Embryology - User contributions [en-gb]

User:Z3251292

2015-10-28T22:09:47Z

Z3251292:

{{StudentPage2015}}

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:47, 6 August 2015 (AEST) Thanks for setting up your page. We will be talking more about this in the [[ANAT2341_Lab_1_-_Online_Assessment|Practical on Friday]].

= Lab Attendance =
--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 13:46, 7 August 2015 (AEST)

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 17:40, 7 August 2015 (AEST) Note Square brackets not curly for links. Curly brackets are for templates.

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 15:43, 10 August 2015 (AEST)Many Thanks

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 13:13, 14 August 2015 (AEST)

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 13:23, 4 September 2015 (AEST)

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 13:23, 11 September 2015 (AEST)

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 12:16, 18 September 2015 (AEST)

--[[User:Z3251292|Z3251292]] ([[User talk:Z3251292|talk]]) 13:29, 16 October 2015 (AEDT)

[[Test student 2015]]

= Week2 Lab1 Reference =

== Summary 1 ==

PMID 26254037

Casillas <ref><pubmed>26254037</pubmed></ref> evaluated the in-vitro maturation, and in-vitro fertilization of cryopreserved immature porcine oocyte with different protocols. Immature porcine oocytes were vitrified with 7.5% dimethylsulphoxide (Me2SO) and 7.5% ethylene glycol (EG), followed by preserving with the cryolock protocol. Two test groups of cryopreserved oocytes are the cumulus-cell oocyte complexes (COCs group), and the denuded oocytes co-cultured with granulose cells (NkO-cc group). The un-vitrified fresh oocytes were used as the control group in experiments.

The in-vitro maturation was achieved by culturing in maturation medium supplemented with Luteinizing hormone(LH) and Follicle-stimulating hormone(FSH). Then the matured oocytes were subjected to in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The capabilities of embryo development of the test oocytes were compared.

Statistical results showed that the NKO-cc group produced higher cleavage rate and blastocyst production than the COCs group, which also suggested a higher embryonic development in NKO-cc group. And there was no significant difference between the control group and the NKO-cc group. Blastocysts were generated by both IVF and ISCI, Whilst IVF resulted in better blastocyst development.

== Summary 2 ==

PMID 26253435

Oxidantive stress caused by high levels of reactive oxygen species (ROS) production is believed to degrade spermatozoa generically and functionally. Crocin, which can quench free radicals were hypothesised to serve as an anti-oxidant protector, so as to improve the quality of sperm, and the in vitro fertilization rate.

Sapanidou<ref><pubmed>26253435</pubmed></ref> investigated whether the addition of Crocin in in-vitro sperm preparation media will improve the sperm quality by preventing them over-oxidated during the freeze-thaw process. Spermatozoa collected from 4 bulls were freezed, thawed and washed, followed by incubation with media supplemented with 3 different concentration of crocin (0.5, 1 and 3mM) up to 240 minutes. The sperm motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation were evaluated. The author also tested the effects of crocin (1mM) in the IVF medium by evaluating the embryo development rate.

Results showed that spermatozoa incubated with 1mM crocin developed lower level of ROS production, lower lipid peroxidant and lower percentage of fragmented cells. They also maintained better motility, viability, and acrosomal function. Furthermore, the addition of 1mM crocin in IVF media significantly increased the embryo development rate. Thus, the author concluded that 1mM crocin improved the sperm quality and fertilization rate by regulating ROS concentration.

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 11:05, 17 September 2015 (AEST) These are good summaries. The second article could have included a description of what "Crocin" actually is. I would have also listed the references at the top as shown below.

<pubmed>26254037</pubmed>
<pubmed>26253435</pubmed>

= Week3 Lab2 Image =

[[File:Embryo and Uterus stained with CD34 at Embryonic day 6.png|300px]]

Embryo and Uterus stained with CD34 at Embryonic day 6 <ref><pubmed> 26247969 </pubmed>| [http://www.mdpi.com/2072-6643/7/8/5284 Nutrients]</ref> PMID 26247969

Embryo and Uterus stained with CD34 at Embryonic day 6. Impaired formation of decidual angiogenesis in the folate-deficient group. N:normal; FD:folate-Deficient group; E:Embryo; L:luminal epithelium; G:glandular epthelium; M:mesometrial; AM:anti-mesometrial; VSF:vascular sinus folding. Scale bar: 500um(left), 100um(right).

