first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA<ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003, scientists at Sun Yat-Sen University in China were the first to attempt this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses<ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010, researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days. This marked the procedure as a success in preventing mitochondrial disease<ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer<ref name=PMID25573721/>.]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote<ref name ='humanmodel2003'/>.
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human embryo model===

A research group at Newcastle University performed pronuclear transfer on human embryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilized human zygotes generated following in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres.

The safety effects of this procedure (zygote mtDNA carryover) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities.

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research<ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice models. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth<ref><pubmed>16275929</pubmed></ref>. Human embryo model studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos<ref name=pmid20393463/>. One disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar body transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amounts of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|An early human zygote<ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>.]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases<ref name=PMID25472922/>.

The advantages of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* Each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significantly increases the efficiency of using a donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer<ref name=PMID25472922/>.]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer<ref name=PMID25472922/>.]]
|}

===Mice model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicates that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
Other research has coupled polar body transfer with pronuclei transfer or spindle-choromosome transfer on a mice model, which increased the yield of reconstructed embryos with low mtDNA carryover<ref name=PMID25573721/>.

==Other approaches==

===Germinal vesicle nuclear transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte<ref><pubmed>19924284</pubmed></ref>.]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocyte arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization<ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal vesicle transfer (GVT)''' is the transfer of a GV from an unfertilized oocyte into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to metaphase II through in-vitro maturation (IVM) and is then fertilized through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before being transferred to a patient<ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for women whose oocytes fail to fertilize, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques, a major concern of GVT is that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T14:46:48Z

Z3292373: /* 2010s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leigh Syndrome.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T14:45:01Z

Z3292373: /* Mitochondrial mutation and benefits of mitochondrial donation */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leighs Disease.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell, although they are also involved in signalling, differentiation, cell cycle, cell development, neuronal function and many other functions<ref name=pmid2830540/>. In mammals, mitochondria contain their own circular genome encoding for 37 genes, of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA, over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number of mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contains mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body, clinical presentations are also wide-ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone who is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot, however, prevent inheritance of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment, any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that, per year, 152 women in the United Kingdom and 778 in the United States are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorders===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells, as described in the table bellow. The higher the level of mtDNA mutation in the parent cell, the greater the likelihood the daughter cell will receive a random distribution mutated mtDNA above the threshold<ref name="PMID1463006"/>. When this occurs during meiotic cell division, the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood, there has been shown to be a selective pressure against germ-line cells with an accumulation of deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for heteroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref>, suggesting some mechanisms of mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes, the mitochondria of the parent distributes randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to completely affected. ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production, but symptoms can include deafness, vision loss, diabetes and organ failure. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers Disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]].
| nDNA gene mutation
| style="background-color: salmon;"|No
|-
| Kearns-Sayre Syndrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| [[2015 Group Project 1#Glossary|Pigmentary retinopathy]], conduction block, ataxia. Can cause mental retardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|20% of Cases
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|No
|-bgcolor="F5FAFF"
| Myoclonus Epilepsy with Ragged Red Fibers (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dementia.
| mtDNA point mutation
| style="background-color: lime;"|Yes
|-
| Neuropathy, Ataxia and Retinitis Pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa.
| mtDNA 6-gene mutation
| style="background-color: lime;"|Yes
|-bgcolor="F5FAFF"
| Pearson Syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre Syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|Yes
|-
| Progressive External Opthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be a distinct syndrome or part of greater mitochondrial disorder.
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|Most Cases
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T14:20:12Z

Z3292373: /* History */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>.
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice<ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified<ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>.

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>.
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer<ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model<ref><pubmed> 9674999 </pubmed></ref>.

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place<ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref>.
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>.

===2010s===
::* '''2010, United Kingdom''' - Craven et al. show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011, United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Leighs Disease.
::* '''2014, United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015, United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref>.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T14:14:43Z

Z3292373: /* 2000s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2001, United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials have been performed. At this time it was estimated from the eight clinics offering the service, 30 live births had taken place <ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref> .
::* '''2002, United States''' - One of the children conceived through ooplasmic transfer was diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the ''Human Fertilization and Embryology Act'' allows research into the techniques of three person IVF.
::*'''2009, United States''' - First successful trials of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys.<ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2010 United Kingdom''' - Craven et al. Show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011 United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Lieghs Disease.
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref> .
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T14:11:32Z

Z3292373: /* 1990s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interest in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment, however, was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized and brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2001 United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials had been preformed. At this time it was estimated from the 8 clinics offering the service 30 live births had taken place <ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref> .
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2010 United Kingdom''' - Craven et al. Show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011 United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Lieghs Disease.
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref> .
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T14:08:01Z

Z3292373: /* 1990s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the ''Human Fertilization and Embryology Act 1990''. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the regulatory body "Human Fertilization and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' -Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment however was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2001 United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials had been preformed. At this time it was estimated from the 8 clinics offering the service 30 live births had taken place <ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref> .
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2010 United Kingdom''' - Craven et al. Show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011 United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Lieghs Disease.
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref> .
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T12:05:05Z

Z3292373: /* 2010s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the reglatory body "Human Ferilisation and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' -Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment however was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2001 United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials had been preformed. At this time it was estimated from the 8 clinics offering the service 30 live births had taken place <ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref> .
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2010 United Kingdom''' - Craven et al. Show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2011 United Kingdom''' - Leading researcher Edward Bernardi dies of mitochondrial disorder Lieghs Disease.
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK<ref> The Human Ferilisation and Embryology (Mitochondrial Donation) Regulation 2015, 29.10.15, UK, sourced http://www.legislation.gov.uk/ukdsi/2015/9780111125816/introduction 02.10.2015</ref> .
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T11:55:43Z

Z3292373: /* 2000s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the reglatory body "Human Ferilisation and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' -Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment however was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2001 United States''' - The FDA announce a stop to cytoplasmic transfer until clinical trials had been preformed. At this time it was estimated from the 8 clinics offering the service 30 live births had taken place <ref> FDA BRMAC Briefing Document for Day 1 May 9. 2002, retrieved from http://www.fda.gov/ohrms/dockets/ac/02/briefing/3855b1_01.pdf 23/10/15</ref> .
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2010 United Kingdom''' - Craven et al. Show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T11:43:10Z

Z3292373: /* 1990s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the reglatory body "Human Ferilisation and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' -Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>. This treatment however was in response to infertility and not directly correlated with mitochondria mutation.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2010 United Kingdom''' - Craven et al. Show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T11:33:08Z

Z3292373: /* 2010s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the reglatory body "Human Ferilisation and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2010 United Kingdom''' - Craven et al. Show that pronuclear transfer could eliminate defective mitochondria<ref><pubmed> 20393463 </pubmed></ref>.
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
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| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
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| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
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=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
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=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
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| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
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| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
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| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
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| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
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| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
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| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
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| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
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| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T11:20:25Z

Z3292373: /* 1990s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) '''Teenage Girl Has Three Biological Parents.''' Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref name=pmid2830540><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implementation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present. This legislation also establishes the reglatory body "Human Ferilisation and Embryology Authority (HEFA)<ref>'''Human Fertilization and Embryology Act 1990 c.37''', retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents at 23 Oct 2015</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref name=pmid19710649><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref name=pmid2830540/>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref name=pmid16814712><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref name=pmid16814712/>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref name=pmid20393463><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref name=pmid24382342><pubmed> 24382342</pubmed></ref> <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref name=pmid24382342/>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref name=pmid24382342/> <ref name='3egirl'> Pritchard, C. (2014). '''The girl with three biological parents''' retrieved from http://www.bbc.com/news/magazine-28986843 at 23 Oct 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref name=pmid6896904/>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref name=pmid6896904/>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref name=pmid9250192/>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref name=pmid9570273><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref name=pmid9250192/> <ref name=pmid9570273/> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref name=PMID23271951/>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref name=pmid23103867><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization, and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721/>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref name=pmid23103867/>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref name=PMID25472922><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) '''Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne''' FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> Connor, S. (2015). '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World.''' retrieved from http://www.geneticsandsociety.org/article.php?id=8314. at 23 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref name=pmid20393463/>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) '''Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research.''' Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref name=pmid20393463/>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref name=pmid24949971><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) '''Embryology Early zygote labelled.jpg.''' retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg at 23 Oct 2015</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922/>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref name=pmid24949971/>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref name=PMID25573721/>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group (2005). '''Germinal vesicle transfer''' SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> Watts, G; Braude, P; Flinter, F; Harding, S; Lewens, T; Parker, M. (2012). '''Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.''' retrieved from http://nuffieldbioethics.org/report/techniques-prevention-mitochondrial-dna-disorders-ethical-review/conclusions-ethical-considerations/. at 23 Oct 2015. </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, J. (2015). '''MPs say yes to three-person babies.''' retrieved from http://www.bbc.com/news/health-31069173 at 09 Oct 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref name=pmid24382342/>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T07:37:48Z

Z3292373: /* 2010s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made on the efficacy, but agreements were made on further practice in animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=PMID19710649><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=PMID19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research. Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref name=PMID25472922><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref name=PMID25472922/>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref name=PMID25472922/> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref name=PMID25472922/> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T07:09:38Z

Z3292373: /* Glossary */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=PMID19710649><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=PMID19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research. Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
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| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
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| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
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| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
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| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''Cortical necrosis''' Break down of the kidney tisssue.

'''Hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''Homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T07:07:11Z

Z3292373: /* 1980s */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref name=pmid6896904><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref name=PMID23271951><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref name=PMID23271951/> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref name=PMID25573721><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref name=PMID25573721/>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=PMID19710649><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=PMID19710649/>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref name=PMID25573721/>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research. Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''cortical necrosis''' Break down of the kidney tisssue.

'''hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

Talk:2015 Group Project 1

2015-10-23T03:00:54Z

Z3292373: /* Useful resources */

{{ANAT2341Project2015discussionheader}}

==Stem cell presentation==
Hi, I have listed some papers which I am interested in doing because it is highly relevant to my own project. But I am more than happy if you post other papers and topics which interest you and we can work on them together and get ready earlier.

PMID 26295456

PMID 26439174

PMID 24837661

PMID 26418893

'''PMID 24981862'''

Hey, my pick would be C. Sturgeon et al. Wnt Signaling. purely on ease of doing a review. PMID 24837661. If that suites people. --[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 15:51, 12 October 2015 (AEDT)
==Useful resources==

Here is a good source for overview and status of 3 Person IVF. http://www.geneticsandsociety.org/article.php?id=6527

first ref = <ref name="PMIDXXXXX"><pubmed>XXXXX</pubmed></ref>

second =<ref name="PMIDXXXXX"/>

==Mitochondria==
*discovered in muscle by Kölliker in 1857
*mitochondria are the "powerhouses" of the cell and the location where respiration occurs at the cellular level.
*mitochondria contain their own DNA (mitochondrial DNA or mtDNA) that has been originally inherited only from the oocyte (maternal inheritance).
*The spermatozoa (paternal) mitochondria- energy for fertilization motility but are generally destroyed during the first mitotic cell divisions.
*This pattern of inheritance has important implications for a variety of mitochondrial associated diseases, usually occurring in tissues requiring lots of energy (muscle, brain).

[[File:Mitochondria EM01.jpg|200px|thumb|Electron micrograph of mitochondria.]]

===Eukaryotic mitochondrial genomes===
*double stranded circular DNA (mitoDNA. mtDNA)
*1981 complete human sequence (16,569 nucleotides)
**37 genes
**encodes 13 polypeptides involved in oxidative phosphorylation
*remaining genes transfer RNA (tRNA) and ribosomal RNA (rRNA)
*multiple copies within the matrix
*maternally inherited
*remainder encoded by nuclear DNA
*proteins made in cytosol and imported into mitochondria

link to Embryology website [[Mitochondria]]

==Chat==

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 11:13, 25 September 2015 (AEST) OK so just text on your page to date and not yet a thorough coverage of the topic. Animal models, timeline, images, diseases.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 16:07, 21 August 2015 (AEST) I think you will have 3 students and therefore will exist as a group.

