Molecular Mechanism Of Prostaglandin E2on The Steroidogenesis In Granulosa Lutein Cells From Women With Endometriosis

Endometriosis is a common, chronic gynecological disorder in women of reproductive age. Fifty percent of women with endometriosis are infertility. However, the the cellular and molecular mechanisms has not yet been understood. Apart from mechanical distortion of normal pelvic anatomy and immunologic deficiencies and so on, the ovarian dysfunction is also an important reason for the endometriosis associated infertility. Women with endometriosis have slowly growth of the follicles and few ova number, more atretic follicles and decreased fertilization rates. Nevertheless, it had been reported that the pregnancy and implantation rates in women with endometriosis undergoing IVF were significantly decreased compared with those submitting to ovum donation. Thus, there must be some dysfunction of the ovary in endometriosis. Granulosa cells, the main functional cells in synthesis and secretion of estradiol and progesterone, play an important role in follicle development and oocyte maturation. Thus, it may be a good method to value ovarian function of women with endometriosis through studying the function of granulosa cells.Some cytokines in follicular fluid also have important regulating function on the development of oocytes and granulosa cells. To date, it has been proved that PGE2plays a number of essential roles during reproductive processe, including follicular development, oocyte maturation, ovulation and the luteinization of granulosa cells and so on. It has been reported that PGE2regulated the production of progesterone through its receptors, and it modulated the cumulus expansion and meiotic maturation of the cumulus-oocyte through PI3K/AKT, ERK1/2and NF-κB pathways. Moreover, a recent report found the levels of PGE2in peritoneal fluid and blood serum were significantly higher in women with endometriosis compared with those with tubal factors. But the levels of PGE2in endometriotic follicular and what effects does PGE2have on the synthesis of steroidogenesis in granulosa cells from women with endometriosis are still unclear, so what are the levels of PGE2in endometriotic follicular fluid? Whether the expression of PGE2is decreased in endometriosis and thus it is not sufficient to enhance the production of steroid hormone, or its function is disturbed, as a result its function of promoting the development of the ovary is damaged? It is as yet unclear.In the first part of the study, we first isolated luteinized granulosa cells from follicular fluid of in vitro fertilization patients due to tubal obstruction. By using Chemoluminescence, Real-time PCR and In-cell Western methods, we detected the effect of PGE2on the production of estradiol and progesterone or the expression of StAR and CYP19A1of human luteined granulosa cells; In the second part of the study, we evalude the function of the PI3K/AKT, ERK1/2and NF-κB pathways within the process of PGE2promoting the production of steroid hormone; In the third part of the study, we detected the levels of PGE2, estradiol and progesterone in follicular fluid from women with endometriosis, evaluated the correlation between PGE2and steroid hormone. Moreover, we evaluated the IVF-ET outcomes as well. In the last part of the study, we detected the different expression and the effect of PGE2on the expression of steroid hormone and the associated enzymes in the endometriotic granulosa cells. Furthermore, we evaluated the alteration of the PGE2pathway in endometriotic granulosa cells. The purpose of this research is to better understand the regulation of PGE2pathways on endometriotic granulosa cell.Part I Effect of PGE2on the expression of StAR and CYP19A1in human granulosa lutein cellsObjects:To investigate the dose-and time-effects of PGE2on the expression of StAR and CYP19A1in human granulosa lutein cells.Methods:Ovarian granulosa lutein cells were obtained from28tubal-factor infertility women underwent IVF-ET, and then the cells were incubated for48hours. Subsequently, we stimulated granulosa cells with50ng/ml PGE2for different time intervals (0h,6h,12h,24h). Then, the cells were treated with different concentrations PGE2(0ng/ml,10ng/ml,50ng/ml,250ng/m) for hours according to the former results. By using Chemoluminescence, Real-time PCR and In-cell Western methods, we detected the effect of PGE2on the production of estradiol and progesterone or the expression of StAR and CYP19A1of human luteined granulosa cells.Results:1Results of ChemoluminescenceWe chose Oh group as the control group, the production of progesterone was significantly enhanced at6h,12h and24h, while there was no difference between the groups of the estradiol levels. Meanwhile, compared with the0ng/ml group,50ng/ml and250ng/ml PGE2significantly increased the production of progesterone, and the levels of estradiol were not different among the groups.2Results of Real-time PCRThe expression of StAR mRNA was significantly enhanced at6h and12h, while there was no difference between the24h group and the0h