P–338 Epigenetic alterations in H19-DMR regulatory region in endometrial tissues of women with endometriosis
Authors: K S , Shahhoseini M , Shahhoseini M , Shahhoseini M , Amirchaghmaghi E , Amirchaghmaghi E , Ghaffari F , Ghaedi K , Ghaedi K , Kamrani S 
Published: August 2021

Abstract

Study question
Is epigenetic modifications pattern in DMR region of H19 gene different in endometrial tissues of women with endometriosis in compare to normal endometrium?

Summary answer
The methylation level in DMR region of H19 gene was significantly lower in the endometriosis group.

What is known already
Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus and has been considered as an epigenetic disease. The lncRNA H19 and insulin-like growth factor–2 (IGF2) genes form a reciprocally imprinted cluster (IGF2/H19). The expression of these two genes is regulated by imprinting control region (ICR). The ICR region is located between these genes and is a differentially methylated region (DMR). The H19 and IGF2 genes are involved in regulating cellular growth and differentiation and might be targeted by MeCP2 (a marker of DNA methylation) for subsequent epigenetic modifications through DMR regulatory region.

Study design, size, duration
In this case-control study, 12 endometrial samples (eutopic) and 12 endometriotic lesions (ectopic) of women with endometriosis and 12 endometrial control samples were analyzed. Control samples were obtained from women who had no evidence of endometriosis during diagnostic laparoscopy. Control and eutopic endometrial samples were obtained by pipelle. Ectopic samples were obtained during laparoscopy. All women signed the informed consent form and did not receive any hormonal treatments during the last three months.

Participants/materials, setting, methods
After endometrial tissues collection, gene expression levels of IGF2 and H19 were evaluated using real-time PCR . The occupancy of MeCP2 on two subregions within DMR region of H19 gene was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR. One-way ANOVA was used for data analysis. P value less than 0.05 was considered statistically significant.

Main results and the role of chance
Gene expression profile of H19 and IGF2 was decreased in eutopic and ectopic endometrial lesions of endometriosis group compared with control ones. The decrease in gene expression of H19 in ectopic samples was significant in compared to the control ones while it was nearly significant in compared to the eutopic samples (p = 0.01, p = 0.056, respectively). The ChIP analysis revealed that MeCP2 incorporation in region II (between −3945 and –3818 bp)within DMR region of H19 gene was significantly decreased in eutopic samples compare to control group (p = 0.02) while its decrease was nearly significant in ectopic samples (p = 0.056). However, this DNA methylation profile was not significantly different between eutopic and ectopic endometrial samples in endometriosis in group. Incorporation of MeCP2 in region I (between −2230 and –2103 bp)within DMR region of H19 gene was not significantly different between the eutopic, ectopic and control samples (p &gt; 0.05). (data was presented at 21th Royan International Congress).

Limitations, reasons for caution
The main limitations of this study is its small sample size. For getting more information, we need to monitor this DNA methylation profile in a large number of women with and without endometriosis. Also, more investigations are required to clarify the further epigenetic modifications in this region.
Wider implications of the findings: It seems that reduced expression of IGF2 may be due to hypomethylation of H19-DMR region II while this hypomethylation has no effect on H19 expression in endometriosis. As previously was shown , hypomethylation of H19-DMR causes decrease of IGF2 expression and increase in H19 expression.

Trial registration number
Not applicable