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 17:29, 21 August 2015 (AEST) hi all,sorry that I still could not make my way to uni today due to illness. I will definitely be back next week. I have added few sub-headings to the points you guys setup, feel free to change them. BTW, would you like to pick one of the 5 topics for now? and start working on it? or there was some good arrangement already? please let me know.

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 20:51, 24 August 2015 (AEST) Hey, ummm sorry i lead us astray putting up those headings ''female fertility'' had been taken so we have to pick another. I put up the list of ones left. My choice would be three parent ivf. So ill do a bit of research and on that now (add some headings)just 'cuase i got some free time, but by all means if you guys would like to do something else that interests you I'm more then happy to change.

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 13:19, 27 August 2015 (AEST) Hi all, I am good with your choice. let's work on 3 person embryo. i have added few papers I find good on this topic.

not sure if youll see this but theres a fantastic review on ethics here http://onlinelibrary.wiley.com.wwwproxy0.library.unsw.edu.au/doi/10.1002/stem.2221/epdf

===General===

Note to self doing history benifits.

===Section===

==Peer Reviews==

===1===
This wiki page does well in covering a lot of areas relating to the topic, however the website does not outline the information found/used quite clear enough or to the right extent. The page would benefit largely in focusing much more attention to the mechanics of the process itself and how it physically works. There is lots of information regarding other various aspects relating to the topic, however the fundamentals of the topic are not clearly discussed on the page, and it is not clear what goes on in the process.

The page should also fix up some grammatical and syntax errors. Read through the page carefully and ensure all paragraphs make sense ensuring that the quality of the information portrayed is fully appreciated. To also make the page clearer, some thought should be given to rethinking the order of the subheadings. Having a natural cohesion throughout the page as a whole is important – some subheadings do not fit into place correctly and could be moved around a little bit. Also having linking sentences within paragraphs – involving each subheading with others and the topic as a whole – will make the page much more cohesive.

The page used a good amount of supporting pubmed articles, hwoever more images/media files could be used to break up the concentrated use of text. The video used is relevant and informative – however there is no copyright information.--[[User:Z5015534|Z5015534]] ([[User talk:Z5015534|talk]]) 22:44, 10 October 2015 (AEDT)

===2===
Hi guys! I'll start of by saying that the images and video you have included are excellent and are relevant to your topic of discussion. Also, you have a large number of reliable references which is good to see. However you have yet to include a hand drawn image, which is required for the wiki page. I feel like you could probably eliminate the typed out 'timeline of mitochondrial donation' and instead use this as an opportunity to use a hand drawn image of the timeline. Furthermore, the timeline under 'cystoplasmic transfer' feels a bit awkward and unnecessary. You could probably include this timeline alongside the 'timeline of mitochondrial donation'.

Under the 'Technical Progression' section it would be best to incorporate human embryo, mouse and human models under a sub-sub heading, as at the moment it feels a bit jumbled.

I would also recommend moving the 'Benefits' heading towards the end of the page. It feels odd reading about the benefits of three person embryos before I gain a proper understanding of how they work. Also it might be worth talking about the disadvantages (if there are any) to three person embryos to balance out the 'benefits' section. As has been previously stated, make sure you sort out the copyright information for your video as it would be a shame to lose marks if it was missing. Also, don't forget to add the 'student template' to the 'Swapping mitochondrial DNA mammalian oocytes' image as it is currently absent.

===3===
The entire project is presented simplistically and all the content is relevant and easy to understand. Majority of the flaws I found were based around poor grammar and syntax which could be fixed up with some editing. Below is a more detailed breakdown of some of the things you could fix.

Firstly, I liked that the introduction was brief and concise and gives the reader a basic understanding of the topic of three person embryo. The video was also informative and provided some background information around the topic. I was informed that mitochondrial DNA was the major factor concerning this topic however there was a lack of information about its importance to the body so a short summary could be included along with some examples of diseases it could cause.

Also, the use of a timeline to present the history is a great idea and I think it could be improved and would look more aesthetically pleasing if it were to be placed into a table. I also think the 1990s, 2000s and 2010s label could be removed to make it look less clustered since they aren’t particularly necessary.

Some information under the heading ‘Technical Progression’ has yet to be filled in but from what is there I’d like to suggest exchanging the bullet points for numbering instead for the information under ‘Pronuclear transfer’ and ‘Polar body transfer’ since they sounded like sequence steps as opposed to separate points.

Finally, I found the layout of the table under the heading ‘Legal status’ to be very well put together. There are however some countries placed under the incorrect continents and I found that the order was easily changed and mixed up. I also noticed that several of the countries were linked to the same sources which made the information very unspecific. Instead of just links I think a few sentences explaining the legislation would be more informative.

===4===

I liked how you guys started the introduction and provides partially a brief overview of what your project is about. But I believe it is not enough to allow the audience an insight to your project page. This is something that needs to be worked on and maybe add some images also. However, the choice of short video used in the introduction is great. This is definitely a benefit for your page as it will reinforce the information you have been trying to get across. Like I mentioned, one thing you could work on is adding images and explaining the content in more depth. There is great amount of reference at the end of the page in the reference list which is fantastic!, however there is no in- text referencing in each section such as introduction or in some of the parts of the “Technical Progression” like “Cytoplasmic transfer” or “Spindle-chromosome transfer”. Having in-text referencing will allow the audience to know exactly where the information was read from and for the interest of the audience can read that specific paper in detail.

I also noticed that there are no information for “Benefits” and “Legal Status” or there is limited information for such headings like “Ethics”. I’m assuming you didn’t get the chance to upload information there or you haven’t had the time. This is something you need to work on so that the audience has some note of what this page is about. Also you need to change the format of the page for example it is to move the 'Benefits' heading towards the end of the page after the audience gained a good level of understanding of the project. You included some great images but be careful with copyright as I didn’t see it. But also consider some more images, tables, diagrams as well as hand drawn images in some sections, to make it more inviting and not overwhelming with just content. I do appreciate that the section of “Technical Progression” is subdivided into “Human Model”, timeline” and etc. But maybe consider adding in the current research, historic research, limitations and disadvantages to ensure that you can get all the marks possible by addressing all the key concepts. The timeline is a great idea that outlines the significant progresses and in turn helps put major events into perspective, making it more effective for students to study and understand.

Well done on making the “Glossary” at the end. This is exactly what I would have expected to see and I used it while I was reading through your page. Also it is great to see the table in the “Prohibited” section but I would suggest you to write some sentences explaining the legislation rather than just pasting the links.

Overall, this project page has room for improvement by giving certain sections of the page the attention they deserve. Images are imperative in allowing a balance between text and the image itself. Diagrams, tables and animations can sometimes be refreshing, and less overwhelming to see them among paragraphs of content. Try and work on time management, or set a group deadline that everyone has to meet so that all the information can be well up before the due date so your group can have time to edit and add images and play around with the page comfortably. Goodluck!

===5===

It would be nice if the history section could start earlier in time e.g. who came up with the idea of 3 person embryos. It kind of feels as though 3 person embryos popped out of nowhere. This is just a small nuance but the first sentence in “Hereditary mitochondrial Disease” doesn't really make sense. It sounds incomplete. I think you have too many timelines going on in your page and it makes it a bit confusing. There is one under “History” and another timeline in “technical progression”. I understand they may be timelines for different things, but it’s all too much history. Maybe the “technical progression” timeline could be simplified into a paragraph?

Some sections have too many subsections e.g. “technical progression”, and this makes the section messy and hard to read. It is however good for the table of contents though as it makes it easier to specify what you want to read on the page so my suggestion would be to keep some subheading but cut down a bit. You guys are listing papers to read too often. People want to have the information summarized for them on a wiki page, not have to outsource all the information themselves. It’s too time consuming and if they wanted to read a bunch of articles, they would go on PubMed themselves. However, I do like that some articles have been listed but maybe cut it down to one or two great ones instead of 4-5 etc.

I really like the table under the subheading “prohibited”, however, it would be nice to have a little summary next to the links about what each countries stance is, because again it’s too time consuming to have to read all those links. I also think some sections need a lot more work e.g. “ethics” and “benefits” and some more words could be added to the glossary. For example, a definition of what the word gamete mean would be good as, whilst we may know what it means, other people who view your page may not.

I really likes the images you used in “technical” progression. They were easy to understand and simplified the text a lot. It would be good however to add a few more images, perhaps to “history”. Some hand-drawn ones would be good. You've got a good amount of references in there, just maybe add a few more. This indicates that you have done significant research and they appear to be correctly cited. The key points of your topic are clearly described and I feel as though your intro., whilst short, really opens up the topic well. Your page relates well to the learning aims of embryology.

To conclude, I think you've got a great framework and some really good information in there. Just makes sure your page doesn't look too busy and is easy to read. A little bit more work needs to be done in some of the sections and a bit more technical touch ups and you should be good!

===6===

Your group’s topic looks very interesting! You have addressed the key points of your topic, and the placement of the video gives the reader a great overview of your project.

'''COMMENDATIONS:'''

• Information has been organised well most of the time. Good use of bullet points and subheadings.

• The table under “Prohibitions” is a great way of summarising information, and it was easy to read.

• I like the addition of a glossary, however, more terms could be added here as a lot of jargon has been used in your text.

• Cytoplasmic transfer images were great as they aided the text well. These images could be re-sized as some of the text is blurry.

'''RECOMMENDATIONS:'''

• Be mindful of spelling and capitalisation, e.g. “Hereditary Mitochondrial Disease” rather than “Hereditory mitochndrial Disease.”

• In terms of formatting, more spacing between major headings will make reading the page easier and will allow your information to flow.

• I recommend adopting a set formatting scheme for each section: i.e. make sure that the subheadings are all the same size, that they are in bold/italic (if that is what you intended).

• Some references and PMIDs are placed throughout the page. These should all be under your References heading at the end of the page.

• Information is missing under certain headings, e.g. “Mitochondria Linked Infertility.” I’m assuming that information from the two links provided will be summarised for the final submission.

• Hand drawn image is absent – maybe you could hand draw one of your timelines? (Seeing that both of them currently take the same format/structure).

Great job so far!

===7===

Thus far, I think this page has a good layout to be a successful page on Three Person Embryos. The headings and subheadings are relevant and show that you have conducted literature searches to deduce what information needs to be covered. I suggest moving “Benefits” below “Technical Progression” as it is important for the reader to understand the process of three person embryos, before learning its advantages. You could also add information about disadvantages and controversial issues.

On a positive note, I am impressed with the way you have set up headings under “Technical Progression”. The consistency of discussing a model and current research provides a systematic approach to the viewing of your page, making it easy to understand. Delving further in each of these subheadings would provide a greater understanding of the current technologies available, such as including limitations and advantages, and statistics of their success rates. The timeline under “Cytoplasmic Transfer” could probably be incorporated with the timeline under “History” to equalize the amount of content under each heading.

The content under each heading still needs work in terms of editing and elaboration. There are quite a lot of grammatical and spelling errors such as “Timeline of Mitocondrial Donation” (missing an ‘h’ in mitochondrial), and some sentences aren’t finished. Proofreading would be key to making the information more understandable and effective to the reader. Information seems to be lacking under a few headings especially “Benefits”, “Hereditary Mitochondrial Disease”, “Mitochondria linked Infertility” and “Other approaches”. To make it a bit easier for yourselves, you may want to consider using a table, flow chart for pathogenesis of the disease, and a detailed diagram of the relevant heading. You have provided a table to explain the "Prohibited Section" however a very short description/summary of each source in the table would be very helpful.

I also noticed you have not included many images, videos or tables. These visual aids really help the reader to understand the content in front of them, and also keep their interest in the topic so it imperative to focus on them as much as the content.

The references have all been cited correctly and have shown you have performed adequate research to cover the important information for this topic. As you add more information, more references should be present within the body of your page.

Overall, I think this page has a really good framework for further information to be added. With more editing, content and diagrams, you are sure to produce a wonderful Wiki page.

