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Journal of Assisted Reproduction and Genetics

, Volume 35, Issue 8, pp 1377–1384 | Cite as

Assessment of developmental potential of human single pronucleated zygotes derived from conventional in vitro fertilization

  • Yoshiteru Kai
  • Hitomi Moriwaki
  • Keitaro Yumoto
  • Kyoko Iwata
  • Yasuyuki Mio
Embryo Biology

Abstract

Purpose

The aim of this study was to non-invasively validate the developmental potential of human single pronucleated (1PN) zygotes derived from conventional in vitro fertilization (c-IVF) at the zygote stage.

Methods

Fifty 1PN zygotes derived from 45 patients undergoing c-IVF were used. Immunohistochemistry and fluorescence live cell imaging were used to confirm normal chromosome segregation during the first mitosis. The usefulness of measuring pronuclear diameter was assessed on the basis of the presence or absence of a proper first cleavage and validated by subsequent development.

Results

Although approximately 80% (15/19) of 1PN zygotes contained a diploid genome, immunohistochemistry revealed an unequal distribution of paternal and maternal genomes at the first mitosis. Fluorescence live imaging revealed that 73% (8/11) of 1PN zygotes formed a functional mitotic spindle at the first mitosis resulting from diploid genomes, with 25% (2/8) of these forming a tripolar spindle. 1PN zygotes in which the pronucleus disappeared and that subsequently underwent cleavage had a pronuclear diameter ≥ 32.2 μm. The selection of 1PN zygotes based on pronuclear diameter resulted in zygotes that all formed mitotic spindles with poles during cleavage. Furthermore, 63% (5/8) of these zygotes reached the blastocyst stage.

Conclusions

This study demonstrates the usefulness of a non-invasive assessment of 1PN zygotes derived from c-IVF as an indicator of developmental potential. Furthermore, diploid 1PN zygotes do not always exhibit normal chromosome segregation at the first mitosis. A pronuclear diameter ≥ 32.2 μm just before PN breakdown might be a useful criterion to assess 1PN zygotes that are capable of further development.

Keywords

Single pronucleus (1PN) Mitotic spindle Chromosome segregation Fluorescence live cell imaging 

Abbreviations

5mC

5-Methylcytosine

5hmC

5-Hydroxymethylcytosine

H3K9me3

Histone H3 trimethyl lysine 9

MAP4

Microtubule associated protein 4

PN

Pronucleus (plural: pronuclei)/pronuclear

PB

Polar body

c-IVF

Conventional in vitro fertilization

ICSI

Intracytoplasmic sperm injection

PGS

Preimplantation genetic screening

Notes

Acknowledgements

The authors wish to thank Dr. Takeshi Matsui for assistance with the plasmid vector constructions. We are grateful to all the staff of the Mio Fertility Clinic for their assistance and encouragement.

Author contributions

Y.K. designed this research, performed experiments, analyzed the data, and wrote the manuscript. H.M. also analyzed the data and wrote the manuscript. K.Y and K.I were involved in the clinical zygote vitrification. Y.M. supervised this research.

Compliance with ethical standards

Ethics approval

This study was approved by the Ethics Committee of JISART (Japanese Institution for Standardizing Assisted Reproductive Technology) and was registered with the Japan Society of Obstetrics and Gynecology.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10815_2018_1241_MOESM1_ESM.pdf (318 kb)
Online Resource 1 (Supplementary Figure 1). Immunofluorescence analysis of 5mC (green) and 5hmC (red) representing three staining patterns in single pronucleated (1PN) zygotes. (a) Representative images showing a mixed 5mC/5hmC-positive genome existing in a 1PN zygote. While the 5mC-positive region is only one part of the pronucleus, the 5hmC-positive region covers a widespread area. (b) Images of a 5mC-strongly-positive pronucleus. While weak 5hmC signals exist, they are not like in Fig. 1a. (c) Images of a 5hmC-strongly-positive pronucleus. Few 5mC signals are detected, while the 5hmC-positive region covers a widespread area of the 1PN zygote. (d) Image of a normally-fertilized two-pronuclear (2PN) zygote stained with 5mC and 5hmC as a control. Scale bar = 10 μm. Abbreviations: DIC, differential interference contrast (PDF 318 kb)
10815_2018_1241_MOESM2_ESM.avi (792 kb)
Online Resource 2. Live-imaging movie of 1PN zygote showing a bipolar spindle formation and cleaved to two cells during the first cleavage (AVI 791 kb)
10815_2018_1241_MOESM3_ESM.avi (599 kb)
Online Resource 3. Live-imaging movie of 1PN zygote showing a tripolar spindle formation leading to aberrant chromosome segregation and aberrant first cleavage (AVI 599 kb)
10815_2018_1241_MOESM4_ESM.avi (1.1 mb)
Online Resource 4. Live-imaging movie of 1PN zygote showing chromosomal condensations but spindles were not formed (AVI 1082 kb)
10815_2018_1241_MOESM5_ESM.avi (6 mb)
Online Resource 5. Live-imaging movie showing the mitotic spindles during development of 1PN zygotes with a pronuclear diameter ≥32.2 μm (AVI 6142 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Fertility Research CentreMio Fertility ClinicYonagoJapan

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