Effects Of Beclin-1 On Progesterone Synthesis In Granulosa Cells Of Patients With Endometriosis And The Mechanism

BackgroundOvarian dysfunction is one of the important causes of poor pregnancy outcomes in patients with endometriosis(EMS).Changes of progesterone levels in follicular fluid of patients with EMS can lead to follicular dysplasia and decreased oocyte quality,which affects the developmental potential of embryos.Previous studies have found that Beclin-1(BECN1),an essential mediator of autophagy,is closely related to the development and progression of EMS.However,the expression of BECN1 in EMS ovarian granulosa cells and its effect on ovarian function are still not clear.This study aimed to investigate the regulation of BECN1 on progesterone synthesis in ovarian granulosa cells of patients with EMS.Methods1.Twenty-six infertile patients diagnosed with EMS by laparoscopy and 29 patients with tubal factor infertility were enrolled in the study.Follicular fluid aspirated by puncture was collected.2.Patients’ characteristics and clinical outcomes such as high-quality embryo rate of the recruited patients were analyzed.3.Granulosa cells were isolated and purified from follicular fluid.The expression of BECN1 and autophagy level were detected by western blot analysis,electron microscopy,quantitative real-time reverse transcription PCR(RT-PCR).4.Progesterone concentrations in follicular fluid were measured by electrochemiluminescence immunoassay(ECLIA).The expression levels of steroidogenic acute regulatory protein(StAR),cholesterol side-chain cleavage enzyme(CYP11A1)and 3β-hydroxysteroid dehydrogenase(HSD3B),the enzymes involved in progesterone synthesis in ovarian granulosa cells,were detected by western blot analysis and real-time RT-PCR.5.The primary granulosa cells cultured in vitro were transfected with BECN1 siRNA to detect the progesterone concentration in the culture medium and the expression of key enzymes in progesterone synthesis.6.Primary granulosa cells were treated with low-density lipoprotein(LDL)three days after transfection with BECN1 siRNA or combined with chloroquine,an autophagy inhibitor,for 24 h.Western blot analysis was used to study the role of BECN1 and autophagy in progesterone synthesis induced by LDL in ovarian granulosa cells.The intracellular and extracellular cholesterol concentrtations were measured by enzyme-linked immunosorbent assay(ELISA).7.The primary granulosa cells were infected with lentivirus to determine the effects of BECN1 overexpression on progesterone production as well as cholesterol levels in granulosa cells.8.Human granulosa-like tumor cell line KGN were transfected with BECN1 overexpression plasmid to further detect the role of BECN1 on progesterone synthesis and the expression of related enzymes.Results1.The clinical data showed that basal follicle-stimulating hormone(FSH)levels and serum progesterone/estradiol ratio on the day of human chorionic gonadotropin(hCG)administration were significantly higher in EMS patients than in controls.However,anti-Müllerian hormone(AMH)levels,the number of retrieved oocytes,the rate of oocyte retrieval and the rate of high-quality embryos in the EMS group were significantly lower than those in the control group,suggesting decreased ovarian reserve and damaged oocyte quality in patients with EMS.2.BECN1 expression and autophagy levels were increased in granulosa cells of EMS patients.3.Progesterone concentration in the follicular fluid of the EMS group was significantly higher than that of the control group.Moreover,the mRNA level of BECN1 in granulosa cells was positively correlated with STAR mRNA abundance,but negatively correlated with the number of oocytes retrieved,suggesting that the abnormal progesterone level in the follicular fluid of EMS is closely related to the expression of BECN1,which could be one of the important causes of ovarian dysfunction in EMS.4.In primary granulosa cells cultured in vitro,BECN1 knockdown reduced progesterone secretion and its enzymatic pathway.5.Inhibition of autophagy by knockdown of BECN1 or chloroquine significantly inhibited LDL-mediated progesterone synthesis and reduced intracellular cholesterol levels,suggesting that BECN1 and autophagy signaling may release free cholesterol by hydrolyzing LDL,thus playing a regulatory role in the synthesis of progesterone.6.In both primary granulosa cells and KGN cells,overexpression of BECN1 induced the expression of steroidogenic enzymes and the production of progesterone.ConclusionsBECN1 expression and autophagy were increased in granulosa cells of patients with EMS.The excessive BECN1 level might contribute to the enhanced progesterone accumulation in follicular fluid by promoting the degradation pathway of LDL for progesterone biosynthesis via lysosome activation in granulosa cells,thus leading to impaired oocyte quality.