===8===
Your project instills a great first impression on a visitor to the page! It is a well designed webpage that doesn’t come across as overwhelming and too wordy encouraging and drawing the reader to explore your page. Your choice of content is relevant and provides a good understanding of the topic so far. The introduction is nice and succinct, explaining easily and clearly what the topic is about with a great video that complements the introduction. Together they give the reader good background information on the topic, and are taught in a way that’s easy for someone with no prior knowledge on the topic or in embryology in general to understand.

Furthermore, the timelines you provided, the ethics section and the table on the legal status of the technique is a good way of showing how far the concept has come and good at placing the technique in the context of how it has been translated into modern society. I really like that you have included animal models in explaining the various techniques, provided the current research available, and included further reading. This is very relevant and interesting for other embryology students and researchers who visit your page! The diagrams you have already chosen are very appropriate and explain the technique clearly to visual learners and are very engaging.

In improving on your page I think the main focus is to elaborate on some key points further and add a few more diagrams and pictures so you can maintain a perfect balance of words and images and the engaging layout you already have begun. For example, maybe for the section “hereditary mitochondrial disease” you can talk about the type of hereditary diseases there are. Also, some of your wording and grammar need further editing so make sure you go through and reread your work.

Other things you should edit include, adding a reference to your introductory paragraph and maybe clarifying that three person embryos are now legal in the UK since your video says “its on the threshold of acceptance”. Also check your copyright on some of the images such as the one that says “Copyright © 2015 BBC. The BBC is not responsible for the content of external sites. Read about our approach to external linking”, I am not sure if this means you are allowed to use it so just clarify this.

Overall, you have great progress on your page so far, it has a great teaching element to it and shows extensive research and citing into the project!

===9===
It’s great to begin with a introductory video which defines your topic. I do suggest finding a reference for the first paragraph for the introduction. Besides that, references have been cited correctly and shows that you have conducted extensive research, but remember to reference as you add information ( [##] ). I think you guys did a great job with the heading and subheadings; it shows us that you have done extensive literature research, and have came to a conclusion as to what information was relevant. Just a grammatical error made in “Timeline of Mitocondrial Donation” which is missing a H in mitochonidral. I suggest proof reading all the text before uploading! This will make it easier for the audience to understand and also for yourself! As to the “Benefit” heading, I think it will be a good idea to add information and case studies on disadvantages towards three person embryos.

Nice to see that you guys have included a timeline, this shows the progress made throughout the years. But I think there is still information that can be added into this area; for example: different possible approaches or more controversial issues that has emerged. “Technical Progression” is an impressive choice of heading; I found it very interesting to read. The cytoplasmic transfer images used were great! They were very easy to understand. The “Timeline” under “Cytoplasmic transfer” could be merged with the history timeline heading above.

Overall, I think more information and content needs to be added under all the headings and subheadings. To make it easier for the audience to read, I suggest adding detailed images, tables and flowcharts. It will be more eye-catching for readers and will keep them interested. I see that you guys have a table under “Prohibited Section” however that just leads to another link, rather than having the link there; I think it would be a great improvement if there is a short summary of all the sources found.

I think your page is organised and formatted very well! With more information/content, detailed diagrams and tables; it will further improve your Wiki Page! Good Luck.

===10===
Firstly, this is a very impressive wiki and I think you guys are setting the bar very high. The resources you have used and referenced are of really high quality and demonstrate that you have carried out extensive research and identified the information that is most relevant and important. The introductory video and all the images you have included are awesome and really help to solidify the information that you are presenting; they also add more depth to the page and provide further explanation and clarification of the information. The "Technical Progression" section is really great, well structured and provides a lot of explanation about the procedure that I found aided my understanding about major the concepts of the page.

My suggestions to improve your wiki page would be to have a second look at the layout and headings; I found they were difficult to follow and disrupted the flow of the page, for example the heading "Benefits" followed immediately by "Mitochondrial linked information" which was followed again almost immediately by "Hereditary Mitochondrial Disease". These headings made me stop and think about whether there was some information missing and I was just a little confused about whether the two latter headings came under the first. I really liked the inclusion of the legislation and ethics surrounding the topic (very interesting reading), however I am a bit concerned that they are the biggest portions of the page; I think this would be easily rectified by simple adding further explanations of the current research and journal articles that you have referred to instead of providing only one or two sentences about each. Lastly, it would be really interesting to present information about the controversial opinions/incidents surrounding the topics and also about the disadvantages of the procedures.

Over all, I think you guys have done an awesome job thus far and with a few minor changes the page will be amazing! Good Luck!

===11===

Firstly, I think your page is very well thought out and includes a lot of relevant information. The sub-headings fit in well with the topic and allow for a coherent flow of information, however it would be better if some of the sub-headings were re-arranged. For example, it might be better if the benefits section is placed after technical progression in order to really emphasize the relevance and value of this procedure. Under some sub-headings, it would be good if you could write 3 or 4 sentences summarising that section instead of having the sub-sub heading right underneath, especially for Benefits and Legal Status. This allows for a better flow of information and also makes it look more organised.

In terms of media content, I think the introductory video is great in providing a brief overview of the whole topic. I also think your choice of images for the technical progression section is great in being able to visually summarise the written information. As I could not see an original picture in your page, I think it would be a good idea to include a more visually appealling hand drawn diagram of one of your timelines. You could have the timeline going horizontally with coloured boxes coming off of it to describe the events. This can be easily hand drawn or done in word.

It's clear a lot of research has been done due to the sheer number of articles that have been referenced, especially in reference to the inclusion of several animal and human models. I like your use of timelines however I think the timeline under prohibited section is quite laden with content and can be presented in a more appealing way.

Overall, I think you guys have done a great job in setting up this page. It has the foundations to becoming a very informative and useful page.

===12===
The group project page ‘Three Persons Embryos’ covers a lot of aspects of this topic. The page incorporates the example of Alana Saarinen. This ‘personal’ case study makes the topic interesting and makes people want to learn more about it. The video is a good way of introducing the reader to the page, but maybe a summary would be useful for people who cannot/ do not want to access the video. Overall, the headings are in a logical order and the subheadings are useful. However, it might make it easier to follow the page if the subheadings for each of the techniques were unified, e.g. “Procedure” “Animal Models” “Current Research” for each procedure. Also, the heading “Benefits” appears to be rather a description of the indications for the procedure, a renaming of this heading might clarify this.

Many key points relating to the topic have already been mentioned, but expanding further on the majority of headings and subheadings would be useful. Several useful pubmed articles are mentioned where the text is only short, so only their summary is missing. The referencing appears to be correct most of the time, however, there are several instances where one paper was referenced several times individually. Check https://embryology.med.unsw.edu.au/embryology/index.php/Help:Reference_Tutorial#Multiple_Instances_on_Page to learn how to avoid this.

In addition, the three uploaded images need to be double checked for their copyrights, as they do not clearly state an allowance for the reuse of their content. In case, they cannot be reused, the images could be self-drawn. The timelines are interesting and necessary components of the project. They could also be displayed as actual timeline-graphs. This would add to the amount of graphs used and illustrate the content nicely. Overall it might be useful to check for spelling and grammar mistakes before the final deadline.

=== 13 ===

From first glance of the page, just by looking at the contents table found at the top of the wikipage, I can already see that all the sections have been planned and well thought out with the appropriate subheadings and sub-subheadings which makes the page a lot easier to navigate if I were to be searching for something in particular on the topic ‘Three Person Embryo’

The introduction is short and concise with the appropriate description of what the topic is about, adding the YouTube video in your introduction was a great touch (props to you for asking the maker permission to re-use as it’s under the YouTube Standard License) however I think this section could be improved by maybe addressing the key points that the wiki page will be covering

It’s great to see some history behind the development and progression of ‘Mitochondrial’ Donation throughout the years!! Is there any more history regarding this topic or is 1997 the first date with historical records?? Tabulation of the data may clean up this section, but otherwise great find!

Under the benefits section, no actual benefits seems to have been listed? I think this section has not yet been completed

The pictures and information under the ‘Technical Progression’ subheading is substantial and informative. The images seem to have been referenced and labelled accordingly but not sure if they are reusable as the sites / locations you obtained them from do not mention permission rights for reuse of their material. Some of the referencing done still shows the blue PMID which disrupts the flow of the project page.

It is clear that a lot of research has been done into making this page what it is currently. I would suggest firstly to check that all material used on your page is re-useable as well as fixing up the minor coding of references and also grammar and spelling mistakes throughout the page. Great work!!

===14===

Your project is looking very promising. You have found great resources in you images and video as well as used an ample amount of valid and reliable PUBMED articles to support your findings. You have found a variety of interesting and relevant topics that you have succinctly communicated. I would recommend reviewing your work for grammatical, punctuation and spelling errors in your content and your headings.
The video and images uploaded are very interesting, please ensue that they are correctly referenced, it would really be a shame if you lost marks on such great parts of your presentation, and more images, to the same quality standard as the Primate model would only improve your project. while the video is a great resource, your should find some journal article evidence to re-affirm its findings, enabling you to not only used the video but also other very reputable sources to validate your statements.

The timelines are a great way to succinctly present a lot, of information, easily. perhaps re-do the Timeline of Mitochondrial Donation as an image, maybe you could incorporate your hand drawn or computer simulated image here. If you wish to reference most of the dates on your timelines - I would suggest either re-referencing the same resource of finding resources to support each date. Endeavour to make all timelines on the wiki page presented in the same manner, ensuring a uniform and cohesive webpage.

Similarly your table under legal status is a great resource, perhaps you could make it a collapsible table, that way it will not take up such a large portion of your page, that could better be used for more content. Perhaps you could review the specific "countries" within the "regions" as some do not match

It would be in your best interest to look into restructuring some of your headings and links especially under Technical progression heading and the Mitochondria linked Infertility heading , and making the effort to present the information in the same manner under each using both animal and human models where applicable. this will make your web page easier to read and understand :) formatting is super important, spacing between subheadings as well as pictures break up the information and make it easier to read.

Certain areas within your report and good but could use a little more content such as the intoduction as well as Hereditory mitochondrial Disease in order to provided a well rounded base level of understanding for all users of this site. Many Pubmed links and references are scattered throughout the web page, these should be correctly coded and placed in the reference list, perhaps instead of having the articles there you could summaries the findings for the readers, it would be easier than having them outsource the information. Be careful of over referencing your pubmed articles and more key word that are being repeated should be incorporated into your glossary.

On the whole good work so far, I look forward to seeing the final result.

===15===

This Group Project has a lot of potential. Information is presented in an engaging way through an introductory video documenting a case, timeline providing retrospective insight as well as diagrams detailing the complex processes in a concise manner. I also found some subsections interesting and relevant, including the discussion on ethics, Current research areas, as well as the glossary which provides a quick reference.

Although I understand you intend to elaborate more on certain areas including current research, the project does seem a little thin in regards to text. There are many papers that are linked with no explanation. For example, with regards to the table under prohibitions, I am confused as to what exactly those countries prohibit (all genetic modification? mitochondrial techniques only?) and also whether there is any more nuance to the discussion (do they ban different things?). Of course, one could click through the links under each country, but i think some text before the table itself would only add to your project and bring more context to the table itself. Diagrams themselves can also be referred to in the text (as you would find in a textbook), to add context and create a more cohesive wikipedia page (e.g. for Pronuclear transfer). In terms of the information provided, a little deeper exploration or discussion about certain issues including the reasoning behind ethics arguments may be relevant. If the articles listed under the headings are anything to go by, it seems you intend to do this.

Finally, some proofreading will be very beneficial in polishing up the wikipedia page. There are some sentences like "And it effectiveness in doing so. And the processes that occur in the oocyte when this method is used" under Human Embryo Model, which may well do with some editing. I would like to add that the referencing has been fantastic and consistent. Good Luck!

===16===

The entire group project is short in comparison to other reports. Adding a video about a teenage girl who has three biological parents is an excellent tool to engage the readers. Regarding the history, the timelines used help structure the changes over time and allow easier reading, although it is reality short for the entire history, possibly add one or two more paragraphs covering all aspects of the history of three-person embryo. Benefits section wasn’t completed equally as some sections such as the mitochondria linked infertility and Hereditary mitochondrial disease are fairly small in comparison to the technical progression.

