Loss of methylation of H19-imprinted gene derived from assisted reproductive technologies can be mitigated by cleavage-stage embryo transfer in mice

  • Shuqiang Chen
  • Meizi Zhang
  • Li Li
  • Ming Wang
  • Yongqian Shi
  • Hengde Zhang
  • Bin Kang
  • Na TangEmail author
  • Bo LiEmail author
Embryo Biology



Studies on rodents have shown that assisted reproductive technologies (ARTs) are associated with perturbation of genomic imprinting in blastocyst-stage embryos. However, the vulnerable developmental window for ART influence on the genomic imprinting of embryos is still undetermined. The purpose of this study was to establish the specific embryonic development stage at which the loss of methylation of H19 imprinting control regions (ICRs) was caused by ART occurrence. Additionally, we explored protocols to safeguard against possible negative impacts of ART on embryo H19 imprinting.


Mouse embryos were generated under four different experimental conditions, divided into four groups: control, in vitro culture (IVC), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). The methylation levels of H19 ICR of the grouped or individual embryos were analyzed by bisulfite-sequencing PCR.


Our data showed that the loss of methylation of H19 ICR in mouse blastocysts was inflicted to a similar extent by IVC, IVF, and ICSI. Specifically, we observed a significant loss of methylation of H19 ICR between the mouse 8-cell and morula stages. In addition, we revealed that the transfer of mouse embryos generated by ARTs in the uterus at the 8-cell stage induced the occurrence of methylation patterns in the blastocysts closer to the in vivo ones.


Our findings indicate that the loss of methylation of H19 ICR caused by ARTs occurs between the 8-cell and the morula stages, and the transfer of cleavage embryos to the uterus mitigates the loss methylation of H19 derived by mice ARTs.


Assisted reproductive technologies (ARTs) Genomic imprinting Imprinting control regions (ICRs) Loss of methylation 



We are grateful to the valuable comments from other members of our laboratory.

Funding information

This study was supported by the grants from the National Natural Science Foundation of China (31640056, 31801250), Shaanxi Natural Science Foundation of China (2016JM8052), Key Research and Development Program of Shaanxi, China (2018SF-258), and Scientific and Technical Innovatory Project of Tangdu Hospital (2017LCYJ001).

Compliance with ethical standards

Ethical approval

The present study was reviewed and approved by the Ethics Committee of Animal and Medicine of the Tangdu Hospital of The Fourth Military Medical University (Approval identification: TDLL-2018-03-47) and was conducted in accordance with the guidelines from the Committee on the Use of Live Animals in Teaching and Research of the Tangdu Hospital of The Fourth Military Medical University.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10815_2019_1575_MOESM1_ESM.docx (17 kb)
ESM 1 (DOCX 16 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Obstetrics and GynecologyTangdu Hospital, the Fourth Military Medical UniversityXi’anChina
  2. 2.Reproductive Medicine CenterTianjin Frist Central HospitalTianjinChina
  3. 3.Shaanxi Institute for Food and Drug ControlXi’anPeople’s Republic of China

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