Effects of peritoneal fluid and tumor necrosis factor-α on granulosa cell function and autophagy in endometriosis patients

Abstract

Objective \nTo investigate the effects of peritoneal fluid of peritoneal type I-II endometriosis (EMS) patients and tumor necrosis factor-α (TNF-α) on granulosa cells function and autophagy, and to investigate the causes of infertility in peritoneal endometriosis. \n \n \nMethods \nHuman ovarian granulosa cell line KGN was cultured in vitro, and cells were treated with peritoneal fluid of EMS patients (EMS group) and recombinant TNF-α protein in different concentrations (1 μg/L, 10 μg/L, 100 μg/L) (TNF-α group), respectively. Peritoneal fluid of non-EMS patients served as control group. Cell proliferation was detected by CCK-8 method, cell apoptosis was detected by flow cytometry and mitochondrial membrane potential, estradiol and progesterone levels were detected by enzyme-linked immunosorbent assay (ELISA), and autophagic markers including LC3-II/LC3-I and p62 were detected by Western blotting. The number of autophagosomes was observed by transmission electron microscopy. \n \n \nResults \nEMS patients’ peritoneal fluid and high concentrations of recombinant TNF-α protein inhibited KGN granulosa cell proliferation (P<0.001), promoted granulosa cell apoptosis (P=0.009), and decreased estradiol and progesterone secretion levels (P<0.001, P=0.05), promoted granulocyte autophagy markers LC3-II/LC3-I ratio increased (P<0.001), the p62 protein decreased (P<0.05), the number of autophagosomes increased (P<0.001), and autophagy was over activated. \n \n \nConclusion \nEMS patients’ peritoneal fluid and TNF-α can affect the function of granulosa cells by inhibiting the proliferation, promoting apoptosis, affecting the secretion of estradiol and progesterone, and causing excessive autophagy. The paracrine and autocrine functions of granulosa cells may affect oocyte quality, fertilization and embryo development and implantation, resulting in infertility in EMS patients. \n \n \nKey words: \nEndometriosis;\xa0Peritoneal fluid;\xa0Tumor necrosis factor-α;\xa0Granulosa cell;\xa0Autophagy