Diagrams, more timelines and human models were used and discusses which is a valuable asset to the project. As well as large tables discussing where this procedure is prohibited. Overall this group project reaches out to different aspects of three person embryos although it still has a long way to go, certain sections need to be updated with information, and some other sections need to be clarified more with the addition of vital information.

Adding further reading clearly shows that the group are interested in the topic they have chosen and have taken the time to add additional links for people who are interested in reading up on three person embryos. Also the glossary is way too small, it needs to include a lot more definitions, which is due to the lack of information in the overall set out of the project. The citations and references are filled out correctly and help with the clarity of the report.

===17===
The topic of three person’s embryo was outlined and described well, with good choice of headings, separating the different key points and areas that you’ve discussed on your page. It’s evident you’ve done extensive research on your topic, and especially going through your reference list, all your research data were extremely recent, which is excellent!

<b>Pros</b>
*I loved that you put an introductory video in the beginning, which is a personal account rather than a scientific stimulation video, giving it a more personal touch on the page, and highlighting the importance of this breakthrough in 3 person’s embryos.
*Because your topic isn’t a disease but a medical breakthrough, your own chosen headings were fantastic and on point, giving a well-rounded and detailed wiki page
*The format of the page is also really good. Its good to use more subheadings than main headings, and you’ve formatted the page really well
*You’ve used very current research, making your page much more credible and updated with the newest information
*The “further reading” section was really good, it extended beyond the normal “teaching” topics, giving the reader a better insight of the topic, if they choose to.
<b>Cons</b>
*The introduction looked a bit short. A more detailed explanation will make the reader understand a lot more on how it works and how the page looks, without the reader having to watch the video if they choose to just skim the page.
*Your heading and subheading of “History” and “Timeline” could’ve been merged into one, as that was your only subheading of that heading,it would’ve looked more readable that way
*Timeline information could be extended more, in terms of date (if found) and details on what occurred on that date
*The heading of “Benefits” also needs to be extended VERY much more, two subheadings an 3 sentences under one, with a link under the other, does not explain any benefits at all
*Pictures in “Technical Progression” could be skewed to the right, so the words all flow, with the HELP of the diagram, instead of the focus being on the photos
*Current research also needs to be expanded more. PMID wouldn’t be needed either if you had expanded the current research, and referenced it in your reference list; section looks a bit messy with bullet points starting with hyperlink
*“Technical Progression” needs to be greatly edited and to have information added
*“Ethics” also needs to be expanded, and again, don’t lead with PMID
*“Glossary” is not really needed if you explain those terms in each words occurrence instead of at the end, but this is a minute point

2015 Group Project 1

2015-10-23T02:47:02Z

Z3292373: /* Inheritance of mitochondrial disorder */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (2013, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
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'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref name="PMID1463006"><pubmed>1463006</pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref name="PMID1463006"/>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research <ref> The Wellcome Trust Centre for Mitochondrial Research, A film by Barry J Gibb. (2012, September 15) Healing broken batteries: The Wellcome Trust Centre for Mitochondrial Research. Retrieved from https://www.youtube.com/watch?v=Sr7Jnr9qn44 </ref>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''cortical necrosis''' Break down of the kidney tisssue.

'''hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

2015 Group Project 1

2015-10-23T02:33:22Z

Z3292373:

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>

===Limitations===

Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>
PMID 23608245

Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>
PMID 24382342

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>
PMID 20933103

Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>
PMID 26020522

The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>
PMID 25421171

Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>
PMID 25807984

=Glossary=
'''cortical necrosis''' Break down of the kidney tisssue.

'''hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

User talk:Z3292373

2015-10-23T02:12:25Z

Z3292373:

Complete and post catia when it goes live.

User:Z3292373

2015-10-23T01:21:37Z

Z3292373:

'''The Completer Finisher'''
is a perfectionist and will often go the extra mile to make sure everything is "just right," and the things he or she delivers can be trusted to have been double-checked and then checked again. The Completer Finisher has a strong inward sense of the need for accuracy, and sets his or her own high standards rather than working on the encouragement of others. They may frustrate their teammates by worrying excessively about minor details and by refusing to delegate tasks that they do not trust anyone else to perform.

I had no idea that Oocytes and spermatozoa were not matured at the time of release, for no reason at all i assumed that it was bucket science and the two just needed mixing and "let them do their thing". Which is ridicules now that i think about it.

=Laboratory Work=

==Lab 1==
A summery of two recent research articles on fertility or fertilization.
=== Article One:Degradation of Paternal Mitochondria by Fertilization-Triggered Autophagy in ''C.elegans'' Embryos <ref><pubmed>21998252</pubmed></ref> ===
Miyuki Sato,Ken Sato

Using ''C.elegans'' as a model organism this paper investigates the possible mechanisms in which paternal mitochondria might be removed from the Oocyte after fertilisation.
Initially mitochondria from the spermatozoa were track through embriogenisis via marking with MitoTracker red (MT) and Heat Shock Green Fluorescence Protein (HS-GFP). This initial tracking showed paternal mitochondria entering the cell at fertilisation and being randomly inherited by blastomeres through to the 4 cell stage then clearing by the 16 cell stage.

To establish a link between this pattern and autophagy, autophagisomes with GFP marked homologue of a protein in their membrane (LGG-1) were fertilised. These autophagisomes were built up around the pronuclear paternal DNA (''C.eleigans'' spermatazoa have their mitochondria distributed around the head of the gamete and posses no tail) and then dispersed around the cytoplasm as the blastocyst developed. Like the paternal mitochondria the autophagisomes then cleared by the 16 cell stage as well as appearing sparadicaly in places related with regular development.

Further fertilisation with mutant sperm line ''spe-9'', that cannot produce the proteins for normal fertilisation, did not produce the initial induction of autophagy. Whilst insertion of sperm line ''spe-11'', that cannot fertilise but permit embryogenesis but show the same patterning of auto phagisomes around the paternal pronuclei. This suggested that induction of autophagy is based on the entry of paternal proteins.
By arresting the Oocyte in metaphase 1 by ''emb-27(RNAi)'' it was also shown that the induction happens independent of progress into anaphase 1.

Confirmation of the role of autophagy in the removal of paternal mitochondria was done by the use of mutants with compromised autophagy regulators. Gamete mutants ''lgg-1(tm3489)'' that could not produce the autophagicyte membrane could proceed through fertilisation at a reduced capacity however could not progress past the L1 larval stage. In these matings the paternal mitochondria persisted past the 16 cell stage.
In contrast if mutant Oocytes were mated with wild type spermatozoa then paternal mitochondria would be present until the Lima Bean stage and then cleared.
Spermatozoa that contained the same ''lgg-1'' mutation however had no change to the regular clearing of paternal mitochondria when mated with wildtype oocytes.
This suggested that the maternal autophagocytes were the main contributors to paternal mitochondrial clearing. However paternal ''lgg-1'' expression could, at a reduced capacity, compensate for compromised maternal autophagy.

=== Atricle Two: Developmental potential of zona pellucida–free oocytes obtained following mild in vitro fertilization <ref><pubmed>25256934</pubmed></ref> ===
Satoshi Ueno et al.

In this research, from a Japanese fertility clinic normal oocyte and zona free (ZF) oocytes where taken from the same patient in the same collection cycle. The ZF oocytes did not have an intrinsic absence but breakages of the zona palucida through which the oocyct was protruding. The oocyte was retrieved from the extrusion. These were then fertilised via intracytoplasmic sperm injection, cultured and followed through cleavage and blastocyst formation.

A comparison of the blastocyst formation from successfully fertilised ZF and normal oocytes showed no statistical difference in viability. The same was found of thoughs ZF derived blasticysts that were carried to full term.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:28, 4 September 2015 (AEST) These are accurate suppers of these 2 papers (5/5)

==Lab 2==
Introduction to addition of images to website.

[[File:Cells_of_the_innate_and_adaptive_immune_system_present_in_the_uterus_at_the_time_of_implantation.jpg|600px]]

Cells of the innate and adaptive immune system present in the uterus at the time of implantation <ref><pubmed>26136750</pubmed></ref>| [http://journal.frontiersin.org/article/10.3389/fimmu.2015.00321/abstract '''frontiers''' in Immunology]

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:30, 4 September 2015 (AEST) Image uploaded with correct reference, copyright and student template. Please in future use a briefer image title for example, File:Cells of the innate and adaptive immune system present in the uterus at the time of implantation.jpg, could have been simply File:Cells of the innate and adaptive immune system at implantation.jpg. (5/5)

==Lab 3==

Brief description of 3 research articles on your subject.

===Article 1:===
PMID 25629662 '''Mitocondrial donation--how many women could benefit?'''<ref><pubmed> 25629662 </pubmed></ref>

This is a statistical analysis of the prevalence of women of child bearing age that have pathogenic mutation to their mitochondria that could benefit from mitochondrial donation in the UK. And the affects of the mitochondrial mutation on fertility as compared to background natural birth rate. They found no difference in fertility rates and 4% of women at risk of passing on symptomatic mitochondrial disease.

===Article 2:===
PMID 20393463 '''Pronuclear tranfer in human embryos to prevent transmition of mitochondrial DNA disease'''<ref><pubmed> 20393463 </pubmed></ref>

As the name suggest this paper looks at pronuclear transfer as way to remove donor mitochondria measured by mt-DNA. And it effectiveness in doing so. And the processes that occur in the oocyte when this method is used.

===Article 3:===
PMID 18674747 '''Pathogenic mitochondrial DNA mutations are common in the general population.'''<ref><pubmed> 18674747 </pubmed></ref>
Another on the prevalence of mitochondrial mutations in the populous. This time via mtDNA sequencing from umbilical samples from live births, looking for ten specific mt-DNA mutations. It found a frequency rate of 0.54% for these mutations. Although they had limited data on the prevalence of these mutations maternally.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:32, 4 September 2015 (AEST) These are relevant references to your group project. (5/5)
==Lab 4==

Three question quiz on Placenta Development
===Take the Quiz===

<quiz display=simple>

{Which of these is '''NOT''' a part of Tertiary Chorionic Villi formation?
|type="()"}
- Mesenchyme differentiating into blood vessels and cells.
- Fusing with placental vessels.
+ Extra-embryonic mesoderm grows into the villi.
- All of the above
|| The ingress of extra-embryonic mesoderm is part of '''Secondary''' [[Placenta - Villi Development#Chorionic Villi|Chorionic Villi]] development.

{Which of these structures make up part of the maternal surface of the placenta?
|type="()"}
- Amniotic membrane.
+ Cotyledons
-Chorionic plate.
-Syncytiotrophoblasts

|| [[C#cotyledon|Cotyledons]] from the greek for "Cup,bowl" named after the proto leaf structure in germinating plants gives the leaf-like or cobblestone like appearance, on the [[Lecture - Placenta Development#Placenta at Birth|maternal surface]] of the placenta.

{What is the name given to the abnormal development of the placenta described by the attachment of the placenta deep into the uterine wall and also penetrating into the uterine muscle but not into the uterine serosa ?
|type="()"}
+ Placenta Increta.
- Placenta Accreta
- Placenta Percreta
- Placenta Previa

|| [[P#placenta accreta|Placenta Accreta]] occurs when the placenta adheres to the myometrium without inter-lying decidua basalis.In [[Lecture - Placenta Development#Placental Abnormalities|Placenta Percreta]] the placental villi penetrate all the way through to the Serosa. [[Lecture - Placenta Development#Placental Abnormalities|Placenta Previa]] occurs when the placenta adheres over the ostium (or external orifice) of the uterus blocking the birth canal.

