Journal of Assisted Reproduction and Genetics

, Volume 32, Issue 11, pp 1589–1595 | Cite as

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

  • Yoshiteru Kai
  • Kyoko Iwata
  • Yumiko Iba
  • Yasuyuki Mio
Embryo Biology



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.


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.


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.


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.

Key words

Abnormal fertilization Monopronuclear (1PN) zygote Tripronuclear (3PN) zygote 5-Hydroxymethylcytosine (5hmC) Centrosome 







Assisted reproductive technologies


Bovine serum albumin


Phosphate-buffered saline


Pronucleus (plural: pronuclei)/pronuclear


Male pronuclei


Female pronuclei

2nd PB

Second polar body


Conventional in vitro fertilization


Intracytoplasmic sperm injection


Metaphase II



We are grateful to Keitaro Yumoto, Minako Sugishima, Chizuru Mizoguchi, Sayako Furuyama, and Yuka Matoba for the collection of the samples. This work was supported by all the staff in the Reproductive Centre, Mio Fertility Clinic, Japan. We also thank S.N. for the technical advice and are particularly grateful for the assistance given by Motokazu Tsuneto.

Supplementary material (848 kb)
Supplementary Movie 1 3D image reconstruction of a 2PN zygote derived from c-IVF. Red signal means 5mC-positive and green signal means 5hmC-positive. It provides images relating to Fig. 1a. This normal 2PN zygote showed a 5mC/5hmC double-positive PN and a 5mC-positive PN. (MOV 847 kb) (1 mb)
Supplementary Movie 2 3D image reconstruction of a 1PN zygote derived from c-IVF. It provides images relating to Fig. 1c. This 1PN zygote showed both 5mC and 5hmC staining in a single PN. (MOV 1074 kb) (857 kb)
Supplementary Movie 3 3D image reconstruction of a 3PN zygote derived from c-IVF. It provides images relating to Fig. 1d. This 3PN zygote showed a single 5mC-positive PN and two 5mC/5hmC double-positive PNs. (MOV 856 kb) (849 kb)
Supplementary Movie 4 3D image reconstruction of a 3PN zygote derived from ICSI. It provides images relating to Fig. 1e. This 3PN zygote showed two 5mC-positive PNs and a 5mC/5hmC double-positive PN. (MOV 849 kb) (288 kb)
Supplementary Movie 5 3D image reconstruction of a 3PN zygote derived from c-IVF at syngamy. It provides images relating to Fig. 2g. This movie shows a Y-shaped metaphase plate and aberrant centrosome positions of this 3PN zygote. (MOV 288 kb)


  1. 1.
    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.CrossRefPubMedGoogle Scholar
  2. 2.
    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.PubMedGoogle Scholar
  3. 3.
    Taylor AS, Braude PR. The early development and DNA content of activated human oocytes and parthenogenetic human embryos. Hum Reprod. 1994;9:2389–97.PubMedGoogle Scholar
  4. 4.
    Palermo GD, Munné S, Colombero LT, Cohen J, Rosenwaks Z. Genetics of abnormal human fertilization. Hum Reprod. 1995;1:120–7.CrossRefGoogle Scholar
  5. 5.
    Balakier H, Cadesky K. The frequency and developmental capability of human embryos containing multinucleated blastomeres. Hum Reprod. 1997;12:800–4.CrossRefPubMedGoogle Scholar
  6. 6.
    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.PubMedGoogle Scholar
  7. 7.
    Mio Y, Iwata K, Yumoto K, Maeda K. Human embryonic behavior observed with time-lapse cinematography. J Health Med Inf. 2014;5:143.Google Scholar
  8. 8.
    Mio Y. Morphological analysis of human embryonic development using time-lapse cinematography. J Mamm Ova Res. 2006;23:27–35.CrossRefGoogle Scholar
  9. 9.
    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.PubMedCentralCrossRefPubMedGoogle Scholar
  10. 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.CrossRefPubMedGoogle Scholar
  11. 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.CrossRefPubMedGoogle Scholar
  12. 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.PubMedCentralCrossRefPubMedGoogle Scholar
  13. 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.CrossRefPubMedGoogle Scholar
  14. 14.
    Munné S, Cohen J. Chromosome abnormalities in human embryos. Hum Reprod Update. 1998;4:842–55.CrossRefPubMedGoogle Scholar
  15. 15.
    Plachot M. Chromosome analysis of oocytes and embryos. In Verlinsky Y, Kuliev A. (eds), Preimplantation Genetics. Plenum Press; New York, 1991: 103–12.Google Scholar
  16. 16.
    Palermo GD, Munné S, Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum Reprod. 1994;9:1220–5.PubMedGoogle Scholar
  17. 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.CrossRefPubMedGoogle Scholar
  18. 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.CrossRefPubMedGoogle Scholar
  19. 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.CrossRefGoogle Scholar
  20. 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.PubMedCentralCrossRefPubMedGoogle Scholar
  21. 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.PubMedCentralCrossRefPubMedGoogle Scholar
  22. 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.PubMedGoogle Scholar
  23. 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.PubMedGoogle Scholar
  24. 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.CrossRefPubMedGoogle Scholar
  25. 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.CrossRefPubMedGoogle Scholar
  26. 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.Google Scholar
  27. 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.CrossRefGoogle Scholar
  28. 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.CrossRefPubMedGoogle Scholar
  29. 29.
    Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of the zygotic paternal genome. Nature. 2000;403:501–2.CrossRefPubMedGoogle Scholar
  30. 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.CrossRefPubMedGoogle Scholar
  31. 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.CrossRefGoogle Scholar
  32. 32.
    Xu Y, Zhang JJ, Grifo JA, Krey LC. DNA methylation patterns in human tripronucleate zygotes. Mol Hum Reprod. 2005;11:167–71.CrossRefPubMedGoogle Scholar
  33. 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.CrossRefPubMedGoogle Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. 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.CrossRefPubMedGoogle Scholar
  37. 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.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Yoshiteru Kai
    • 1
  • Kyoko Iwata
    • 2
  • Yumiko Iba
    • 2
  • Yasuyuki Mio
    • 1
    • 2
  1. 1.Fertility Research CentreMio Fertility ClinicYonagoJapan
  2. 2.Reproductive CentreMio Fertility ClinicYonagoJapan

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