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

Journal of Assisted Reproduction and Genetics volume 32, pages 1589–1595 (2015)Cite this article

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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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

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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

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
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  2. Kyoko Iwata
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  3. Yumiko Iba
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  4. Yasuyuki Mio
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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

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)

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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

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

Key words

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