</quiz>

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:33, 4 September 2015 (AEST) Only 2 questions. I will come back later. [[ANAT2341 Student 2015 Quiz Questions]]

{{Template:Quizzes}}

==Lab 5==

===Brief Overview of '''Hirschsprung's disease'''===

Hirschsprung's disease (HSCR) also know as ''congenital aganglionic megacolon'' or ''intestinal aganglionosis'' is a disorder of the gastrointestinal tract characterised by a lack of neurons in the intestinal tract (IT). Most commonly affecting regions of the colon and more distal sections of the hindgut although can be prevalent from the stomach to the rectum. This causes the inability of the enteric nervous system (ENS) to control secretions and blood-flow in the affected area as well as maintain peristalsis leading to sustained contraction of the smooth muscle and hence obstruction and distension of the bowel <ref><pubmed>17514199</pubmed></ref>. Clinically this is displayed by the absence of a meconium stool in the first 48 hours after birth and confirmed by radiological examination with a barium enema <ref><pubmed>6691093</pubmed></ref>. Treatment requires removal of the defective region via surgery and has many possible complications <ref><pubmed>9722005</pubmed></ref>.t

The ENS is derived from the Neural Crest. Vagal neural crest cells (NCC) contributing to the fore-,mid- and hind-gut<ref><pubmed>8565847</pubmed></ref>, Sacral NCC's contributing to the distal hindgut<ref><pubmed>9753687</pubmed></ref>. Malformations of it's development such as HSCR are termed neurocristopathies. In the case of HSCR, most commonly it a restriction in the migration and proliferation of the neural crest cells in early development (weeks 4-7) impeding their colonisation of the gut. The cause of this impediment is varied. Around half of cases can be linked to the GDNF/RET (glial cell line derived neurotrophic factor/receptor tyrosine kinase)gene families that regulate to progression of the NCC cells through the mesoderm <ref><pubmed>12399307</pubmed></ref>. Mutations in any number of these genes can lead to delay or inability of the NCCs in their progress rostro-cordauly.

Another cuase comes from disruption of Endothelin pathways that although also control migration, maintain the enteric NCC progenitors cells in their proliferative state <ref><pubmed>16624853</pubmed></ref>. Mutations in this pathway have shown to stop differentiation of the NCC cells at the distal bowel <ref><pubmed>16339294</pubmed></ref>, meaning although fully colonised the ENS cells have reduced excitatory fibres and abnormal neurotransmitter release.

Outside of genetic mutations retinal (vitamin A) deficiency has been link to HSCR <ref><pubmed>12702665</pubmed></ref>. Although vitamin A deficiency has been linked to numerous congenital defects of which HSCR is a small part. The complexity of systems that contribute the enteric nervous system mean that HSCR, although fairly understood as a disease, has many possible causes not yet linked.

==Lab 6==
Complete work on group project.

==Lab 7==
A brief description of the findings of a recent research paper on Odontogenisis.

=== Enamel hypomineralisation due to endocrine disruptors.===
jedeon K et al.<ref><pubmed>25158179</pubmed></ref>.

Endocrine disruptors and their possible health effects have been gaining a lot of traction in the media in the past decade. Most touted of these being the estrogen-like Bisphenol A (BPA) found in many plastic products. This study compared the affects of BPA and two other endocrine disruptors (EDs) genistein (G) and vinclozolin (V) on tooth development by use of mouse models and cell culture.

Their results showed that phenotypicaly rats exposed to BPA devolop the greatest degree of hypomineralisation, 75% of rats treated. Compared to lowest reading of 35% for though exposed to GV excluding the control group.
Further more in HAT-7 rat ameloblast-like cell culture and analysis with RT-qPCR it was shown that BPA was up-regulating enamelin mRNA levels and down-regulating klk4 mRNA. The reson that mixtures of EDs were not as disruptive as BPA alone was discovered to be link to V which had a down-regulation effect on enamelin mRNA promoters only.
It was suggested that there was most likely other target genes that the EDs affect that gave rise to there results.

==Lab 9==
2.Great stuff guys,
Good layout, nice flow of topics and comprehensive. Language is easy to follow. Well research and supported. There are couple of parts that need some references WIKI original recommends that if you make a statement of fact or something that can be disputed you should add a reference ie. last statement of epidemiology . But, work in progress, i understand.
You could hyperlink some of the more unfamiliar words to the UNSW embryology glossary and other pages to get the wiki "click through" effect.
I hate to recommend it because i really like how clean and "wiki" like your page is but we have to add images so perhaps a map of the genes and mutations, show the promoters and such?
The symptoms section has some repetition to its structure i think you should condense it all into the table then write a lead in paragraph to the table. Lead in could have a bit about when the symptoms usually come on in life?
I would recommend moving diagnosis to above treatment and after pathology to help with flow. This would also semi-separate the page into theory and clinical.
For the pathology image if you make a one by one table and put the image into it, it should sit in alignment on the page. Its just my browser but on a smaller screen it cuts out to the left. Not a big deal. try to have the images on a line to them selves or at the end of paragraphs rather then word wrapping the text. Makes it look neater no matter how big you have the window.
That's all i can think of. Other wise looks like it going to be one of the best of the class. Very professional.

3.This is really good and basically finished,
Images are relevant and informative, then drawn image is especially great. Flow and headings are spot on, nice table. Realy professional.
Not a huge amount to say on this one just a bit of housework to do before you publish.
Make sure any factual statements you have are refrenced
Id move the first image of the FRS so your not leading in with it. Drop the Definition subheading and just make in your introduction section. Makes it more wiki like. For a style guide have a look at the wiki page https://en.wikipedia.org/wiki/Ovarian_hyperstimulation_syndrome. Taking the underline from under the subheading will make it read nicer, but then I have a person hate of underlines.
You could add an epidemiology section to move to world map to and there’s lots information you could put in such a section.
Your image under pathogenesis is word wrapped to the left. I would change that to in line or word wrap to the right. Given the detail of the image I would suggest in line. Likewise with your rat specimen image bring it down under the heading. If you put it in its own box you could add some details about what we’re looking at in the image.
Topics like Environmental factors could use some more detail. Although I know were all still working on our pages.
Make sure to hyperlink unusual words or other topics mentioned to the sites glossary or pages for that lost clicking through wiki effect.
Apart from that another top notch page, great detail, really interesting and direct. Fantastic work guys.

4.
Great work guys, a lot of information in there.
Well referenced and easily readable. Nicely broken up with some informative images. Tables are great and make it easy to find information on a big page. And the first embedded video I’ve seen which is nice.
Looks like it all the information is there already just a couple of heading to finish off so well done.
Stylistically the only things ill pick on are,
The numbered list in treatments, doesn’t fit with the formatting of the rest of the page. Just turn them into subheading or tables like the others.
Some of the tables are standalone ie. Male infertility disorder that should really have some intro text to them. Also make sure they have a description underneath each one describing what’s in table. And reference of where the information’s coming from.
You could add a section on epidemiology/prevalence of types of infertility. But the intro could be enough if you flesh something out in there.
Don’t forget to hyperlink some of the important words to the sites glossary and other pages so people can find extra reading or background. And I think you’ll have a really informative page.
Fantastic work guys.

5.
Hey guys been working hard I see,
Lots of information, well referenced (chemotherapy section pending) and really interesting stuff.
The layouts a bit hard to follow, I’d suggest dropping things down a heading level so that, example, infertility was the heading, with page beak. And targeted drugs surgery etc. wear a third level head and just bold.
At times the page seems to go off on a tangent such as how chemo is administered. Try to tie it in with what relation it has on onco-fertility or consider leaving it out and just linking to further information on the subject.
For the above to points have a look at a wiki page that also deals with a dense subject like the world war 2 page https://en.wikipedia.org/wiki/World_War_II look at what they’ve done to group the information and break out blocks of text.
The list of drugs at the end would go much nicer into a table after leading in with the text. I recommend you rotate you tables too so that they read left to right rather than top to bottom ie switch rows and columns and then put the data into bullet list in each table area rather than dashing out the excess space.
Most of the images are great and interesting (oocytes and tissues), Some don’t add much to the page (DNA breaks, Lady get intravenous). Have a look through them and think about whether the image provides any extra information, assists understanding of topic or is eye-catching, attention grabbing, if it doesn’t do one of the three grab another image.
Consider adding some hyperlinks to the sites glossary and other pages of site key words.
It’s all there and the information’s really good. If you work out the formatting so the text is easily digestible you’ll have a great page.

6.
Fantastic job guys, this ones hard to pick on.
Layout is great, flows, logical topics, easy to read, nicely broken up with informative or interesting images. Just smashing. References appear to all be solid.
So what can I suggest? You could try making up a table for the advantages and diss advantages so it’s easy to compare each tech. The PCR Cycle copies table is just a list of exponential growth and can go.
Hyperlink some of your key words to the glossary or other pages so people can get background or further reading on topics involved.
Change the heading to spell out Assisted Repro… etc. so that its stated before you abbreviate. Collect up the info to make your intro and so far you have the best project of the group. Pretty flawless guys.

==Lab 10==
===Hyaloid Vascular System===

Link to permalink image: [https://embryology.med.unsw.edu.au/embryology/Slides/Embryo_Stages/Stage22/08-eye/Stage22-08-eye.html?zoom=5&lat=-3273&lon=4370&layers=B | Hyaloid Vascular System]

The hyaloid vascular system (HVS) is a network of blood vessels, supplied by the hyaloid artery and extending from the optic disk to the superior part of the retina. These nourish the eye during development . This structure is transient disappearing before birth, but is commonly present in premature infants<ref><pubmed> 23772130 </pubmed></ref>. Persistence post nataly leads to severe blindness. It is a common target of studies into the signaling pathways of vascuralisation and regression.

'''Embryology link''' [[Vision - Retina Development #Week 8]]

==Lab Attendance==
--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:45, 7 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:31, 14 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:43, 21 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:36, 28 August 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:20, 4 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:08, 18 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 14:00, 25 September 2015 (AEST)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:55, 9 October 2015 (AEDT)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 13:45, 16 October 2015 (AEDT)

--[[User:Z3292373|Z3292373]] ([[User talk:Z3292373|talk]]) 12:21, 23 October 2015 (AEDT)

=Notes=
[[Test student 2015]]

{{Uploading Images in 5 Easy Steps table}}

[http://www.biomedcentral.com]

{{StudentPage2015}}

=References=

2015 Group Project 1

2015-10-23T00:45:16Z

Z3292373: /* Glossary */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
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'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=
'''cortical necrosis''' Break down of the kidney tisssue.

'''hetroplasmy''' When a cell line contains two dissimilar mitochondrial DNA elements

'''homoplasmy''' When a cell line contains only one mitochondrial DNA

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Myopathy''' A disease of the muscle tissue

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''pigmentary retinopathy''' Migration and proliferation of the retinal pigment cell into the retina. Produces blindness.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:58:08Z

Z3292373: /* Under Discussion */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven members of the public who had contacted the FDA in advance spoke in favor of the techniques. There was no formal decision made on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:53:34Z

Z3292373: /* Permitted */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country that legally allows the inheritable genetic modification of humans. On February 24 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would allow the creation of an embryo with genetic material from three different people and result in inheritable genetic modification. It was passed with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:50:14Z

Z3292373: /* Ethics */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
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'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. They argue that unlike the use of donor eggs or embryos, children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques. Thus for these intending mothers, using egg or embryo donation is not a suitable alternative. From the childrens point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical well-being such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country in the world legally allows the inheritable genetic modification of humans. On February 24, 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would create an embryo with genetic material from three different people and result in inheritable genetic modification, with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:44:20Z

Z3292373: /* Other Approaches */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is the large nucleus of an immature oocytes arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise, arrest during development or are associated with aneuploidy. Studies in humans have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation progresses<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. Unlike the use of donor eggs or embryos, they think that children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques; thus, for these intending mothers using egg or embryo donation is not a suitable alternative. From the children’s point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents function. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country in the world legally allows the inheritable genetic modification of humans. On February 24, 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would create an embryo with genetic material from three different people and result in inheritable genetic modification, with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:38:42Z

Z3292373: /* Polar Body Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. They contain complementary chromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygote <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. It contains a haploid set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually.