Copyright © 2015 by the authors; licensee MDPI, Basel, Switzerland.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 11:10, 17 September 2015 (AEST) Image uploaded OK, file name could have identified the species (mouse). You do not need the copyright information on your student page here, but in the image summary box, which does not contain the reference, copyright or student image template as requested. I have now added these to the image summary box and you should see what you should have done. (3/5)

= Week 4 Lab 3 Paper=

'''Topic: 3 person IVF'''

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>

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 11:14, 17 September 2015 (AEST) Thes papers relate to your group project, I hope they are useful for your final submission. (5/5)

= Week 5 Lab 4 Assessment=

==Quiz==

<quiz display=simple>

{From Implantation onward, Which of the following are not right:
|type="()"}
- Cytotrophoblast cells fuse and form a multinucleated cytoplasmic mass called syncitiotrophoblasts.
- Cytotrophoblast cells lay around the blastocyst, proliferates and extends behind syncitiotrophoblasts.
- Syncitiotrophoblasts invade the decidua.
+ Cytotrophoblast secrets hCG and support Corpus Luteum.
||Syncitiotrophoblasts secrets hCG and support Corpus Luyteum.

{Implantation of human embryos typically occurs:
|type="()"}
- 1 day after fertilization.
+ about one week after fertilization.
- about two weeks after fertilization.
- During fertilization.
||The implantation of human embryo usually occur around 7 days after implantation.

{Which of the following are not derived from mesoderm:
|type="()"}
+ Neural crest
- Sclerotome
- Dermatome
- Myotome
|| Neural crest are derived from ectoderm

</quiz>

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 11:16, 17 September 2015 (AEST) You have not given your quiz questions a title (perhaps, Implantation and Mesoderm). Q1 " Which of the following are not right" should be written "from the following options select the INCORRECT answer". The question looks as if it is designed to test the difference between syncitiotrophoblasts and cytotrophoblasts. Your revealed answer should have provided more information and links to resources. Q2 is not a very well designed question as it does not really test a developmental concept, some of your options are also easily excluded. Q3 is far to simplistic for an assessment item. (7/10)

= Week 6 Lab 5 Assessment=

Discuss how aganglionic colon is a gastrointestinal tract abnormality related to neural crest migration.

Aganglionic colon is also called Hirschsprung’s disease(HD), or congenital megacolon. It is the most frequent congenital disorder of intestinal motility and the most significant entity of pediatric intestinal motility abnormality, caused by the lack of enteric neurons in the distal intestine.<ref><pubmed>26361414</pubmed></ref>

Most entreric neurons arise from a multipotent cell population named neural crest cells, Which first migrate from the caudal hindbrain to the cranial end of gastrointestinal tract, and then migrate caudally along the entire gut during embryo development. A failure of neural crest-derived cells to colonize the affected gut regions will then cause the absence of enteric neurons from variable lengths of the bowel. <ref><pubmed>10917288</pubmed></ref>

Extensive research has identified a number of key genes that regulate neural crest cells' survival, proliferation, differentiation, and migration in the pathogenesis of HD.<ref><pubmed>23799632</pubmed></ref> In addition, the micro-environmental factors also affect the development of the neural crest cells. For example, a small variance of different factors (eg. GDNF, NTN) in the hindgut can cause an inactivation of the receptor system and result in arrest of the neural crest migration.<ref><pubmed>19196962</pubmed></ref>

--[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 11:16, 17 September 2015 (AEST) You have simply listed references not answered my question. If you fix this before the lab this week I can give you a mark.

= Week 8 Lab 7 Assessment=

'''Identify and write a brief description of the findings of a recent research paper on development of one of the endocrine organs covered in today's practical.'''

