Elevated IL-8/TNF-α in follicle fluid of infertile women with endometriosis decrease LHCGR expression in cumulus cells

Abstract Background: To study whether ILs/TNFs in the follicular fluid (FF) of women with EMs are responsible for impaired follicular development or (and) ovulation or not, and then to explore the underlying mechanisms. Methods: follicular fluid (containing cumulus granulosa cells) was collected from women with EM and male factor infertility at our Clinical Reproductive Medicine Center, and peritoneal fluid was collected from the above patients with EMs. The expression of ovulation-related genes in cumulus cells was analysed by RT-PCR. Mouse cumulus cells expansion degree was assessed after cultured in follicle fluid from infertile women. Follicle fluid was detected by ELISA. Oocytectmized complex cell model was established, and cultured in vitro medium with addition of 100 IU/ml FSH. TUNEL staining was used to determine the apoptosis of cumulus cells. Then, we explored expression of P-SMAD2/3，key enzyme for retinoic acid metabolism, and methylation of SP1 binding sites in Lhcgr promoter region. Meanwhile, the P-AKT and P-catenin were assessed by Western blot. All experiments were performed independently at least three times, and data are presented as mean ± SEM. Statistical analyses were performed using Graphpad Prism 5 software p<0.05 (* and different letters) were defined as significant differences. Results: In cumulus cells, expression of genes related to ovulation decreased significantly than that in controls (P < 0.05), especially starting from LHCGR . The concentrations of IL-8 and TNF-α in follicle fluid were significantly higher in infertile women with endometriosis than in controls (P < 0.05). The function of follicle fluid and pelvic fluid of endometriosis women have changed. Addition of 500 pg/mL IL-8/TNF-α to medium did not cause significant apoptosis of cumulus cells, but inhibited P-AKT and P-β-catenin. On the other hand, expression of P-SMAD2/3 and retinoic acid production were reduced, while hypermethylation of the Sp1 binding sequence on Lhcgr promoter was identified, and Lhcgr expression was significantly reduced compared to control (P<0.05). Conclusion: Elevated IL-8/TNF-α in follicular fluid of women with endometriosis indirectly maintains Lhcgr promoter hypermethylation through activation of P-SMAD2/3, while inhibiting AKT and β-Catenin phosphorylation, which together reduce LHCGR mRNA expression.