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soon after birth. The frequency of ... sperm during ICSI (or the penetration of the ... generates triploidy after fertilization (3N with. 69, XXX or 69, ... zygotes? In vitro correction of 3PN ... Human embryos' pronuclear transfer procedure. “to correct ...
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Ahmad Mustafa Metwalley1 Mohamed Fawzy2 Abdalla Mohamed Ahmed3 Ali Hellani4 Saleh Ghaith Ban5 Ahmed Barakat6 1

As approved by Dyban et al. (1, 2), about 1518% of abortions are caused by triploidy fertilization. One of the sources of maternal triploidy is the failure of the first meiotic division (3). The incidence of triploid pregnancy that can develop to live birth is about 1/10.000 (4); otherwise, the variety of abnormalities die soon after birth. The frequency of triploidy in IVF is about 4% of inseminated cycles, and at least one triploid pre-zygote was found in 60% of all IVF cycles (5). The incidences of paternal triploid or diandrous zygotes associated with severe sperm abnormalities are about 33% (6). Diandric zygote develops by either the injection of nucleated sperm during ICSI (or the penetration of the same sperm during IVF) or the penetration of two sperms during conventional IVF (7). Digenic triploidy that originated from extramaternal nuclei developed by either the division failure of meiosis (I / II) or the reduction of tetraploid precursor cells (8). An acquired digenic triploidy is developed by a fertilized giant oocyte (9). A giant oocyte develops by the nuclear but not cytoplasmic division of an oogonium or by the cytoplasmic fusion of two oogonia (10). A giant oocyte is characterized as bigger in diameter (>200 mm in diameter) and will distinguish two polar bodies at metaphase II. Cytogenetical studies (11) have shown that an extra haploid maternal set of chromosomes in giant metaphase II oocytes (MII) (46, XX/ 2N) generates triploidy after fertilization (3N with 69, XXX or 69, XXY). International guidelines suggest not using these oocytes for clinical purposes (12). The hypothesis of the first mitotic division plane with polar axes studies by Lynette Scott and the postulations done using immunostaining procedures with fluorescence cytostaining of zygote achieved by Khursheed Iqbal confirmed to us that PN developed closer to the 2nd polar body is the maternal origin PN (13, 14). Dependant on these scientific considera-

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MSc. Molecular Pathology, Al Baraka Fertility Hospital, Manama, Bahrain 2 Ibn Sina IVF Center, Sohag, Egypt 3 Clinical Embryologist at Al Baraka Fertility Hospital, Manama, Bahrain 4 Founder of Viafet company, Viafet, Sydney, Australia 5 IVF specialist at Barakat Fertility Hospital, Manama, Bahrain 6 IVF consultant at Barakat Fertility Hospital, Manama, Bahrain

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Any possibility of treating abnormally fertilized zygotes? In vitro correction of 3PN

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Address for correspondence: Ahmad Mustafa Metwalley, MD MSc. Molecular Pathology Senior clinical embryologist and Cytogenetist Al Baraka Hospital IVF Unit Director Al Baraka Fertility Hospital Manama, Bahrain Phone: 0097333461318 E-mail: [email protected]

Summary

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Video attached with this study shows the manipulation procedure can be used for treating abnormally fertilized oocytes at different ART procedures. Cytogenetic studies for these treated oocytes describe the safety and accuracy of treatment. KEY WORDS: abnormal zygote fertilization, 3PNs correction, giant oocyte management, triploidy fertilization, maternal nucleus removal.

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Table 1 - PN removal from abnormally fertilized oocyte. 22

Successful Fertilization with 3PNs

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Successful maternal PN removal

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Degenerated zygotes after PN removal

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Total Injected Giant Oocyte with MII Stage

Embryos developed to Day3 and Biopsied

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Blastocyst Developed

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Day 5 FISH study - Normal Male - Normal Female - Diploidy but Abnormal

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tions, we organized the penetration site for PN removal without disturbing the mitotic plan and facilitating a target PN for removal (maternal PN). Giant oocytes that had been collected from IVF cycles were prepared for injection with normal sperm using intracytoplasmic sperm injection (ICSI). Fertilization checks were done 17 hours +/-1 post-ICSI, and on a micromanipulator using an ICSI needle with the maternal PN’s removal from the fertilized oocyte before the syngamy starts. Videos attached to show the processes of zygote manipulation, by the way, avoid the division axis and focus on the extramaternal PN targeted for aspiration. Human embryos’ pronuclear transfer procedure “to correct mitochondrial disease” invented the idea of using the same technique of pronuclear manipulation, so the possibility of utilizing the 3PNs correction had been developed as a research tool. We arranged to study available received giant oocytes during IVF cycles. We arranged for pronuclear removal followed by FISH evaluation for targeting normal male embryos that ensure proper extramaternal pronucleus removal. Successful trials of maternal PN removal for giant oocytes collected from COS cases are summarized in Table 1. All blastocysts developed trophectoderm (TE) arranged for FISH, so all embryos were used for cytogenetic evaluation.