PMID 26348989 '''Hes1 and Hes5 are required for differentiation of pituicytes and formation of the neurohypophysis in pituitary development.''' <ref><pubmed>26348989</pubmed></ref>

The pituitary gland is essential endocrine organ which involves in homeostasis, metabolism, reproduction, and growth. It composed of two main parts, adenohypophysis and neurohypophysis. The adenohypophysis derived from Rathke’s pouch, and consists of the anterior and intermediate lobes, where the neurohypophysis contains the posterior lobe, and originated from the infundibulum, which is an evagination of the ventral diencephalon. The neurohypophysis mainly composes two cell types, pituicytes and axons. The pituicytes in neurohypophysis are specially classified as glial cells, which are non-neural cells but regulate homeostasis, and support neurons in the central and peripheral nervous system.

The auther’s research group focused on studying the molecular mechanisms that regulate neurohypophysis development in pituitary, especially the of role Hes1 and Hes5 genes in pituicytes’ differentiation. Hes gene is a repressor type of basic helix-loop-helix genes. It maintains stem cells and progenitors, as well as the differentiation timing of them. Hes1 and Hes5 are important effectors for the famous ‘Notch signalling’.

By examine the expression pattern of Hes1 and Hes5 gene in mutant mice embryo (Hes1-null mice and wild-type mice), the Arthur hypothesis that Hes1 may control the evagination of the ventral diencephalon and the neurohypophysis development, and Hes5 effects as a compensation for Hes1 in this development. Further study with mutant mice embryo (Hes1-/- Hes5+/-, Hes1-/- Hes5-/-) at E12.5 revealed that in the absence of Hes genes, progenitor cell may differentiate into neurons but in the loss of pituicytes and astrocytes. This result is also comfirmed by immunostaining with anti-Sox2 antibody at E12.5 mutant mice. The infundibulum and neurobypophysis were found to be lost in Hes1 and Hes5 mutant embryo at E12.5 and E16.5 respectively. Taking together all the above results, the author commented that the Hes genes are essential for the development of pituicytes and eventually posterior pituitary.

'''Identify the embryonic layers and tissues that contribute to the developing teeth.'''

Teeth consists of three different types of hard tissue, enamel, dentine, and cementum. Both ectoderm and mesoderm layers contributed to the developing teeth ([[Integumentary_System_-_Tooth_Development|Tooth Development]] '''UNSW embryology'''). Ectodermal epithelum give rise to ameloblasts which then forms the enamel. Mesoderm develops into the endothelial cells lining the blood vessels. Neural crests derived ectomesenchyme has the major contribution in the teeth development. It differentiates into odontoblasts and cementoblasts, and eventually form the dentine and cementum. All the connective tissues in teeth (eg. periodontal ligament which holds tooth in bone socket) are also originated from neural crest cells. <ref><pubmed>17209531</pubmed></ref> <ref><pubmed>12640730</pubmed></ref>

This Summary is also referenced from:

'''Textbook''': '''Larsen's Human Embryology''' (7th ed.) Chapter17 pp466-469

=Peer Review=
==Group 2==
This Wiki covers the topic well. The content is very well written and easy to understand. Images and texts are correctly cited and referenced. In some of the sections, eg, ‘Ovulation Induction’, ‘Avoiding hCG during Luteal Phase Support’, more in-text reference will need to be added.

It is a great idea to have some bold texts in lines, which highlight the main points of paragraphs, and help readers to understand when skimming.

The hand-draw diagram of ‘pathogenesis of OHSS’ is excellent. It is well structured, and easy to understand and memorize. It will be great if more images, diagrams, videos can be added to the other sections.

Overall, the project page is very well developed. Some of the sections need to have more work on though. It would be nice if more graphs and tables can be added to balance the texts.

==Group 3==
This project page is nicely organized, and well balanced with graphs, tables, and diagrams. It is wise to narrow down the topic and focus on the female infertility caused by polycystic Ovarian Syndrome. But it will be better if the other possible causes are mentioned at the beginning.

The image ‘ PCOS Ovary vs. Non-PCOS Ovary’ explains the differences between normal and PCOS Ovary very well. It will be better if the image is inserted after the texts which define PCOS as it causes confusion about your topic at the current location.