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalities. More recently the role of polar bodies in assisted reproductive technology are: single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer have been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimum mitochondria but carries the entire genome.
* Polar body 1 and 2 are separate from the oocyte, thus can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted in mice models to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on a mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is a large nucleus of the immature oocytes arrested naturally in the first meiotic prophase. the oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise or arrest during development or are associated with aneuploidy. Studies using human oocytes have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation ensues<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. Unlike the use of donor eggs or embryos, they think that children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques; thus, for these intending mothers using egg or embryo donation is not a suitable alternative. From the children’s point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents function. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country in the world legally allows the inheritable genetic modification of humans. On February 24, 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would create an embryo with genetic material from three different people and result in inheritable genetic modification, with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:29:27Z

Z3292373: /* Spindle-Chromosome Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction in heteroplasmy risk, thus offering a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. The chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to asses the safety of this procedure. Tachibana et al(2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome transfer is a safe procedure. Because defects may develop later in life, or in their offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are now testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. they contains complementary choromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygot <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. it contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. it contains a haploit set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalitiesg. More recently, the new roles of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and the polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer has been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimunmitochondria but carries the entire genome.
* Polar body 1 and 2 are seperate from the oocyte thus it can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted on mice model to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is a large nucleus of the immature oocytes arrested naturally in the first meiotic prophase. the oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise or arrest during development or are associated with aneuploidy. Studies using human oocytes have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation ensues<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. Unlike the use of donor eggs or embryos, they think that children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques; thus, for these intending mothers using egg or embryo donation is not a suitable alternative. From the children’s point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents function. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country in the world legally allows the inheritable genetic modification of humans. On February 24, 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would create an embryo with genetic material from three different people and result in inheritable genetic modification, with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:17:46Z

Z3292373: /* Risk of Cytoplasmic Transfer -- Heteroplasmy */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- Heteroplasmy===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction of heteroplasmy risk, thus offer a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>.This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. Similarly, the chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome tranfer, experiments in non-human primates are required to access the safety of this procedue. Tachibana et al(2009) have carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome tranfer is a safe procedure. Because defects may develop later in life, or in their own offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. they contains complementary choromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygot <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. it contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. it contains a haploit set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalitiesg. More recently, the new roles of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and the polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer has been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimunmitochondria but carries the entire genome.
* Polar body 1 and 2 are seperate from the oocyte thus it can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted on mice model to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is a large nucleus of the immature oocytes arrested naturally in the first meiotic prophase. the oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise or arrest during development or are associated with aneuploidy. Studies using human oocytes have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation ensues<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. Unlike the use of donor eggs or embryos, they think that children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques; thus, for these intending mothers using egg or embryo donation is not a suitable alternative. From the children’s point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents function. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country in the world legally allows the inheritable genetic modification of humans. On February 24, 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would create an embryo with genetic material from three different people and result in inheritable genetic modification, with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T18:15:45Z

Z3292373: /* Cytoplasmic Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor [[Oocyte Development|oocyte]] or [[zygote|zygote]] into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8‐cell stage in humans, where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still investigating the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many additional factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- '''Heteroplasmy'''===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. Previously it was believed to have been a rare heteroplasmic mutation in healthy individuals . However, human mtDNA sequencing has now shown that each person has some low-frequency, variant mtDNA types, mixed with the maternally inherited dominant type. These low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

The low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold will defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction of heteroplasmy risk, thus offer a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>.This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. Similarly, the chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome tranfer, experiments in non-human primates are required to access the safety of this procedue. Tachibana et al(2009) have carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome tranfer is a safe procedure. Because defects may develop later in life, or in their own offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. they contains complementary choromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygot <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. it contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. it contains a haploit set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalitiesg. More recently, the new roles of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and the polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer has been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimunmitochondria but carries the entire genome.
* Polar body 1 and 2 are seperate from the oocyte thus it can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted on mice model to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is a large nucleus of the immature oocytes arrested naturally in the first meiotic prophase. the oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise or arrest during development or are associated with aneuploidy. Studies using human oocytes have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation ensues<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. Unlike the use of donor eggs or embryos, they think that children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques; thus, for these intending mothers using egg or embryo donation is not a suitable alternative. From the children’s point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents function. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country in the world legally allows the inheritable genetic modification of humans. On February 24, 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would create an embryo with genetic material from three different people and result in inheritable genetic modification, with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=

2015 Group Project 1

2015-10-22T17:59:45Z

Z3292373: /* Mitochondrial mutation and benefits of mitochondrial donation */

{{ANAT2341Project2015header}}

=Three Person Embryos=

'''Three Person Embryos''' are embryos from oocytes that contain maternal and paternal DNA, and [[mitochondria]] from a third donor. Collectively, the techniques for the creation of Three Person Embryos are referred to as Mitochondrial Donation or Mitochondrial replacement-assisted IVF. Mitochondrial donation is used for the prevention of maternal inheritance of [[2015 Group Project 1#Hereditory mitochndrial Disorders|Mitochondrial disorders]] that occur due to the mutation of mitochondrial DNA (mtDNA). It is considered a germ-line therapy, with the donated mitochondria being passed maternally to the next generation. Because of this it has generated debate in the media and scientific community over the [[2015 Group Project 1#Ethics|ethics]] of its use, since the first techniques were developed in the 1980s. Recently, with the development of safer techniques, the United Kingdom and United States have begun the process of [[2015 Group Project 1#Legal Status|legalizing]] its clinical use.

<html5media width="560" height="315">https://www.youtube.com/embed/0Zs2KntZ7vU</html5media>

Teenage Girl Has Three Biological Parents <ref> GeoBeats News. (20131, December 19) Teenage Girl Has Three Biological Parents. Retrieved from https://youtu.be/0Zs2KntZ7vU </ref>

=History=
<div class="NavFrame">
<div class="NavHead">'''
'''Timeline Of Mitochondrial Donation'''
'''</div>
<div class="NavContent">
===1980s===
::* '''1981, United Kingdom''' - Complete sequencing of human mitochondrial genome<ref><pubmed> 7219534 </pubmed></ref>
::* '''1982, United Kingdom''' - Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian [[2015 Group Project 1#Cytoplasmic Transfer|cytoplasmic transfer]] in mice <ref><pubmed> 6896904 </pubmed></ref>.
::*'''1984, United Kingdom''' - Publication of the Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation worldwide.
::*'''1988, US and UK''' - First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1990, United Kingdom''' - Implamentation of the Human fertilization and embryology act 1990. Governs the legal requirements around research and clinical use of IVF technologies until present.<ref>Human Fertilisation and Embryology Act 1990 c.37, retrieved from http://www.legislation.gov.uk/ukpga/1990/37/contents 23/10/15</ref>
::*'''1996, United Kingdom''' - Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.
::* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
::*'''1998, United States''' - FDA ban use of cytoplasmic transfer techniques.
::*'''1998, United States''' - First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===
::* '''2002 United States''' - One of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
::*'''2008, United Kingdom''' - Changes to the Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009, United States''' - First success-full trails of [[2015 Group Project 1#Spindle-Chromosome Transfer|spindle transfer]] in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::* '''2014 United States''' - Public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 United Kingdom''' - Regulations to allow the open use of three person IVF via [[2015 Group Project 1#Pronuclear transfer|pronuclear transfer]] in fertility clinics comes into affect in the UK.
</div>

=Mitochondrial mutation and benefits of mitochondrial donation=
[[Mitochondria]] are generally known as the ATP production sites of the cell. Although they are also involved in signalling, differentiation, cell cycle, cell development, Neuronal function and many other functions <ref><pubmed> 2830540 </pubmed></ref>. In mammals mitochondria contain their own circular genome encoding for 37 genes of which 13 are vital to oxidative phosphorylation and hence the respiratory chain. The remainder of mtDNA encodes for tRNAs and rRNAs<ref><pubmed> 16814712 </pubmed></ref>. Each mitochondria contain 2-10 identical copies of their mtDNA at birth in a healthy person and average 100 mitochondria per cell. In addition to mtDNA over 1000 nuclear DNA (nDNA) encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </pubmed></ref>. In normal mammalian mating all mtDNA is maternally inherited<ref><pubmed> 17506638 </pubmed></ref>.

Mutations in mtDNA or nDNA mitochondrial genes can lead to abnormalities in normal function. The level of dysfunction in non-X-linked maternally inherited disorders is related to the copy number mutated mtDNA molecules in individual mitochondria and the percentage of mitochondria in a cell that contain mutated mtDNA. Because mitochondria cover a wide range of functions in varying regions of the body clinical presentations are also wide ranging<ref><pubmed> 16814712 </pubmed></ref>.

Mitochondrial donation can benefit anyone that is at risk of passing on to their offspring a mitochondrial disorder that is caused by a mtDNA mutation. It cannot however prevent inheratence of nDNA derived disorders. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations.

Extrapolation from small studies estimate that per year 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>. Prevalence in the population of mtDNA associated disorders is estimated to be 1 in 10,000.<ref><pubmed> 20393463 </pubmed></ref>.

===Inheritance of mitochondrial disorder===
Although mtDNA is entirely maternally inherited, offspring of a pathogenic mother may have substantially different pathology and level of mutated mtDNA. Clinical presentation of disease only occurs once levels of mutated mtDNA pass a threshold within a cell<ref><pubmed> 1463006 </pubmed></ref>. Fission and fusion of mitochondria inside of the cell leads to transmission of copies of mtDNA and hence an uneven distribution on mutated mtDNA. This then leads to a distribution of functional, semi-functional and dysfunctional mitochondria within each cell. During cell division these mitochondria are then randomly distributed among the daughter cells as described in the table bellow. The higher the level of mtDNA mutation in the parent cell the greater the likelihood of the daughter cell to receive a random distribution mutated mtDNA above the thresh hold<ref><pubmed> 1463006 </pubmed></ref>. When this occurs during meiotic cell division the mtDNA in the daughter cell will go on to form the entire mtDNA of the offspring.
Although poorly understood there has been shown to be a selective pressure against germ-line cells with an accumulation deleterious mutations<ref><pubmed> 18695671 </pubmed></ref> as well as a tendency for hetroplasmic blastomeres to shift towards homoplasmy before implantation<ref><pubmed> 22701816 </pubmed></ref> suggesting some mechanisms mtDNA selection post division.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Mitochondrial_DNA_Inheritance.jpg|600px|thumb|left|In mammals mitochondria may have between two and ten copies of their genome. Mitochondria may have any ratio of mutated mtDNA. In the production of gametes the mitochondria of the parent distribute randomly. Therefore a partially affected mother may produce a spectrum of gametes with mitochondrial disorders from unaffected to totally affected ]]
|}

