P-180 Alterations of methylation level of H19/IGF2 DMR differentially methylated region(DMR) in human blastocysts following re-vitrification
Authors: Movahedin M , Daneshvar M , Salehi M , Noruzinia M 
Published: June 2022

Abstract

Study question
Does re-vitrification affect on methylation level of H19/IGF2 DMR differentially methylated region(DMR) in human in vitro-produced blastocysts?

Summary answer
The results of this study showed that re-vitrification had no significant effect on the methylation level of H19 / IGF2 DMR region in human blastocysts.

What is known already
Nowadays, re-vitrification seems to be a successful and useful method to prevent the discarding of extra embryos. Several reports have indicated that the embryo manipulations during ART may result in epigenetic errors. This has been attributed to the concurrency of the ART manipulations and epigenetic events after fertilization. The effect of embryo re-vitrification on epigenetic changes has not been studied so far. The aim of this study is to evaluate the effect of embryo re-vitrification on methylation level of H19/IGF2 differentially methylated region (DMR) in donated human blastocysts.

Study design, size, duration
The effect of re-vitrification on methylation level of H19/IGF2 DMR was evaluated in 9 ICSI-derived human embryos individually and the expressions of H19 and IGF2 were assessed in 15 embryos individually. Embryos were donated from fertile couples referring for family balancing program. Donated embryos were cultured to blastocyst stage and high quality blastocyst (AA-AB) assigned to three groups: fresh, vitrified and re-vitrification. Scoring of blastocysts were according to Gardner et al. (2012) grading system.

Participants/materials, setting, methods
Collected blastocysts were vitrified on Cryotech carriers, with the method described by Kuwayama. After warming of blastocysts and 4-5 hours culturing of them, expression of H19 and IGF2 genes in blastocysts evaluated with Real time PCR and methylation levels of H19/IGF2 DMR in blastocysts were analyzed by Bisulfite Sequencing PCR (BSP) technique. 10 clones were sequenced per replicate. Methylation status of lymphocyte was used as control to verify the credibility of our analysis system.

Main results and the role of chance
The results showed that the overall percentage of methylated CpGs of H19/IGF2 DMR in the control, vitrification, re-vitrification groups and lymphocyte were 42.71, 40.90, 39.23% and 49.30 %. respectively. There was no significant difference in methylation level of H19/IGF2 DMR between the groups. H19 and IGF2 expression did not show a significant difference in vitrified group compared to the control and in re-vitrified group compared to vitrified.

Limitations, reasons for caution
- Small number of samples examined due to difficult access to human blastocysts donated by fertile couples.
- Evaluation of high-quality embryos donated by healthy fertile couples while embryos of infertile couples may respond differently.

Wider implications of the findings
According to the obtained results, embryo re-vitrification did not have a significant change on the methylation level of H19/IGF2 DMR and expression of H19 and IGF2 genes. However, that further studies on the effect of re-vitrification on epigenetic errors in low-quality embryos and the embryos of infertile couples are needed.

Trial registration number
not applicable