Skip to main content
SpringerLink
Log in

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

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

5mC:

5-Methylcytosine

5hmC:

5-Hydroxymethylcytosine

ART:

Assisted reproductive technologies

BSA:

Bovine serum albumin

PBS:

Phosphate-buffered saline

PN:

Pronucleus (plural: pronuclei)/pronuclear

mPN:

Male pronuclei

fPN:

Female pronuclei

2nd PB:

Second polar body

c-IVF:

Conventional in vitro fertilization

ICSI:

Intracytoplasmic sperm injection

MII:

Metaphase II

References

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  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.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  PubMed Central  PubMed  Google Scholar 

  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.

    Article  PubMed Central  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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. 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.

    Article  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Fertility Research Centre, Mio Fertility Clinic, 2-1-1, Kuzumo-Minami, Yonago, Tottori, 683-0008, Japan

    Yoshiteru Kai & Yasuyuki Mio

  2. Reproductive Centre, Mio Fertility Clinic, 2-1-1, Kuzumo-Minami, Yonago, Tottori, 683-0008, Japan

    Kyoko Iwata, Yumiko Iba & Yasuyuki Mio

Authors
  1. Yoshiteru Kai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kyoko Iwata
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yumiko Iba
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yasuyuki Mio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yasuyuki Mio.

Additional information

Heading

Identifying the fertilization status in abnormal human zygotes

Capsule

A novel method was established to identify PN origin based on the epigenetic status of the genome utilizing a molecular biological technique. The results were also used to analyze the centrosome numbers in human zygotes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

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)

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)

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)

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kai, Y., Iwata, K., Iba, Y. et al. Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number. J Assist Reprod Genet 32, 1589–1595 (2015). https://doi.org/10.1007/s10815-015-0568-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-015-0568-1

Key words

Search

Navigation