P-331 Gene expression and epigenetic modifications of Cumulus expansion genes PTGS2, HAS-2, and GREM1 in the follicular fluid and Cumulus cells of endometriosis patients

Abstract Study question Can evaluation of Cumulus expansion genes (PTGS2, HAS-2, and GREM1) in follicular fluid (FF) and Cumulus cells (CCs) predict oocyte status in women with endometriosis? Summary answer Endometriosis affects the expression and epigenetic modifications of PTGS2, HAS-2, and GREM1, which can reduce oocyte quality and maturation. What is known already Endometriosis is an epigenetic disorder in women, characterized by endometrial-like tissue outside the uterus. This disease causes infertility through several mechanisms, including the reduction of oocyte quality. Evaluation of oocyte status in women with endometriosis should be done using indirect methods. FF and CCs are the best options that affect the quality and maturation of the oocytes. Cumulus expansion genes, such as PTGS2, HAS-2, and GREM1, are introduced as indicators of oocyte maturation and affect oocyte quality. Therefore, it seems that the evaluation of the expression and epigenetic modifications of these genes can help to predict the oocyte status. Study design, size, duration In this study (October 2023 - February 2025), according to ethical standards of the Medical Ethics Committee of Royan Institute and with informed consent, FF and CCs were obtained from 30 women with stage III and IV of endometriosis and 30 women in the control group (male factor infertility, oocyte freezing, pre-implantation genetic diagnosis). Women with polycystic ovary syndrome, diminished ovarian reserve, human papillomavirus, and herpes simplex virus 2 were not present in this study. Participants/materials, setting, methods To evaluate the expression of PTGS2, HAS-2, and GREM1 for each patient individually RNA extraction, cDNA synthesis, and qRT-PCR were performed. Chromatin immunoprecipitation/qRT-PCR techniques were also used to evaluate the H3K9ac, H3K9me2, and DNA methylation in the regulatory region of candidate genes for pooled CC samples (n = 24). Quantitative mRNA expression and epigenetic modifications were analyzed by the parametric independent t-test and non-parametric Mann-Whitney U test, and P ≤ 0.05 was considered statistically significant. Main results and the role of chance In this study, the expression levels of PTGS2, HAS-2, and GREM1 in FF and CCs of women with endometriosis decreased significantly compared to the control group (p-value<0.001). The level of H3K9 acetylation in the regulatory region of candidate genes in women with endometriosis showed a decreasing pattern compared to the control group, and this difference was significant for PTGS2 and HAS-2 (p-value=0.0045 and p-value=0.0157, respectively). The levels of H3K9 methylation and DNA methylation in the regulatory region of PTGS2, HAS-2, and GREM1 in the endometriosis group showed a significant increase compared to the control group (p-value<0.05). Among the studied demographics (age, body mass index, luteinizing hormone (LH), Follicle-stimulating hormone (FSH) and Anti-Mullerian Hormone (AMH) levels, duration of ovarian stimulation, and history of abortion), the history of gravidity in women with endometriosis has decreased significantly compared to the control group (p-value=0.026). In addition, the number of retrieved oocytes and metaphase II (MII) oocytes in the group of endometriosis patients decreased significantly compared to the control group (p-value=0.0553 and p-value=0.0205, respectively). Limitations, reasons for caution More samples are needed to confirm these data, especially regarding epigenetic alterations. Another limitation was the pairing of FF and CCs for the same study participant. In addition, evaluation of embryo status and other epigenetic modifications related to the regulatory region of PTGS2, HAS-2, and GREM1 is recommended. Wider implications of the findings The integration of clinical data with molecular findings in this study confirms the reduction in the number and quality of MII oocytes in endometriosis patients. These findings offer an improved understanding of the molecular mechanisms that may affect the acquisition of oocyte developmental competence in patients suffering from severe endometriosis. Trial registration number No