The use of colour highlighting is very impressive. It do make the important messages stand out.
Pages are correctly referenced. Current scientific researches are nicely summarized and fitted into the context. It is impressive to include animal and cell culture models in the pathogenesis section.

Overall, the wiki page has covered the topic well. Contents are concise and easy to understand. It would be better if a ‘glossary’ can be added to explain some of the terminologies for readers.

==Group 4==
The topic is well investigated in this project wiki. It is excellent that you also include some background information on the structure and development of spermatozoa, which would help readers without knowledge in this field.

Images are properly cited and referenced. They make the page looked refreshing. Tables are used wisely to summarize information on ‘male infertility disorders’, but will need some more in-text referencing with the table contents.

Paragraphs are written very well. There are great efforts in rewriting and summarizing. It will be better if the texts can be simplified by some diagrams or lists, which will be easier for readers to get through.
Overall, this project wiki is an excellent work.

==Group 5==
This is an excellent group project wiki. The content covers the topic in all aspects. However, there might be excessive effort in the investigation of ‘infertility’, ’Fertility Drugs’ and ‘Chemotherapy’, which occupied more than half of your project page. They are relevant to this topic, but might need to be consolidated to balance the page.

Images and videos are good choice in your page. It would be better if more images, diagrams, tables are added into your page to balance the texts.

Referencing and citing are excellent in most section, although some sections seem to be lack of in-text references. You might still want to work on them.

Overall, this wiki is an excellent work in investigating oncofertility. It is relevant to the aim of learning embryology.

==Group 6==
This project page is very well done. Clearly, you have done huge amount of research on this topic. And all sections of the page are well balanced.

I appreciate that you include the advantages and disadvantages of each diagnostic methods, which not only indicates your thorough understanding of the topic, but also make it easier for readers to compare each methods.

Images and tables are well chosen in your page. It would be great if you can add more visual aids in some sections because there are large amount of texts in some section.

==Week11==
link to permalink image [https://embryology.med.unsw.edu.au/embryology/Slides/Embryo_Stages/Stage22/11/Stage22-11.html?zoom=6&lat=-3948&lon=6149&layers=B|Cochlear Duct]
'''cochlear duct'''
The cochlear duct is an fluid filled cavity inside the cochlea. It located between the tympanic duct and the vestibular duct, and between the basilr membrane and reissner's memebrane. It derived from otic placode, otic vesicle, and originated from surface ectoderm.

=Stem cell presentation=
PMID 26295456
PMID 26439174
PMID 24837661

=Reference list=
<references/>

User talk:Z3251292

2015-10-28T22:08:55Z

Z3251292: /* Week11 */

=Week11=
link to permalink image [https://embryology.med.unsw.edu.au/embryology/Slides/Embryo_Stages/Stage22/11/Stage22-11.html?zoom=6&lat=-3948&lon=6149&layers=B|Cochlear Duct]
'''cochlear duct'''
The cochlear duct is an fluid filled cavity inside the cochlea. It located between the tympanic duct and the vestibular duct, and between the basilr membrane and reissner's memebrane. It derived from otic placode, otic vesicle, and originated from surface ectoderm.

==embryology link==
[[Sensory - Hearing and Balance Development]] --Inner Ear

=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>

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 gray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|200px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

=== 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>PMID 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>PMID 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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, spindle transfer can reduce the risk of heteroplasmy, thus offer a better reproductive option to prevent mtDNA disease transmission in affected families <ref><pubmed>23103867</pubmed></ref>. has better potential in 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>.

PMID 25229667
PMID 23103867
PMID 24382342

==='''What is the procedure?'''===
#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother and will develop unaffected by inherited mitochondrial disease.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of spindle-chromosome transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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|600px|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>.

==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===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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:#E0CEF2">'''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.]

==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>.
* 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

It has been reported with mice modle that coupling Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer may increase the yield of reconstructed embryos with low mtDNA carryover. <ref><pubmed> 25573721 </pubmed></ref>

===What is the procedure===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===Mice Model===

PMID 24949971 '''Polar body genome transfer for preventing the transmission of inherited mitochondrial diseases''' <ref><pubmed> 24949971 </pubmed></ref>
The authur adopts polar body transfer 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.

