Decreased expression of EZH2 in granulosa cells contributes to endometriosis-associated infertility by targeting IL-1R2 [RNA-Seq]

The mechanism by which endometriosis, a common gynecological disease characterized by chronic pelvic pain and infertility, causes infertility remains elusive. Luteinized unruptured follicle syndrome, the most common type of ovulatory dysfunction, is a cause of endometriosis-associated infertility involving reduced numbers of retrieved and mature oocytes. Ovulation is controlled by luteinizing hormone and paracrine signals produced within the follicle microenvironment. Generally, interleukin (IL)-1β is elevated in endometriosis follicular fluid, whereby it amplifies ovulation signals by activating extracellular-regulated kinase 1/2 and CCAAT/enhancer binding protein β pathways. However, this amplification of ovulation by IL-1β does not occur in patients with endometriosis. To illuminate the mechanism of ovulatory dysfunction in endometriosis, we analyzed the impact of oxidative stress and IL-1β expression in endometriosis follicles. We found that oxidative stress decreased EZH2 expression and reduced H3K27Me3 levels in endometriosis ovarian granulosa cells (GCs). Selective Ezh2 depletion in mice ovarian GCs reduced fertility by disturbing cumulus-oocyte complex expansion and reducing epidermal growth factor-like factor expression. Gene expression and H3K27Me3 ChIP-sequencing of GCs revealed IL-1 receptor 2 (IL-1R2), a high-affinity IL-1β-receptor that suppresses IL-1β-mediated inflammatory cascades during ovulation, as a crucial target gene of the EZH2-H3K27Me3 axis. Moreover, IL-1β addition did not restore ovulation upon Ezh2 knockdown, indicating a vital function of IL-1R2 in endometriosis. Thus, our findings show that reducing EZH2 and H3K27Me3 in GCs suppressed ovulatory signals by increasing IL-1R2 expression, which may ultimately contribute to endometriosis-associated infertility. To compare global gene expression profiled between WT-GCs (n = 4) and KO-GCs (n = 3), WT and KO mice were injected with 5 IU PMSG, and 48h later 5 IU HCG was injected to induce ovulation. No less than 2 × 10^6 mGCs were collected from WT and KO mice ovaries, and these cells were further used to perform RNA-seq. According to the results of quality control measurements by Biomarker Technologies (BMKCloud, Beijing, China), 2.5 ug RNA from WT-GCs and 2.5 ug RNA from KO-GCs were ultimately used for transcriptome analysis. The process of RNA-seq mainly includes sample detection, library construction, quality control, and sequencing on an high-throughput sequencing platform (Illumina, San Diego, CA, USA). Approximately 24008 genes were successfully annotated.