The hypomethylation of imprinted genes in IVF/ICSI placenta samples is associated with concomitant changes in histone modifications

ABSTRACT

Although more and more children are born by Assisted Reproductive Technologies (ART), ART safety has not fully been demonstrated. Notably, ART could disturb the delicate step of implantation, and trigger placenta-related adverse outcomes with potential long-term effects, through disrupted epigenetic regulation. We have previously demonstrated that placental DNA methylation was significantly lower after IVF/ICSI than following natural conception at two differentially methylated regions (DMRs) associated with imprinted genes (IGs): H19/IGF2 and KCNQ1OT1. As histone modifications are critical for placental physiology, the aim of this study was to profile permissive and repressive histone marks in placenta biopsies to reveal a better understanding of the epigenetic changes in the context of ART. Utilizing chromatin immunoprecipitation (ChIP) coupled with quantitative PCR, permissive (H3K4me3, H3K4me2, and H3K9ac) and repressive (H3K9me3 and H3K9me2) post-translational histone modifications were quantified. The analyses revealed a significantly higher quantity of H3K4me2 precipitation in the IVF/ICSI group than in the natural conception group for H19/IGF2 and KCNQ1OT1 DMRs (P = 0.016 and 0.003, respectively). Conversely, the quantity of both repressive marks at H19/IGF2 and SNURF DMRs was significantly lower in the IVF/ICSI group than in the natural conception group (P = 0.011 and 0.027 for H19/IGF2; and P = 0.010 and 0.035 for SNURF). These novel findings highlight that DNA hypomethylation at imprinted DMRs following ART is linked with increased permissive/decreased repressive histone marks, altogether promoting a more permissive chromatin conformation. This concomitant change in epigenetic state at IGs at birth might be an important developmental event because of ART manipulations.

KEYWORDS:

• Assisted reproductive technologies
• epigenetics
• histone modifications
• in vitro fertilization
• DNA methylation
• placenta

Declarations

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

PF, DM, and CC were the principal investigators and take primary responsibility for the paper. PF, DM, PP, and CC were responsible for the study design. CC and PF recruited the patients. CC, DM, PP, MS, JH, and AM were involved in experiments. PF, DM, and CC coordinated the research. CC performed the statistical analyses. DM, PF, and CC drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank the midwives and nurses of Dijon University Hospital for their help in collecting samples. We thank Benjamin Tournier and Laurence Jego for their help in optimizing protocols. We thank Imprinting and Cancer group, Cancer Epigenetic and Biology Program, Bellvitge Biomedical Research Institute for their help in realizing experiments. We thank Sandrine Daniel, Marie-Laure Humbert-Asensio and Lydie Rossye (member of the “Centre d’Investigation Clinique-Epidémiologie Clinique/essais cliniques” of Dijon) for their precious help in monitoring and analysing the data. We thank Maud Carpentier of the “Direction de la Recherche Clinique et de l’Innovation” (DRCI) of Dijon University Hospital for the promotion and the management of the study. We thank Philip Bastable for his help in writing the manuscript.

Consent for publication

Not applicable

Disclosure statement

The authors declare that they have no competing interests.

Ethics approval and consent to participate

All women had given written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Board and the Ethics Committee of Dijon University Hospital (Comité de Protection des Personnes [CPP] Est I, n°2012-A01010-43).

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by Besançon and Dijon University Hospitals under grant [‘appel à projet Dijon-Besançon 2013’], by the Agence Nationale pour la Recherche under grant [‘ANR-17-CE12-0014’] and by the Fonds pour la santé des femmes. The Monk laboratory was supported by the Spanish Ministry of Economy and Competitiveness under grant [‘MINECO; BFU2014-53093-R’ and ‘BFU2017-85571-R’] and by the European Union Regional Development Fund (FEDER). AMS is a recipient of an FPI PhD studentship from MINECO.