==Other approaches==

===germinal vesicle nuclear transfer===

=ETM transition=
PMID 20943648
PMID 21559368
PMID 21447558

=Talk=

*Timeline of The artificial reproductive technology revolution

::*1984 donor egg pregnancies
::*1984 frozen embryos
::*1960 Ovulation induction
::*1960 Donor sperm
::*1978 IVF
::*1988 Pre-implantation genetic diagnosis
::*1992 Sperm injection
::*1994 Blastocyst development
::*1998 Reliable sperm sex selection
::*2000 Stem cells and cloning and gene transfer
::*2005 Ovarian reserve testing
::*2008 Oocyte freezing, social freezing
::*2013 Whole genome screening of embryos

*Process of IVF
::* Ovarian hyperstimulation
::**two phase during menstrual cycle: lacteal phase and follicular phase
::**About 30 Follicles grows to large size in every mentrual cycle, but only one survived to the ovulation.
::** FSH drove the growth of follicles, also control the growth of follicles (pituitary down regulation), used to stimulate the mature of multiple follicles (10-30).
::** Estrogen....
::** Monitoring by transvirgena ultrasound.
::** LH containing hormones injected to women to stimulate ovulation.

::* Ooocyte Pick-Up(OPU)
::* Sperm retrieval via ejaculation or surgery
::* In Vitro Fertilisation
::** intra-cytoplasmic sperm injection (ICSI)
** Embryo culture
::** day1- form Zygote
::** day3- divide to 8 blastomeres
::** day5- frozen, and transfer to uterus
::* Transfer of the embryo(s)

* New technologies
** Preimplantation genetic diagnosis(PGD) of embryo. eg, fibrosis-premature death
*** Embryo Biopsy for PGD
*** analysis of blastomeres for PGD
****FISH
****PCR
****
*** What to be detected in an embryo
****Aneuploidy
****trisomy 21
****translocations
****single gene defects(eg cystic fibrosis)
** Ovarian reserve and the natural cycle
***blood test measure hormones(AMH, AFC)
***Egg banking ('social freezing')
** Fertility preservation for cancer
** Oocyte in vitro maturation (IVM)
*** putting oocytes in vitro and culture 24 hours before IVF (Complex medium , protein, FSH, EGF, LH, E2 cysteamine)
*** comparing to conventional IVF, no Hormones required to give to mother for the maturation of oocyte.

=Reference=
{{reflist}}

2015 Group Project 1

2015-10-23T11:18:17Z

Z3251292: /* 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. (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''' - 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 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

2015-10-23T11:14:50Z

Z3251292: /* Key Events of Cytoplasmic Transfer */

2015 Group Project 1

2015-10-23T11:08:04Z

Z3251292: /* Spindle-Chromosome Transfer */

2015 Group Project 1

2015-10-23T11:01:38Z

Z3251292: /* 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''' - 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 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 (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/>. 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

2015-10-23T02:59:22Z

Z3251292: /* 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=6896904><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><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

2015-10-23T02:42:06Z

Z3251292: /* 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. (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><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 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

2015-10-23T02:36:34Z

Z3251292: /* Why do 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. (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><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 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

2015-10-23T02:28:22Z

Z3251292: /* Limitations */

{{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

<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:

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/>

=External Links=

2015 Group Project 1

2015-10-23T02:24:22Z

Z3251292: /* Human Embryo Model */

{{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

<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:

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/>

=External Links=

File:77254175 embryo repair 624 method 2.gif

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==Diagram for cytoplasmic transfer for Embryo repair==
*Both the mother's and donor's eggs are fertilised with the father's sperm to create two embryos
*The pronuclei, the nuclei during the process of fertilisation, contain the majority of the genetic material. They are removed from both embryos. The donor's is destroyed
*A healthy embryo is created by putting the parents' pronuclei into the donor embryo

===Reference===
[http://www.bbc.com/news/magazine-28986843] The girl with three biological parents

====Copyright====
Copyright © 2015 BBC. This is an open source material.