===Hereditary Mitochondrial Disorders===
Mitochondrial disorders cover a broad range of clinical symptoms and affected organs. Predominantly they present as neurologic and [[2015 Group Project 1#Glossary|myopathic]] diseases owing to the retardation of ATP production but symptoms can include deafness, vision loss, diabetes and organ failure among others. The following is an inexhaustive list of the most notable disorders.
{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Disease Type
! Clinical Pathology
! Mutation
! Preventable with mitochondrial donation
|-bgcolor="F5FAFF"
| Alpers disease<ref><pubmed>20220442</pubmed></ref>
| Degenerative disease of CNS
| [[2015 Group Project 1#Glossary|Psychomotor retardation]], epilepsy, liver failure, [[2015 Group Project 1#Glossary|cortical necrosis]]
| nDNA gene mutation
| style="background-color: salmon;"|"No"
|-
| Kearns-Sayre Sydrome (KSS)<ref><pubmed>25539952</pubmed></ref>
| Mitochondrial Myopathy
| Causes [[2015 Group Project 1#Glossary|pigmentary retinopathy]], conduction block, ataxia. Can cause mental reardation/deterioration, delayed sexual maturation.
| mtDNA deletion
| style="background-color: lime;"|"Yes"
|-bgcolor="F5FAFF"
| Leigh Syndrome<ref><pubmed>18651330</pubmed></ref>
| Mitochondrial Myopathy
| Necrotizing lesions in the brain-stem, developmental delays, muscle weakness, [[2015 Group Project 1#Glossary|hypotonia]], respiratory distress and death before the age of five.
| 30 X-linked Recessive genes. mtDNA mutation.
| style="background-color: LightBlue;"|"20% of Cases"
|-
| Mitochondrial DNA Depletion Syndrome (MDS)<ref><pubmed>23385875</pubmed></ref>
| Mitochondrial Myopathy
| Muscle weakness, liver failure and developmental retardation. Can cause brain abnormalities, pigmentary retinopathy and seizures.
| nDNA mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stoke-like episodes (MELAS)<ref><pubmed>25038129</pubmed></ref>
| Mitochondrial Myopathy
| Prolonged focal seizures and epilepsia. Pigmentary retinopathy, muscle weakness, hearing loss,diabetes.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE)<ref><pubmed>26264513</pubmed></ref>
| Mitochondrial Myopathy
| Gastrointestinal disorders, diarrhea, abdominal pain. Peripheral neuropathy.
| nDNA TYMP gene mutation
| style="background-color: salmon;"|"No"
|-bgcolor="F5FAFF"
| Myoclonus epilepsy with ragged red fibres (MERFF)<ref><pubmed>12876264</pubmed></ref>
| Mitochondrial Myopathy
| Seizures, ataxia, myopathy linked to diabetes, optic atrophy peripheral neuropathy, hearing loss and dimentia.
| mtDNA point mutation
| style="background-color: lime;"|"yes"
|-
| Neuropathy, ataxia and retinitis pigmentosa (NARP)<ref><pubmed>11730668</pubmed></ref>
| Mitochondrial Myopathy
| Rod-Cone dystrophy of the eye, muscle weakness, ataxia and retinitis pigmentosa
| mtDNA 6-gene mutation
| style="background-color: lime;"|"yes"
|-bgcolor="F5FAFF"
| Pearson syndrome<ref><pubmed>25691415</pubmed></ref>
| Mitochondrial Myopathy
| Bone marrow failure and pancreatic insufficiency. If survival past childhood develops into Kearns-Sayre syndrome.
| mtDNA rearrangement, deletion.
| style="background-color: lime;"|"yes"
|-
| Progressive external ophthalmoplegia (PEO)<ref><pubmed>26251896</pubmed></ref>
| Mitochondrial Myopathy
| Progressive paralysis of the eye muscles. Can be distinct syndrome or part of greater mitochondrial disorder
| mtDNA and nDNA mutations
| style="background-color: LightBlue;"|"Most Cases"
|}

=Technical Progression=
Three-person ''in-vitro'' fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos <ref><pubmed> 24382342</pubmed></ref> <ref><pubmed> 25573721 </pubmed></ref>.

*The first proposed treatment is '''cytoplasmic transfer''', which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes <ref><pubmed> 24382342</pubmed></ref>.
*New emerged approaches of mitochondrial transmission are '''pronuclear transfer(PNT)''', '''spindle transfer (ST)''' and '''Polar body transfer (PBT)'''. however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide <ref><pubmed>24373414</pubmed></ref>.
*Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) <ref><pubmed> 25229667 </pubmed></ref>, early stage human embryo and stem cell studies <ref><pubmed> 23103869 </pubmed></ref>.

Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

==Cytoplasmic Transfer==

'''Cytoplasmic transfer''' is also named '''Ooplasmic transfer'''. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor oocyte or zygote into compromised oocyte or zygote from patients.

===Why do cytoplasmic transfer?===
The quality of the oocyte cytoplasm is critical for the future of the embryo <ref><pubmed> 15140871 </pubmed></ref>.Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. it is until the maternal-to-zygotic transition (MZT) stage during the 4–8‐cell stage in humans where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development <ref><pubmed> 3352746 </pubmed></ref> . the maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome <ref><pubmed> 10429238 </pubmed></ref>.

Researches are still underway to investigate the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many factors yet to be determined. '''The benefits of ooplasm transfer''' are revealed by the following two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed> 23602680 </pubmed></ref>.

===What is the procedure?===
Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo <ref><pubmed> 24382342</pubmed></ref> <ref name='3egirl'> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

{| style="border-spacing: 2px; border: 1px solid white;"
| [[File:77260486_cell_structure_304.gif|100px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|400px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|380px|thumb|right|repair by Cytoplasmic transfer <ref name='3egirl'/>]]
|}

=== Key Events of Cytoplasmic Transfer ===
* '''1982, United Kingdom''' - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice <ref><pubmed> 6896904 </pubmed></ref>.
* '''1982 United Kingdon''' - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier <ref><pubmed> 6896904 </pubmed></ref>.
* '''1997, United States''' - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer <ref><pubmed> 9250192</pubmed></ref>.
* '''1998 United States''' – The US Food and Drug Administration (FDA) banned the procedure.
* '''2002 United States''' - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
* '''2014 United States''' - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.

{|
|+ style="text-align: center;" | '''Cytoplasmic transfer cases in human'''<ref name="Barritt2001">J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>
|-bgcolor="CEDFF2"
! Type of Cytoplasm Transferred to recipient oocytes
! No. of Procedures
! Pregnancies achieved
! Offspring delivered
|-
|Synchronized fresh oocytes by electrofusion <ref><pubmed> 9570273 </pubmed></ref>
| 3
| 0
| 0
|-bgcolor="F5FAFF"
| Synchronized fresh oocytes by injection (USA) <ref><pubmed> 9250192 </pubmed></ref> <ref><pubmed> 9570273 </pubmed></ref> <ref><pubmed> 10973657 </pubmed></ref> <ref><pubmed>11228222 </pubmed></ref> <ref><pubmed>11041526</pubmed></ref>
| 30
| 13
| 16
|-
| Synchronized fresh oocytes by injection (Israel) <ref name="Barritt2001"/>
| 15
| 5
| 6
|-bgcolor="F5FAFF"
| Synchronized frozen oocytes by injection <ref><pubmed>10065803</pubmed></ref>
| 4
| 1
| 2
|-
| Asynchronous 3-PN zygotes by injection <ref><pubmed>10521114</pubmed></ref>
| 9
| 4
| 5
|}

===Risk of Cytoplasmic Transfer -- '''Heteroplasmy'''===

'''Heteroplasmy''' is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual <ref><pubmed>20735895</pubmed></ref> <ref><pubmed>24135157</pubmed></ref>. It is believed to have rare heteroplasmic mutation in healthy individuals previously. however, human mtDNA sequencing has now showed that each person has some low- frequency, slightly different mtDNA types mixed with the maternally inherited dominant type. and this low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy <ref><pubmed>23271951</pubmed></ref> .
[[File:Gmb-35-886-g001.jpg|600px|thumb|right| An example of sequence heteroplasmy visualized by partial mtDNA sequencing <ref><pubmed>23271951</pubmed></ref> ]]

*'''Length heteroplasmy''' is the presence of mtDNA molecules that differ in length.

*'''Sequence (site) heteroplasmy''' is the presence of mtDNA molecules that have different nucleotides at the same site.

Although the low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA <ref><pubmed>23077218</pubmed></ref>. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold, defects will be induced<ref><pubmed>23271951</pubmed></ref>.

'''Cytoplasmic transfer in IVF procedure''' has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure <ref><pubmed>16939888</pubmed></ref>. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system <ref><pubmed>26281784</pubmed></ref>. They could also result in mitochondrial disease developing either in the child or in future generations <ref><pubmed>24709341</pubmed></ref>.

==Spindle-Chromosome Transfer==

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background <ref><pubmed>25444504</pubmed></ref>. Comparing to cytoplasmic transfer, the '''advantage''' of spindle transfer is the reduction of heteroplasmy risk, thus offer a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>.This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection <ref><pubmed> 25573721 </pubmed></ref>. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy <ref><pubmed> 25973765 </pubmed></ref>.

===What is the procedure?===
{| style="border-spacing: 2px; border: 1px solid white;"
|-
|[[File:MT transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]
|Assisted reproductive technologies are used to extract the patient’s egg from her ovaries. The cytoplasm of the egg contains the unhealthy mitochondria. Chromosomes, the nuclear DNA material, are found in the patient’s eggs are grouped together in a spindle-like formation.

#The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
#Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. Similarly, the chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
#The spindle-like chromosomes previously taken from the patient's egg are inserted into the enucleated donor’s egg.
#The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
#The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the patient and will develop unaffected by inherited mitochondrial disease.
|-
|}

===Primate model===

{| style="border-spacing: 2px; border: 1px solid white;"
|-
|Due to the uncertainty of the health risks related to spindle-chromosome tranfer, experiments in non-human primates are required to access the safety of this procedue. Tachibana et al(2009) have carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref><pubmed> 19710649 </pubmed></ref>.
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome tranfer is a safe procedure. Because defects may develop later in life, or in their own offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref><pubmed> 19710649 </pubmed></ref>]]
|-
|}

===Current Research===

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are testing the maternal spindle transfer technique on human oocytes. ''Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA''. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization<ref><pubmed> 23103867</pubmed></ref>.

===Limitations===
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities <ref><pubmed> 25472922</pubmed></ref> <ref><pubmed> 23254936</pubmed></ref>.

==Pronuclear transfer==

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed <ref><pubmed>25573721</pubmed></ref>.

*pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA <ref name='Vande2012'>Mado Vandewoestyne , Jitesh Neupane , Björn Heindryckx , Sylvie Lierman ,Dieter Deforce and Petra De Sutter (2012) Pronuclear transfer in mice yields minimal mitochondrial DNA carry-over Mado Vandewoestyne FERTILITY AND STERILITY. 98(3, suppl.). p.S289-S289 </ref>.

*In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses <ref name='humanmodel2003'> '''Three-Parent Baby Pioneer Jamie Grifo: The Brits Will be Ahead of the World''' 16 January 2015 http://www.geneticsandsociety.org/article.php?id=8314. Retrived 15 Oct 2015</ref>.

*In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease <ref><pubmed> 20393463</pubmed></ref>.

===What is the procedure?===

[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref><pubmed> 25573721 </pubmed></ref>]]
The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote <ref name ='humanmodel2003'/>
# It begins with creating an embryo using the parents’ sperm and eggs.
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
# The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
# The pronuclei of the second embryo are removed and discarded.
# The parents’ pronuclei can be placed into the second embryo for development.
# The developed embryo will then be transferred into the mother.

===Human Embryo Model===

Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref><pubmed> 20393463 </pubmed></ref>:

* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
*Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
*Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities

<span style="color:blue">'''Current research on pronuclear transfer''' </span> [https://www.youtube.com/watch?v=Sr7Jnr9qn44| Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.]

===Limitations===

pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice model. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth <ref><pubmed>16275929</pubmed></ref> . Human embryo model study showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos <ref><pubmed>20393463</pubmed></ref>. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.

==Polar Body Transfer==

'''Polar bodies''' are small cells formed during the meiotic reductive division of the oocyte. they contains complementary choromosomes (to the mature oocyte) and small amount of cytoplasmic segregation<ref><pubmed> 24949971 </pubmed></ref>. [[File:Early zygote labelled.jpg|250px|thumb|an early human zygot <ref name = earlyzygot>Hill, M.A. (2015) Embryology Early zygote labelled.jpg. Retrieved October 16, 2015, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Early_zygote_labelled.jpg</ref>]]

* Polar body 1 is formed and released during ovulation. it contains a diploid set of chromosomes.
* Polar body 2 is formed during fertilization and can be identified in the zygote. it contains a haploit set of chromosomes.
* Both polar bodies are unable to be fertilized and disintegrate eventually

Due the unique feature of polar bodies, which can provide beneficial information about the genetic background of the oocyte without potentially destroying it, polar body biopsies have been applied in preimplantation genetic diagnosis to detect inheritable chromosomal or genetic abnormalitiesg. More recently, the new roles of polar bodies in assisted reproductive technology are single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and the polar body transfer to prevent the transmission of mtDNA-associated diseases <ref><pubmed> 25472922 </pubmed></ref>.

The '''advantages''' of polar body transfer has been reported as<ref><pubmed> 25472922 </pubmed></ref>:

* Polar body 1 and 2 contain minimunmitochondria but carries the entire genome.
* Polar body 1 and 2 are seperate from the oocyte thus it can be easily manipulated without damage to the chromosome.
* each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significant increase the efficiency of using donor egg.