References

Dyban AP and Baranov VS. Cytogenetics of mammalian embryonic development. Clarendon Press, Oxford, UK, 1990. 2. McFadden DE, Robinson WP. Phenotype of triploid embryos. J Med Genet. 2006 Jul;43(7):609-12. 3. Jacobs PA, Angell RR, Buchanan IM, Hassold TJ, Matsuyama AM, Manuel B. The origin of human triploids. Ann Hum Genet. 1978 Jul;42(1):49-57. 4. Jacobs PA, Szulman AE, Funkhouser J, Matsuura JS, Wilson CC. Human triploidy: relationship between parental origin of the additional haploid complement and development of partial hydatidiform mole. Ann Hum Genet. 1982;46:223-231. 5. Escriba M, Martin J, Rubio C, et al. Heteroparental Blastocyst Production From Mivrosurgially Corrected Tripronucleated Embryos. Fertil Steril. 2006;86:1601-1607. 6. Macas E, Imthurn B, Keller PJ. Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI. Hum Reprod. 2001;16:115-120. 7. Golubovsky MD. Postzygotic diploidization of triploids as a source of unusual cases of mosaicism, chimerism and twinning. Hum Reprod. 2003 Feb;18(2):236-42. 8. Doshi N, Surti U, Szulman AE. Morphologic anomalies in triploid liveborn fetuses. Hum Pathol. 1983;14:716723. 9. Dyban AP, Baranov VS. Cytogenetics of Mammalian Embryonic Development. Clarendon Press, Oxford, UK. 1987; pp. 40-59. 10. Austin CR. Anomalies of fertilization leading to triploidy. J Cell Comp Physiol.1960:56 (Suppl. 1):1-15. 11. Rosenbusch B, Schneider M, Gläser B, Brucker C. Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of embryonic genome expression in human preimplantation of digynic triploidy. Hum Reprod. 2002;17:2388-2393. 12. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group Embryology. Reproductive Bio-

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Evaluations using STRs (short tandem repeats) are recommended as tools for maternalpaternal genome differentiation. An NGS study is under evaluation for developed embryos for full CCS reporting and more genetic integrity. An epigenetic evaluation study is recommended for triploidy corrected embryos for genetic expressions and early embryo developments and differentiations between paternal and maternal genomic activity. Nucleus manipulation was done for cases suffering from mitochondrial DNA disorders (15-20).

Registration NCT02358759 at Home - ClinicalTrials.gov. 116

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Any possibility of treating abnormally fertilized zygotes? In vitro correction of 3PN

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implantation. Fertil Steril. 2006 Feb; 85(2):384-90. 17. Rosenbusch B. The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities. J Genet. 2012;91(3):397404. Review. 18. Scott L, Alvero R, Leondires M, Miller B. The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum Reprod. 2000;15:2394-2403. 19. Vogel G. Assisted reproduction. FDA considers trials of ‘three-parent embryos’. Science. 2014 Feb 21;343 (6173):827-8. 20. Wolf DP, Mitalipov N, Mitalipov S. Mitochondrial replacement therapy in reproductive medicine. Trends Mol Med. 2014 Dec 10. pii;S1471-4914(14)00215-9. doi: 10.1016/j.molmed.2014.12.001. [Epub ahead of print] Review.

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Medicine Online. 2011;22(6):632-646. 13. Scott L, Alvero R, Leondires M, Miller B. The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum Reprod. 2000;15:2394-2403. 14. Khursheed Iqbal, Seung-Gi Jin, Gerd P Pfeifer, Piroska E Szabó. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci. U.S.A. 2011; 108 (9):3642-7. 15. Amato P, Tachibana M, Sparman M, Mitalipov S. Threeparent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases. Fertil Steril. 2014 Jan;101(1):31-5. Review. 16. Rosen MP, Shen S, Dobson AT, Fujimoto VY, McCulloch CE, Cedars MI. Triploidy formation after intracytoplasmic sperm injection may be a surrogate marker for

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