{{Student Image}}

File:77266645 embryo repair 624 method 1.gif

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===Cytoplasmic transfer for egg repair===

*Eggs from a mother with unhealthy mitochondria and a donor with healthy mitochondria are collected
*The nucleus, containing the majority of the genetic material, is removed from both eggs. The donor nucleus is destroyed
*The mother's nucleus is inserted into the donor egg - it now has healthy mitochondria. The egg is then fertilised by the father's sperm

===Reference===
[http://www.bbc.com/news/magazine-28986843] The girl with three biological parents

====Copyright====
Copyright © 2015 BBC. This is an open source material.

{{Student Image}}

File:77260486 cell structure 304.gif

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Simplified structure of the human cell for explaining mitochondria

Nucleus: Where the majority of our DNA is held and genetic information inherited
Mitochondria: create the energy to make the cell function
Cytoplasm: contains the nucleus and mitochondria

===Reference===
[http://www.bbc.com/news/magazine-28986843] The girl with three biological parents

====Copyright====
Copyright © 2015 BBC. This is an open source material.

{{Student Image}}

2015 Group Project 1

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Z3251292: /* 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. (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

<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

2015-10-22T06:00:59Z

Z3251292: /* Cytoplasmic Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.
</div>

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:53:55Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.
</div>

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:52:12Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.
</div>

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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|100x400px|thumb|Left|Simplified Cell Structure <ref name='3egirl'/> ]]
| [[File:77266645 embryo repair 624 method 1.gif|500px|thumb|middle|Egg repair by Cytoplasmic transfer <ref name='3egirl'/> ]]
| [[File:77254175 embryo repair 624 method 2.gif|500px|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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:47:37Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.
</div>

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:46:07Z

Z3251292: /* Key Events of Cytoplasmic Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.
</div>

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

a

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:44:13Z

Z3251292: /* Key Events of Cytoplasmic Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.
</div>

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

{|
|+ 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="CEDFF2"
| 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="CEDFF2"
| 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.

a

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:41:07Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

a

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:39:08Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
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| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
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| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:26:57Z

Z3251292: /* Polar Body Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===
::* '''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>.
::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===
::*'''1996''' 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>.
::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>
::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' 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 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.
::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<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 base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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|700px|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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:16:18Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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|700px|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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:14:24Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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|700px|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;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 25472922 </pubmed></ref>
| [[File:PB1 transfer.jpg|600px|thumb|left| Polar Body 1 Transfer ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Polar Body 2 Transfer ]]
|}

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:12:48Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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|700px|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: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 25472922 </pubmed></ref>
| [[File:PB1 transfer.jpg|600px|thumb|left| Polar Body 1 Transfer ]]
| [[File:PB2 transfer.jpg|600px|thumb|right|Polar Body 2 Transfer ]]
|}

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:10:56Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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|700px|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: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 25472922 </pubmed></ref>
| [[File:|600px|thumb|left| Polar Body 1 Transfer ]]
| [[File:|600px|thumb|right|Polar Body 2 Transfer ]]
|}

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
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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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
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| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
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| 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]
|-
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| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
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| 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]
|-
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| 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]
|-
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| 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]
|-
|
| 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]
|-
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| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:06:34Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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|700px|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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T05:00:01Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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|700px|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?===

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.

[[File:Pronuclear transfer.jpg|600px|thumb|left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:58:11Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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|700px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:56:09Z

Z3251292: /* Spindle-Chromosome Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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 intending mother 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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:53:04Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[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 intending mother 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|400px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:51:31Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|800px|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 intending mother 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|400px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:50:32Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|700px|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 intending mother 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|400px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:49:07Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|800px|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 intending mother 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|400px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:45:50Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|300x1200px|thumb|Left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother 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|400px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:44:16Z

Z3251292: /* Spindle-Chromosome Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|300X1200px|thumb|Left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother 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|400px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:42:35Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|100x400px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1500px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1500px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|400px|thumb|Left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother 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|600px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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==
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=== 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]
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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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:41:03Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|200x600px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|300x1000px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|290x1000px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|400px|thumb|Left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother 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|600px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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=

File:MT transfer.jpg

2015-10-22T04:39:19Z

Z3251292: /* Reference */

=Diagram of spindle-chromosomal transfer=

* Chromosomes, the nuclear DNA material, are found in the intending mother (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.
* 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 intended mother’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 intending mother and will develop unaffected by inherited mitochondrial disease.

