2015 Group Project 1: Difference between revisions

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


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


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


===Why do cytoplasmic transfer?===
===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>.
The quality of the oocyte cytoplasm is critical for the development of the embryo<ref><pubmed> 15140871 </pubmed></ref>. Following ovulation, the survival of the zygote depends almost exclusively on maternal messenger RNA and proteins that accumulate during oocyte growth and maturation within the ooplasm. It is not until the maternal-to-zygotic transition (MZT) stage, during the 4–8 cell stage in humans, where the new zygote genome is activated and replaces the maternal cytoplasm to become predominant in regulating the zygote development<ref><pubmed> 3352746 </pubmed></ref>. The maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome<ref><pubmed> 10429238 </pubmed></ref>.
   
   
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>.
Researchers are still investigating the molecular and cellular mechanisms by which ooplasm regulates the maturation and activation of human oocytes and zygotes<ref>J A.Barritt, S Willadsen '''Epigenetic and experimental modifications in early mammalian development: part II Cytoplasmic transfer in assisted reproduction''' Human Reproduction Update 2001 Vol.7, No.4 pp.428-435 </ref>. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production  components and many additional factors yet to be determined. '''The benefits of cytoplasm transfer''' are revealed by two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential<ref><pubmed>  23602680 </pubmed></ref>.


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


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


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


=== 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"  
|-bgcolor="CEDFF2"  
!  Type of Cytoplasm Transferred to recipient oocytes   
!  Type of Cytoplasm Transferred to recipient oocytes   
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===Risk of Cytoplasmic Transfer -- Heteroplasmy===
===Risks of cytoplasmic transfer – Heteroplasmy===






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




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


==Spindle-Chromosome Transfer==
==Spindle-chromosome transfer==


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




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|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/>.  
|Due to the uncertainty of the health risks related to spindle-chromosome transfer, experiments in non-human primates are required to assess the safety of this procedure. Tachibana et al. (2009) carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)<ref name=pmid19710649/>.  
*a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
*a - Removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to threadlike spindle fibers (the spindle–chromosomal complex).
*b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
*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.
*c - Fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
*d - developing blastocyst was implanted in a surrogate mother.
*d - Developing blastocyst was implanted in a surrogate mother.
*e - mother gave birth to a healthy baby.
*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.  
Some of the resulting embryos were successful and resulted in healthy offspring with low mtDNA carryover. However, it is still too early to determine whether spindle-chromosome transfer is a safe procedure because defects may develop later in life, or in subsequent generations. Thus long-term studies are required to assess the effects of this procedure, which includes life-long monitoring and multi-generational tracking.  


| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer <ref name=pmid19710649/>]]
| [[File:Swapping mitochondrial DNA mammalian oocytes.jpg|thumb|right|500px|Primate model of spindle-chromosome transfer<ref name=pmid19710649/>.]]
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===Current Research===
===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/>.
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===
===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>.
Experiments showed minimal mutated mtDNA carryover in nonhuman primate offspring and human pre-implantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces premature activation of oocytes and results in karyotype abnormalities<ref name=PMID25472922><pubmed> 25472922</pubmed></ref><ref><pubmed> 23254936</pubmed></ref>.


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


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


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


[[File:Pronuclear transfer.jpg|600px|thumb|right|Diagram of pronuclear transfer <ref name=PMID25573721/>]]
[[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'/>
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.  
# 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.  
# At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.  
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# The developed embryo will then be transferred into the mother.
# The developed embryo will then be transferred into the mother.


===Human Embryo Model===
===Human embryo model===
 






Research Group in New Castle University performed pronuclear transfer on human mebryo model<ref name=pmid20393463/>:  
A research group at Newcastle University performed pronuclear transfer on human embryo model<ref name=pmid20393463/>:  


* Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).  
* Pronuclear transfer was performed using abnormally fertilized human zygotes generated following in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).  
*Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.  
*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.  
*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'.  
*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  
The safety effects of this procedure (zygote mtDNA carryover) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data  provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities.


<html5media width="560" height="315">https://www.youtube.com/embed/Sr7Jnr9qn44</html5media>
<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>
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===
===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.
Pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes in mice models. However, it is reported that PNT-generated mice possessed 6%–21% heteroplasmic mtDNA at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA at Day 300 after birth<ref><pubmed>16275929</pubmed></ref>. Human embryo model studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (<2.0%) in early embryos<ref name=pmid20393463/>. One disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos.


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


* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.  
* Polar body 1 is formed and released during ovulation. It contains a diploid set of chromosomes.  
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* Both polar bodies are unable to be fertilized and disintegrate eventually.
* 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/>.
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/>:
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 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.
* 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.
* Each donor will have three offers (polar body 1, body 2, maternal pronucleus), which significantly increases the efficiency of using a donor egg.




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


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


==Other Approaches==
==Other approaches==


===Germinal Vesicle Nuclear Transfer===
===Germinal vesicle nuclear transfer===
[[File:Human-oocyte.jpg|200px|thumb|right|Germinal vesicle oocyte <ref><pubmed>19924284</pubmed></ref>]]
[[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>.
The '''germinal vesicle''' (GV) is the large nucleus of an immature oocyte arrested naturally in the first meiotic prophase. The oocyte undergoes GVBD soon after MPF activation, and its material (or nucleoplasm) mixes with the cytoplasm (or ooplasm) of maturing oocytes. The germinal vesicle contains a number of proteins, such as histones and DNA polymerases, that are used immediately after fertilization<ref><pubmed> 12193404 </pubmed></ref><ref><pubmed> 21234179 </pubmed></ref>.


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


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


=Ethics=
=Ethics=

Revision as of 02:55, 24 October 2015

2015 Student Projects 
2015 Projects: Three Person Embryos | Ovarian Hyper-stimulation Syndrome | Polycystic Ovarian Syndrome | Male Infertility | Oncofertility | Preimplantation Genetic Diagnosis | Students
2015 Group Project Topic - Assisted Reproductive Technology
This page is an undergraduate science embryology student and may contain inaccuracies in either description or acknowledgements.

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 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 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 legalizing its clinical use.


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Teenage Girl Has Three Biological Parents [1]

History