===What is the procedure?===

{| style="border-spacing: 1px; border: 1px solid white;"
| [[File:PB1 transfer.jpg|600px|thumb|left|Diagram of Polar body 1 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Diagram of Polar body 2 transfer <ref><pubmed> 25472922 </pubmed></ref> ]]
|}

===Mice Model===

Polar body transfer has been adopted on mice model to prevent the transmission of mtDNA variants. They also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases<ref><pubmed> 24949971 </pubmed></ref>.
The other group coupled Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer on mice model which increased the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

==Other Approaches==

===Germinal Vesicle Nuclear Transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]

The '''germinal vesicle''' (GV) is a large nucleus of the immature oocytes arrested naturally in the first meiotic prophase. the oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization <ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.

'''Germinal Vesicle Transfer (GVT)''' is the transfer of a GV from an unfertilised into an enucleated recipient oocyte. Following reconstruction, the GV is allowed to develop to Metaphase II through in vitro maturation (IVM) and is then fertilised through either IVF or ICSI. The resultant zygotes are then allowed to develop in culture before transfer to patients <ref name = 'SCAG2005'> Scientific and Clinical Advances Group 24 Nov 2005 Germinal vesicle transfer SCAG(11/05)04 retrieved from http://www.hfea.gov.uk/docs/SCAG_Germinal_vesicle_transfer_nov05.pdf at 16 Oct 2015</ref>. These procedures have been proposed as potential treatments for those women whose oocytes fail to fertilise or arrest during development or are associated with aneuploidy. Studies using human oocytes have shown that GVT from aged oocytes introduced into the enucleated ooplasm of young oocytes or sibling oocytes can overcome oocyte aneuploidy, and produced the majority of reconstructions with normal karyotypes<ref><pubmed>25985993</pubmed></ref> <ref><pubmed>25515532</pubmed></ref>.

Similar to the other techniques,'''A major concern of GVT ''' is still that the transferred GV is still surrounded by a population of tightly packed mitochondria which will also be introduced into the donor ooplasm. These mitochondria remain close to center of the immature reconstruction and disperse throughout the cytoplasm as maturation ensues<ref name = 'SCAG2005'/>.

=Ethics=

Some people believe that the Mitochondrial Gene Transfer techniques are ethical. The Nuffield Council on Bioethics in the UK examined the ethical issues and wrote in a report that “Due to the health and social benefits to individuals and families of living free from mitochondrial disorders, … we believe that if these novel techniques are adequately proven to be acceptably safe and effective as treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.”. <ref> http://nuffieldbioethics.org/project/mitochondrial-dna-disorders/ </ref>

Others hold an opposite opinion. They doubt the safety of Mitochondrial Gene Transfer techniques and believe that other safe means of reproduction already exist can be used instead. Unlike the use of donor eggs or embryos, they think that children born with Mitochondrial Gene Transfer techniques would have a genetic connection to three parents due to the fact that such therapies involve modification of the germline. Some mothers may feel that it is important to have a genetic link with their future child and that having this genetic link outweighs most disadvantages (e.g. health risks and high financial cost) associated with Mitochondrial Gene Transfer techniques; thus, for these intending mothers using egg or embryo donation is not a suitable alternative. From the children’s point of view, there are also two concerns. First, children may have a troubled relationship with their parents or struggle to develop their identity they are aware that they share a mitochondrial genome with a donor. Second, Mitochondrial Gene Transfer conceived children may be exposed to some risks to their physical wellbeing such as the failure of donor’s mtDNA to function properly with the nuclear genes contributed by the intending parents function. <ref><pubmed>26239841</pubmed></ref>

=Legal Status=
==Permitted==

Britain is the only country in the world legally allows the inheritable genetic modification of humans. On February 24, 2015, the House of Lords approved regulations. Earlier in the month, the UK House of Commons also approved the techniques that would create an embryo with genetic material from three different people and result in inheritable genetic modification, with 382 votes in favor and 128 against. <ref> Gallagher, James (03 February 2015) [http://www.bbc.com/news/health-31069173 MPs say yes to three-person babies] ''BBC News'' Retrieved 09 October 2015. </ref>

==Under Discussion==

In USA, the legality of mitochondrial manipulation techniques is still under discussion. On February 25 and 26, 2014, public meetings that included discussion of mitochondrial manipulation techniques were held by The US Food and Drug Administration (FDA). None of the seven public spoke who had contacted the FDA in advance in favor of the techniques. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data. On January 27 2015, the Institute of Medicine (IOM) held the first in a series of meetings to fulfill the FDA’s request to consider the Ethical and Social Policy of Novel Techniques for Prevention of Maternal Transmission of Mitochondrial DNA Diseases.

==Prohibited==
{| class="wikitable sortable" style="text-align:left"
|-bgcolor="CEDFF2"
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-bgcolor=white
|

=== Asia ===

| Cyprus* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Georgia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| India || [http://www.icmr.nic.in/art/art_clinics.htm National Guidelines for Accreditation, Supervision & Regulation of ART Clinics in India]

[http://india.gov.in/ethical-policies-human-genome-genetic-research-and-services-department-biotechnology Ethical Policies on the Human Genome, Genetic Research and Services by Department of Biotechnology]

[http://www.icmr.nic.in/stem_cell/stem_cell_guidelines.pdf Guidelines for Stem Cell Research]
|-bgcolor="F5FAFF"
|
| Japan || [http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf Act on Regulation of Human Cloning Techniques (Act No. 146 of 2000) / ヒトに関するクローン技術等の規制に関する法律（平成十二年十二月六日法律第百四十六号）]
|-bgcolor=white
|

=== Oceania ===

| Australia || [https://www.comlaw.gov.au/Details/C2006A00172 Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006]
|-bgcolor="F5FAFF"
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-bgcolor=white
|

=== Europe ===

| Austria || [http://www.ris.bka.gv.at/Dokumente/BgblPdf/1992_275_0/1992_275_0.pdf The Act on Reproductive Medicine / Bundesgesetz, mit dem Regelungen über die medizinisch unterstützte Fortpflanzung getroffen (Fortpflanzungsmedizingesetz — FMedG)]
|-bgcolor="F5FAFF"
|
| Belgium || [http://www.lachambre.be/FLWB/pdf/50/2182/50K2182001.pdf Law on Research into Embryos in Vitro / PROJET DE LOI relatif à la recherche sur les embryons in vitro / WETSONTWERP betreffende het onderzoek op embryo’s in vitro]

[http://www.lachambre.be/FLWB/pdf/51/2567/51K2567005.pdf Law on Medically Assisted Reproduction and the Disposition of Supernumerary Embryos and Gametes / PROJET DE LOI relatif à la procréation médicalement assistée et à la destination des embryons surnuméraires et des gamètes / WETSONTWERP betreffende de medisch egeleide voortplanting en de bestemming van de overtallige embryo's en de gameten]
|-bgcolor=white
|
| Bosnia and Herzegovina* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Bulgaria* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Croatia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Czech Republic* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Denmark* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Estonia* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| France || [http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000441469&dateTexte= Bioethics Law No. 2004-800 / Loi n° 2004-800 du 6 août 2004 relative à la bioéthique]

[http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000549618&dateTexte= Law on the Donation and Use of Elements and Products of the Human Body, Medically Assisted Procreation, and Prenatal Diagnosis, No. 94-654 / Loi n° 94-654 du 29 juillet 1994 relative au don et à l'utilisation des éléments et produits du corps humain, à l'assistance médicale à la procréation et au diagnostic prénatal]
|-bgcolor="F5FAFF"
|
| Germany || [http://www.auswaertiges-amt.de/cae/servlet/contentblob/480804/publicationFile/5162/EmbryoProtectionAct.pdf Act for Protection of Embryos(The Embryo Protection Act) / Gesetz zum Schutz von Embryonen (Embryonenschutzgesetz – ESchG)]

[http://www.gesetze-im-internet.de/advermig_1976/BJNR017620976.html Adoption Brokerage Law 2006 / Gesetz über die Vermittlung der Annahme als Kind und uber das Verbot der Vermittlung von Ersatzmüttern ]
|-bgcolor=white
|
| Hungary* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| Iceland* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-bgcolor="F5FAFF"
|
| Lithuania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-bgcolor="F5FAFF"
|
| Moldova* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-bgcolor="F5FAFF"
|
| Norway || [http://app.uio.no/ub/ujur/oversatte-lover/data/lov-20031205-100-eng.pdf Act of 5 December 2003 No. 100 relating to the application of biotechnology in human medicine, etc]
|-bgcolor=white
|
| Romania* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor="F5FAFF"
|
| San Marino* || [http://conventions.coe.int/Treaty/en/Treaties/Html/164.htm Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine]
|-bgcolor=white
|
| Spain || [http://www.boe.es/buscar/doc.php?id=BOE-A-2006-9292 Law on Assisted Human Reproduction Techniques, No. 14/2006 / Ley 14/2006, de 26 de mayo, Sobre Téchnicas de Reproducción Humana Asistida.]

[http://www.boe.es/boe/dias/2007/07/04/pdfs/A28826-28848.pdf Biomedicine Law 14/2007. Ley 14/2007, de 3 de Julio, de Investigación Biomédica]
|-bgcolor="F5FAFF"
|
| Sweden || [https://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/Lag-2003460-om-etikprovning_sfs-2003-460/ Act on Ethics Review of Research Involving Humans, Law No. 460 (2003). Law (2003: 460) / om etikprövning av forskning som avser människor. Svenska författningssamling 2003: 460]

[http://www.riksdagen.se/sv/Dokument-Lagar/Lagar/Svenskforfattningssamling/sfs_sfs-2006-351/ Genetic Integrity Act, Law No. 351 (2006). Law (2006: 351) / om genetisk integritet m.m. Svenska författningssamling 2006: 351]
|-bgcolor=white
|
| Switzerland || [https://www.admin.ch/opc/en/classified-compilation/20001938/index.html Federal Act on Medically Assisted Reproduction / Bundesgesetz über die medizinisch unterstützte Fortpflanzung]

[https://www.admin.ch/opc/en/classified-compilation/20022165/index.html Federal Act on Research Involving Embryonic Stem Cells / Federal Act on Research Involving Embryonic Stem Cells]
|-bgcolor="F5FAFF"
|

=== Americas ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-bgcolor=white
|
| Uruguay || [http://www.ninrial.com.uy/de-interes/legislacion/leyes/ley-no-19167/ Law Regulating Human Assisted Reproductive Techniques No.19167 / Ley 19.167 – Técnicas de reproducción humana asistida. Regulación]
|-bgcolor="F5FAFF"
|

=== Africa ===

| South Africa || [https://www.capetown.gov.za/en/CityHealth/Documents/Legislation/Act%20-%20National%20Health%20Act%20-%2061%20of%202003.pdf National Health Act 2003 (ACT NO.61, 2003)]
|-bgcolor=white
|

|-bgcolor="F5FAFF"
! '''Region''' !! '''Country''' !! '''Laws'''
|}

* These countries have the same law "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" due to their ratification of the Council of Europe's Convention

=Further Reading=

useful publications:

PMID 23608245
The ethics of creating children with three genetic parents. <ref><pubmed> 23608245</pubmed></ref>

PMID 24382342
Three-Parent IVF: Gene Replacement for the Prevention of Inherited Mitochondrial Diseases.<ref><pubmed> 24382342</pubmed></ref>

PMID 20933103
Mitochondrial function in the human oocyte and embryo and their role in developmental competence.<ref><pubmed> 20933103 </pubmed></ref>

PMID 26020522
Mitochondrial reshaping accompanies neural differentiation in the developing spinal cord.<ref><pubmed> 26020522 </pubmed></ref>

PMID 25421171
The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility.<ref><pubmed> 25421171</pubmed></ref>

PMID 25807984
Risks inherent to mitochondrial replacement.<ref><pubmed> 25807984</pubmed></ref>

=Glossary=

'''Maternal Spindle Transfer:''' The transfer of nuclear DNA from a patient egg into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

'''Ooplasmic Transfer:''' The injection of ooplasm from a donor egg into a patient egg. Leads to mitochondrial heteroplasmy.

'''Pronuclear Transfer:''' The pre-fertilized nuclear DNA form a patient is transferred into a donor egg with its nuclear DNA removed, which is then fertilized and implanted via standard IVF.

=References=

<references/>

=External Links=