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Journal of Assisted Reproduction and Genetics

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01-11-2015 | Embryo Biology

Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number

Authors: Yoshiteru Kai, Kyoko Iwata, Yumiko Iba, Yasuyuki Mio

Published in: Journal of Assisted Reproduction and Genetics | Issue 11/2015

Abstract

Purpose

Normally fertilized zygotes generally show two pronuclei (2PN) and the extrusion of the second polar body. Conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI) often result in abnormal monopronuclear (1PN), tripronuclear (3PN), or other polypronuclear zygotes. In this study, we performed combined analyses of the methylation status of pronuclei (PN) and the number of centrosomes, to reveal the abnormal fertilization status in human zygotes.

Method

We used differences in DNA methylation status (5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)) to discriminate between male and female PN in human zygotes. These results were also used to analyze the centrosome number to indicate how many sperm entered into the oocyte.

Result

Immunofluorescent analysis shows that all of the normal 2PN zygotes had one 5mC/5hmC double-positive PN and one 5mC-positive PN, whereas a parthenogenetically activated oocyte had only 5mC staining of the PN. All of the zygotes derived from ICSI (1PN, 3PN) had two centrosomes as did all of the 2PN zygotes derived from c-IVF. Of the 1PN zygotes derived from c-IVF, more than 50 % had staining for both 5mC and 5hmC in a single PN, and one or two centrosomes, indicating fertilization by a single sperm. Meanwhile, most of 3PN zygotes derived from c-IVF had a 5mC-positive PN and two 5mC/5hmC double-positive PNs, and had four or five centrosomes, suggesting polyspermy.

Conclusions

We have established a reliable method to identify the PN origin based on the epigenetic status of the genome and have complemented these results by counting the centrosomes of zygotes.

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Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod. 1987;37:395–401.CrossRefPubMed

Staessen C, Janssenwillen C, Devroey P, Van Steirteghem AC. Cytogenetic and morphological observations of single pronucleated human oocytes after in-vitro fertilization. Hum Reprod. 1993;8:221–23.PubMed

Taylor AS, Braude PR. The early development and DNA content of activated human oocytes and parthenogenetic human embryos. Hum Reprod. 1994;9:2389–97.PubMed

Palermo GD, Munné S, Colombero LT, Cohen J, Rosenwaks Z. Genetics of abnormal human fertilization. Hum Reprod. 1995;1:120–7.CrossRef

Balakier H, Cadesky K. The frequency and developmental capability of human embryos containing multinucleated blastomeres. Hum Reprod. 1997;12:800–4.CrossRefPubMed

Rosenbusch B, Schneider M, Kreienberg R, Brucker C. Cytogenetic analysis of human zygotes displaying three pronuclei and one polar body after intracytoplasmic sperm injection. Hum Reprod. 2001;16:2362–7.PubMed

Mio Y, Iwata K, Yumoto K, Maeda K. Human embryonic behavior observed with time-lapse cinematography. J Health Med Inf. 2014;5:143.

Mio Y. Morphological analysis of human embryonic development using time-lapse cinematography. J Mamm Ova Res. 2006;23:27–35.CrossRef

Iqbal K, Jin SG, Pfeifer GP, Szabó PE. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci U S A. 2011;108:3642–7.PubMedCentralCrossRefPubMed

10.

Wossidlo M, Nakamura T, Lepikhov K, Marques CJ, Zakhartchenko V, Boiani M, et al. 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat Commun. 2011;2:241.CrossRefPubMed

11.

Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature. 2011;477:606–10.CrossRefPubMed

12.

Sathananthan AH, Kola I, Osborne J, Trouson A, Ng SC, Bongso A, et al. Centrioles in the beginning of human development. Proc Natl Acad Sci USA. 1991;88:4806–10.PubMedCentralCrossRefPubMed

13.

Sathananthan AH, Ratnam SS, Ng SC, Tarín JJ, Gianaroli L, Trouson A. The sperm centriole: its inheritance, replication and perpetuation in early human embryos. Hum Reprod. 1996;11:345–56.CrossRefPubMed

14.

Munné S, Cohen J. Chromosome abnormalities in human embryos. Hum Reprod Update. 1998;4:842–55.CrossRefPubMed

15.

Plachot M. Chromosome analysis of oocytes and embryos. In Verlinsky Y, Kuliev A. (eds), Preimplantation Genetics. Plenum Press; New York, 1991: 103–12.

16.

Palermo GD, Munné S, Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum Reprod. 1994;9:1220–5.PubMed

17.

