Journal of Assisted Reproduction and Genetics

, Volume 34, Issue 9, pp 1189–1197 | Cite as

Time-lapse observation and transcriptome analysis of a case with repeated multiple pronuclei after IVF/ICSI

  • J. Dai
  • L. Z. Leng
  • C. F. Lu
  • F. Gong
  • S. P. Zhang
  • W. Zheng
  • G. X. Lu
  • G. LinEmail author
Assisted Reproduction Technologies



The purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).


This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient’s oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n = 1; donors, n = 4). Oocyte-specific genes with differential expression were selected by the Amazonia! database.


From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR.


We identified several candidate genes affecting pronucleus formation as a new cause of infertility.


Multipronuclear Time-lapse Oocyte Single-cell transcriptome 



We thank to the patients and staff at the Reproductive and Genetic Hospital of CITIC-Xiangya and LetPub ( for its linguistic assistance during the preparation of this manuscript. This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81222007 to L.G. and 81471510 to L.G.), the Program for New Century Excellent Talents in University (No. 907010003), and the Fundamental Research Funds for the Central Universities of Central South University (No. 2015zzts101).

Compliance with ethical standards

This study was approved by the Ethics Committee of the Reproductive and Genetic Hospital of CITIC-Xiangya (reference LL-SC-SG-2013-012).

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary Table 1 (DOCX 15 kb)
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Supplementary Table 2 (DOCX 14 kb)
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Supplementary Table 3 (DOCX 14 kb)
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Supplementary Table 4 (DOCX 26 kb)
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Supplementary Figure 1 (DOCX 12337 kb)
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Supplementary Figure 2 (DOCX 2398 kb)
Supplementary video 1

Formation of multiple pronuclei (MP4 10320 kb)

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Supplementary video 2 Mitosis in embryos containing multiple pronuclei (MP4 4597 kb)


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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • J. Dai
    • 1
    • 2
  • L. Z. Leng
    • 2
  • C. F. Lu
    • 1
    • 2
    • 3
  • F. Gong
    • 1
    • 2
    • 3
  • S. P. Zhang
    • 1
    • 2
  • W. Zheng
    • 1
  • G. X. Lu
    • 1
    • 3
    • 4
  • G. Lin
    • 1
    • 2
    • 3
    • 4
    Email author
  1. 1.Reproductive and Genetic Hospital of CITIC-XIANGYAChangshaChina
  2. 2.Institute of Reproductive and Stem Cell Engineering, Basic Medicine CollegeCentral South UniversityChangshaChina
  3. 3.National Engineering and Research Center of Human Stem CellChangshaChina
  4. 4.Key Laboratory of Reproductive and Stem Cell EngineeringMinistry of HealthChangshaChina

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