group. Meanwhile, compared with the Ong/ml group,50ng/ml and250ng/ml PGE2significantly increased the expression of StAR mRNA. However, the expression of CYP19A1mRNA was not different among the groups.3Results of In-cell westernThe expression of StAR protein was significantly enhanced at6h, while both the12h and24h groups were not increased compared to the0h group. Meanwhile, compared with the0ng/ml group,50ng/ml and250ng/ml PGE2significantly increased the expression of StAR protein. However, the expression of CYP19A1mRNA was not different amone these groups.4Results of Immuocytochemistry After intervention of50ng/ml PGE2for6hours, both StAR and CYP19A1mainly expressed in cytoplasm, furthermore, the expression was increased, while there was not any difference of the CYP19A1expression between the two groups.Conclusions:1PGE2can increase the production of progesterone and the expression of StAR, but not the production of estradiol and the expression of CYP19A1.2PGE2(50ng/ml and250ng/ml) can significantly increase the production of progesterone and the expression of StAR after6hours intervention.Part ⅡEffect of PGE2pathway on promoting the production of progesterone and expression of StAR in human granulosa lutein cellsObjects:To investigate the effect of ERK1/2, AKT and NF-κB pathways within the process of PGE2promoting the production of progesterone and the expression of StAR.Methods:Ovarian luteinized granulosa cells were obtained and incubated for48hours. Subsequently, cells were treated with U0126and divided into four groups:Control group, U0126group, PGE2group and PGE2+U0126group, or cells were treated with LY294002and divided into four groups:Control group, LY294002group, PGE2group and PGE2+LY294002group, or cells were treated with PDTC and divided into four groups:Control group, PDTC group, PGE2group and PGE2+PDTC group. By using Chemoluminescence, Real-time PCR and In-cell Western methods, we have detected the effect of PGE2on production of progesterone and the expression of StAR.Results:1Results of ChemoluminescenceAfter treatment with U0126, the production of progesterone in U0126group was increased significantly compared with the control group, and the production of progesterone in PGE2+U0126group was also increased than the PGE2group; After treatment with LY294002, the production of progesterone in LY294002group was decreased significantly compared with the control group, and the production of progesterone in PGE2+LY294002group was also decreased than the PGE2group; After treatment with PDTC, the production of progesterone in PDTC group was decreased significantly compared with the control group, however, there was not any difference between PGE2+PDTC group and the PGE2group.2Results of Real-time PCRAfter treatment with U0126, the expression of StAR mRNA was significantly enhanced in U0126group compared with the control group, while there was not any difference between PGE2+PDTC group and the PGE2group; After treatment with LY294002, the expression of StAR mRNA in LY294002group was decreased significantly compared with the control group, and the StAR mRNA expression in PGE2+LY294002group was also decreased than the PGE2group; After treatment with PDTC, the expression of StAR mRNA in PDTC group was decreased significantly compared with the control group, however, there was not any difference between PGE2+PDTC group and the PGE2group.3Results of In-cell westernAfter treatment with U0126, the expression of StAR protein was significantly enhanced in U0126group compared with the control group, and the expression of StAR protein in PGE2+U0126group was also increased than the PGE2group; After treatment with LY294002, the expression of StAR protein in LY294002group was decreased significantly compared with the control group, and the StAR protein expression in PGE2+LY294002group was also decreased than the PGE2group; After treatment with PDTC, the expression of StAR protein in PDTC group was decreased significantly compared with the control group, while the StAR protein expression in PGE2+PDTC group was not decreased significantly.Conclusions: 1PGE2can increase the production of progesterone and the expression of StAR mainly through PI3K/AKT pathway.2ERK1/2and NF-κB pathways can regulate the production of progesterone and the expression of StAR, but PGE2promotes the production of progesterone and the expression of StAR not mainly through the two pathways.PartⅢ levels of PGE2and the associated steroidogenesis in follicular fluid from women with moderate and severe endometriosisObjects:To investigate levels of PGE2, estradiol and progesterone, and the correlation between PGE2and the steroid hormone in follicular fluid and envalue the influence of PGE2on the ovarian function.Methods:Follicular fluid was obtained from40tubal-factor infertile women and33endometriosis infertile women underwent IVF-ET. Then levels of PGE2, estradiol and progesterone, and the correlation between PGE2and the steroid hormone in follicular fluid were detected, in addition, the outcomes of IVF-ET in women with endometriosis were also evalued.Results:1Clinical characteristics