=Reference=
<pubmed> 25573721 </pubmed>

=copyright=

Copyright © z3251292. permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

{{Student Image}}

2015 Group Project 1

2015-10-22T04:39:08Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|200x600px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|500x1300px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|480x1300px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===

[[File:MT transfer.jpg|400px|thumb|Left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother 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|600px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:37:01Z

Z3251292: /* Spindle-Chromosome Transfer */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|200x600px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|500x1300px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|480x1300px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===
#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother and will develop unaffected by inherited mitochondrial disease.

[[File:MT transfer.jpg|400px|thumb|Left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

===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|600px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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

2015-10-22T04:34:59Z

Z3251292: /* What is the procedure? */

{{ANAT2341Project2015header}}

=Three Person Embryos=
'''Three Person Embyo''' is a form of germline fertility treatment by which an oocyte are formed containing maternal and paternal DNA and donated mitochondrial DNA (mtDNA). This can be with the presence or absence of maternal mtDNA. The purpose of this treatment is to prevent the maternal inheritance of hereditary mitochondrial diseases and treat some forms of infertility caused by mtDNA mutation. It is also referred to as Mitochondrial Donation and Mitochondrial replacement-assisted IVF.

<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=
==Timeline of Mitochondrial Donation==
===1980s===

::*'''1984''' Publication of the UKs Warnock Report on IVF technologies and embryo research in reaction to 1978s first IVF baby. Becomes blueprint for regulation.
::*'''1988''' First pathogenic mitochondrial mutations in humans identified <ref><pubmed> 3201231 </pubmed></ref><ref><pubmed> 2830540 </pubmed></ref>

===1990s===

::*'''1996''' Dolly the sheep born from nuclear transfer. Generates public interests in genetic modification and clinical embryology<ref><pubmed> 9039911 </pubmed></ref>.

::* '''1997''' St Barnabas Hospital announces it has achieved a live birth from mitochondrial donation via Ooplasmic transfer <ref><pubmed> 9250192 </pubmed></ref>

::*'''1998''' FDA ban use of Ooplasmic transfer techniques in the USA
::*'''1998''' First oocyte with DNA transferred from a first polar body fertilized brought to term in a mouse model. <ref><pubmed> 9674999 </pubmed></ref>

===2000s===

::*'''2008 Nov 13th''' The Human Fertilization and Embryology Act allows research into the techniques of three person IVF.

::*'''2009''' First success-full trails spindle transfer in rhesus monkeys <ref><pubmed> 19710649 </pubmed></ref>

===2010s===
::*'''2010''' Craven et al pronuclear transfer and mitochondrial DNA disorder prevention.<ref><pubmed>20393463</pubmed></ref>
::*'''2015 Oct 29th''' regulations to allow the open use of three person IVF via pronuclear transfer in fertility clinics comes into affect in the UK.

=Benefits=
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 encoded genes have so far been identified as involved in the life-cycle and function of mitochondria<ref><pubmed> 19651984 </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. Because mitochondrial replacement is a germ line treatment any future generations will also be free from mtDNA mutations. Extrapolation from small studies estimate that 152 women in the UK and 778 in the United State, are at risk of passing on mtDNA disorders<ref><pubmed> 25629662 </pubmed></ref>.

''cad pic of disease and heredisity''

===Risks of passing on a mitochondrial disorder===
''Mitochondria genome passage between generations''

===Mitochondria linked Infertility===
''can they affect fertility''
https://embryo.asu.edu/pages/ooplasmic-transfer-technology
http://www.ncbi.nlm.nih.gov.wwwproxy0.library.unsw.edu.au/pubmed/12470582

===Hereditory mitochndrial Disorders===

{|
|-bgcolor="CEDFF2"
! Mitochondrial disorder
! Description
! Mutaion
! Preventable with mitochondrial donation
|-
| test
| test
|
|
|-bgcolor="F5FAFF"
|
| test
|
| test
|}

=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>

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 lightgray;"
|+ style="text-align: center;" |Diagram of cytoplasm transfer <ref> Charlotte Pritchard '''The girl with three biological parents'''1 September 2014 http://www.bbc.com/news/magazine-28986843 retrieved September 2015</ref>
| [[File:77260486_cell_structure_304.gif|200x600px|thumb|Left|Simplified Cell Structure]]
| [[File:77266645 embryo repair 624 method 1.gif|500x1300px|thumb|middle|Egg repair by Cytoplasmic transfer]]
| [[File:77254175 embryo repair 624 method 2.gif|480x1300px|thumb|right|repair by Cytoplasmic transfer]]
|}

=== 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.