Levron J, Munné S, Willadsen S, Rosenwaks Z, Cohen J. Male and female genomes associated in a single pronucleus in human zygotes. Biol Reprod. 1995;52:653–7.CrossRefPubMed

18.

Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum Reprod. 1997;12:321–7.CrossRefPubMed

19.

Mio Y, Maeda K. Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos. Am J Obstet Gynecol. 2008;199:1–5.CrossRef

20.

Mio Y, Iwata K, Yumoto K, Kai Y, Sargant HC, Mizoguchi C, et al. Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography. J Assist Reprod Genet. 2012;29:951–6.PubMedCentralCrossRefPubMed

21.

Iwata K, Yumoto K, Sugishima M, Mizoguchi C, Kai Y, Iba Y, et al. Analysis of compaction initiation in human embryos by using time-lapse cinematography. J Assist Reprod Genet. 2014;31:421–6.PubMedCentralCrossRefPubMed

22.

Balakier H, Squire J, Casper RF. Characterization of abnormal one pronuclear human oocytes by morphology, cytogenetics and in-situ hybridization. Hum Reprod. 1993;8:402–8.PubMed

23.

Nagy ZP, Liu J, Joris H, Devroey P, Van Steirteghem A. Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum Reprod. 1994;9:1743–8.PubMed

24.

Payne D, Flaherty SP, Barry MF, Matthews CD. Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum Reprod. 1997;12:532–41.CrossRefPubMed

25.

Plachot M, Mandelbaum J, Junca AM, De Grouchy J, Salta-Baroux J, Cohen J. Cytogenetic analysis and development capacity of normal and abnormal embryos after IVF. Hum Reprod. 1989;4:99–103.CrossRefPubMed

26.

Parelmo G, Joris H, Derde MP, Camus M, Devroey P, Van Steirteghem A. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril. 1993;59:826–35.

27.

Grossman M, Calafell JM, Brandy N, Vanrell JA, Rubio C, Pellicer A, et al. Origin of tripronucleate zygotes after intracytoplasmic sperm injection. Hum Reprod. 1997;12:2762–5.CrossRef

28.

Rosenbusch B, Schneider M, Kreienberg R, Brucker C. Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of digynic triploidy. Hum Reprod. 2002;17:2388–93.CrossRefPubMed

29.

Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of the zygotic paternal genome. Nature. 2000;403:501–2.CrossRefPubMed

30.

Santos F, Hendrich B, Reik W, Dean W. Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol. 2002;241:172–82.CrossRefPubMed

31.

Beaujean N, Harttshorne G, Cavilla J, Taylor J, Gardner J, Wilmut I, et al. Non-conservation of mammalian preimplantation methylation dynamics. Curr Biol. 2004;14:266–7.CrossRef

32.

Xu Y, Zhang JJ, Grifo JA, Krey LC. DNA methylation patterns in human tripronucleate zygotes. Mol Hum Reprod. 2005;11:167–71.CrossRefPubMed

33.

Nakamura T, Arai Y, Umehara H, Masuhara M, Kimura T, Taniguchi H, et al. PGC7/Stella protects against DNA demethylation in early embryogenesis. Nat Cell Biol. 2007;9:64–71.CrossRefPubMed

34.

Guo H, Zhu P, Yan L, Li R, Hu B, Lian Y, et al. The DNA methylation landscape of human early embryos. Nature. 2014;511:606–10.CrossRefPubMed

35.

Zhao XM, Du WH, Hao HS, Wang D, Qin T, Liu Y, et al. Effect of vitrification on promoter methylation and the expression of pluripotency and differentiation genes in mouse blastocysts. Mol Reprod Dev. 2012;79:445–50.CrossRefPubMed

36.

De Munck N, Petrussa L, Verheyen G, Staessen C, Vandeskelde Y, Sterckx J, et al. Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification. Mol Hum Reprod. 2015;21:535–44.CrossRefPubMed

37.

Otsu E, Sato A, Nagaki M, Araki Y, Utsunomiya T. Developmental potential and chromosomal constitution of embryos derived from larger single pronuclei of human zygotes used in in vitro fertilization. Fertil Steril. 2004;81:723–4.CrossRefPubMed

Title: Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number
Authors: Yoshiteru Kai
Kyoko Iwata
Yumiko Iba
Yasuyuki Mio
Publication date: 01-11-2015
Publisher: Springer US
Published in: Journal of Assisted Reproduction and Genetics / Issue 11/2015
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI: https://doi.org/10.1007/s10815-015-0568-1

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number

Abstract

Purpose

Method

Result

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Method

Result

Conclusions

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