for women with endometriosis and tubal factor infertilityThere were no difference between the two groups about the ages, body mass index, levels of estradiol and FSH on the third day of the cycle, and the total amount of GnRH, FSH and hMG.2IVF outcomesWomen with endometriosis developed fewer follicles, and picked-up less oocytes, had lower concentrations of E2in serum on the day of hCG injection compared to the control. However, the fertilization rate was similar in both endometriosis and control group.3Follicular fluid concentrations of PGE2, estradiol and progesteroneIn follicular fluid, the concentration of PGE2in the women with endometriosis was significantly higher than the control. Meanwhile, the women with endometriosis had significantly elevated production of progesterone. In contrast, the concentration of oestradiol was decreased in the women with endometriosis in follicular fluid compared to the control. PGE2concentrations displayed a positive association with progesterone concentrations in follicular fluid at oocyte retrieval both in the women with or without endometriosis. However, no significant correlations were observed between PGE2and estradiol in the two groups.Conclusions:1PGE2and progesterone concentrations are increased in follicular fluid in endometriotic women compared with control patients, and they display a positive association both in the women with or without endometriosis, indicating high levels of PGE2in follicular fluid promoting the lutinization of granulosa cells of women with endometriosis.2Women with endometriosis develope fewer follicles, pick-up less oocytes, and have lower concentrations of E2in serum on the day of hCG injection compared to the control, which suggeste the decreased responsiveness of the ovary in women with endometriosis.PartIV The effects and molecular mechanisms of PGE2on steroidogenesis in granulosa lutein cells in women with moderate and severe endometriosisObjects:To detected the effects of PGE2on the expression of steroid hormone and the associated enzymes in the endometriotic granulosa cells, and to evaluat the alteration of PGE2pathways in endometriosis granulosa cells.Methods:Ovarian granulosa lutein cells were obtained from40tubal-factor infertile women and33endometriotic infertile women underwent IVF-ET. Cells were incubated for48hours. Then, these cells were divided into four groups:Control group, PGE2group, EMs group and EMs+PGE2group. By using Chemoluminescence, Real-time PCR and In-cell Western, we detected the effect of PGE2on the production of progesterone and the expression of StAR in endometriotic granulosa cells. Subsequently, the ERK1/2, AKT and NF-κB protein expression were detected in both control and endometriotic granulosa cells.Results:1Results of ChemoluminescenceThe EMs group had a reduced production of estradiol but an elevated level of progesterone compared with the control. The EMs+PGE2group had an increased production of progesterone than the EMs group, and the production of progesterone in EMs+PGE2group was elevated compared to the PGE2group as well. However, there was not any difference between the groups about the production of estradiol.2Results of Real-time PCRThe EMs group had a reduced expression of StAR mRNA, but an elevated expression of CYP19A1mRNA compared with the control. The EMs+PGE2group had an increased expression of StAR mRNA than the EMs group, and the expression of StAR mRNA in EMs+PGE2group was also elevated compared to the PGE2group. However, there was not any difference between the groups about the expression of CYP19A1mRNA.3Results of In-cell westernThe EMs group had a reduced expression of StAR protein, but an elevated expression of CYP19A1protein compared with the control. The EMs+PGE2group had an increased expression of StAR protein than the EMs group, and the expression of StAR protein in EMs+PGE2group was also elevated compared to the PGE2group. However, the expression of CYP19A1protein was not different between the groups.Moreover, the expression of ERK1/2and p-ERK1/2protein were significantly decreased in endometriotic granulosa cells compared with the control; the expression of AKT was significantly increased in endometriotic granulosa cells, but the p-AKT protein was not significantly different between the endometriotic granulosa cells and the control; the expression of NF-κB P65and p-NF-κB protein were significantly decreased in endometriotic granulosa cells compared with the control.Conclusions:1There are abnormal production of steroid hormone in endometriotic granulosa cells, and PGE2can further promote the production of progesterone and the expression of StAR, suggesting the ability of the endometriotic granulosa cells on steroidogenesis was not damaged.2In the pathways of PGE2, the ERK1/2and NF-κB pathways are down regulated, while the PI3K/AKT pathway is significantly increased in endometriotic granulosa cells, which suggeste that the function of PGE2promoting the expansion of granulosa cells is damaged, rather than promoting the production of progesterone in granulosa cells from women with endometriosis.