{| class="wikitable"
|+ 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>
|-
! 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
|-
| 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
|-
| 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?===
#Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
#Chromosomes, the nuclear DNA material, are found in the intending mother’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 intended mother’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 intending mother and will develop unaffected by inherited mitochondrial disease.

[[File:MT transfer.jpg|400px|thumb|Left|Diagram of spindle-chromosomal transfer <ref><pubmed> 25573721 </pubmed></ref>]]

===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|600px|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?===

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.

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of pronuclear transfer <ref><pubmed> 25377180</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
|}

'''(reproduced diagrams to be uploaded)'''

PMID 25763399

===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?===

PMID 25472922
PMID 16317618

{| style="border-spacing: 2px; border: 1px solid gray;"
|+ style="text-align: center;" |Diagram of Polar body transfer <ref><pubmed> 24949971</pubmed></ref>
| [[File:|200px|thumb|Left|Simplified Cell Structure]]
| [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]]
| [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]
|}

'''(reproduced diagrams to be uploaded)'''

===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 believe 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.

1.PMID 26239841 '''The ethical challenges of the clinical introduction of mitochondrial replacement techniques.''' <ref><pubmed>26239841</pubmed></ref>
The first part of the paper evaluates the three concerns about the safety of mitochondrial replacement techniques including whether it is ethical; persons with three genetic contributors and the trust of society. And then, two recommendations are made.

2.PMID 21059727 '''Ethics of mitochondrial gene replacement: from bench to bedside.''' <ref><pubmed>21059727</pubmed></ref>
Both of the risks and benefits are accessed in this paper after the briefly introduction of mitochondrial replacement techniques. And then the question of when are enough safeguards made to justify introducing mitochondrial gene replacement into the clinic is discussed.

3.PMID 25888328 '''Mitochondrial replacement to prevent the transmission of mitochondrial DNA disease.''' <ref><pubmed>25888328</pubmed></ref>
This paper discussed about the ethics and feasibility of mitochondrial replacement techniques. The possibility of preventing the transmission of mtDNA disease by MRT is first discussed. Moreover, the four big challenges mainly ethics are discussed.

=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"
|-
! style="width:120px;"| '''Region''' !! '''Country''' !! '''Laws'''
|-
|

=== 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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）]
|-
|

=== 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]
|-
|
| New Zealand || [http://www.legislation.govt.nz/act/public/2004/0092/latest/DLM319241.html Human Assisted Reproductive Technology Act 2004]

|-
|

=== 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)]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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 ]
|-
|
| 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]
|-
|
| 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]
|-
|
| Italy || [http://www.salute.gov.it/imgs/C_17_normativa_454_allegato.pdf Medically Assisted Procreation Law / Norme in materia di procreazione medicalmente assistita]
|-
|
| 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]
|-
|
| Malta || [http://justiceservices.gov.mt/DownloadDocument.aspx?app=lom&itemid=11960&l=1 Embryo Protection Act]
|-
|
| 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]
|-
|
| Netherlands || [http://wetten.overheid.nl/BWBR0013797/geldigheidsdatum_08-10-2015 Act Containing Rules Relating to the Use of Gamete and Embryos / Embryowet]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|
| 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]
|-
|

=== America ===

| Canada || [http://laws-lois.justice.gc.ca/eng/acts/A-13.4/ Assisted Human Reproduction Act (S.C. 2004, c. 2)]
|-
|
| 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]
|-
|

=== 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)]
|-
|

|-
! '''Region''' !! '''Country''' !! '''Laws'''
|}

=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=

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=External Links=