Effect Of GOLPH3 On Apoptosis Of Granulosa Cells And Controlled Ovarian Hyperstimulation Outcomes In Patients With Polycystic Ovary Syndrome

Object: The present study was designed with aims to explore the effect of Golgi phosphoprotein-3(GOLPH3)in granulosa cells of patients with polycystic ovary syndrome(PCOS)during controlled ovarian hyperstimulation(COH)cycles on apoptosis of granulosa cells and pregnancy outcomes,so as to provide theoretical and experimental evidence for increasing the success rate of pregnancy with PCOS patients through the investigations on the biological behavior of GOLPH3 and apoptosis of granulosa cells.Methods: Seventy-eight eligible IVF-ET cases with PCOS owing to the tubal factors and 78 corresponding cases with non-PCOS were included.Follicular fluid containing granulosa cells were collected after oocyte retrieval followed by controlled ovarian hyperstimulation.Immunocytochemistry was used to detect localization and the protein expression of GOLPH3,apoptosis-related BCL-2 family(BCL-2,BAX),Caspase family(Caspase-3)in granulosa cells.Real-time PCR and Western blotting were used to detect the m RNA and protein expression of GOLPH3,BCL-2,BAX and Caspase-3 in granulosa cells of the two groups,respectively.Meanwhile,the correlations of GOLPH3 expression with cells apoptosis-related factors and pregnancy outcomes were analyzed.Receiver operating characteristic curve was analyzed to evaluate the accuracy of GOLPH3 in predicting clinical pregnancy.Results: 1.There were no significant differences seen in female age,duration of sterility,basal serum FSH,E2 and PRL level between control and PCOS group(all P values>0.05).BMI,number of baseline antral follicles count,basal serum LH,T and serum AMH level in PCOS were significantly higher than those of control group(all P values<0.05).There were no significant differences seen in serum LH and T level between two groups after treatment of Diane-35 and metformin(all P values>0.05).Althrough the number of oocytes retrieved and the rate of embryo implantation cancelling in PCOS were significantly higher(all P values<0.01),the oocyte retrieved rate,fertilization rate,high-quality embryos rate,embryo implantation rate and clinical pregnancy rate were lower than those of control group(all P values>0.05).The rate of early abortion was higher than that of the control group(P value>0.05).2.GOLPH3 protein was found to be predominantly expressed in cytoplasm.The expression of GOLPH3 protein and GOLPH3 m RNA in the PCOS group was reduced comparing to control group(all P values<0.05).Compared with the control group,the expression of BCL-2 protein and BCL-2 m RNA in PCOS group was decreased,while the expression of BAX protein,Caspase-3 protein,BAX m RNA and Caspase-3 m RNA levels were increased(all P values<0.05).3.There were 12 cases of clinical pregnant and 13 cases of non-pregnant in PCOS group,and a higher GOLPH3 protein and m RNA level was detected in pregnant group than in non-pregnant group(all P values<0.05).Compared with the non-pregnant group,the expression of BCL-2 protein and m RNA level in pregnant group was increased(all P values<0.05).However,a decrease in the expression of BAX,Caspase-3 protein and m RNA level were observed in pregnant group(all P values<0.05).There were 23 cases of clinical pregnant and 22 cases of non-pregnant in control group,and a higher GOLPH3 protein and m RNA level of granulosa cells was detected in pregnant group than in non-pregnant group(all P values<0.05).Compared with the non-pregnant group,the expression of BCL-2 protein and m RNA level in pregnant group was increased(all P values<0.05).However,a decrease in the expression of BAX,Caspase-3 protein and m RNA level were observed in pregnant group(all P values<0.05).Compared with the pregnant group in control group,a lower GOLPH3 protein and m RNA level of granulosa cells was detected in pregnant group of PCOS group(all P values>0.05).The expression of GOLPH3 protein and m RNA level was lower in non-pregnant group of PCOS group than in non-pregnant group of control group(all P values<0.05).4.Pearson correlation analyses revealed that the expression of GOLPH3 protein and m RNA level in granulosa cells was correlated positively with number of zygotes,number of cleavage-stage embryos,number of top-quality embryos,number of top-quality blastocysts number of frozen embryos(all P values<0.05).The expression of GOLPH3 protein level was correlated positively with the expression of BCL-2 protein(P value<0.001),while was correlated negatively with the expression of BAX and Caspase-3 protein(all Pvalues<0.001).Multivariate linear regression analysis showed that a higher GOLPH3 protein expression was associated with more top-quality embryos(P<0.001).Receiver operating characteristic curve discovered that both GOLPH3 protein and GOLPH3 m RNA in granulosa cells can predict clinical pregnency(AUC were 0.808,0.706,respectively,all Pvalues<0.01).Conclusions: 1.The results of the study demostated that the rate of normalfertilization,high quality embryo,the rate of embryo implantation and clinical pregnancy of PCOS group were lower than those in control group,while the expression level of apoptotic factor in granulosa cells of PCOS group were higher,which indicates the oocytes quality decline in PCOS group.2.The expression of GOLPH3 protein and GOLPH3 m RNA in the PCOS group were reduced comparing to control group,the expression of BCL-2 protein and m RNA in PCOS group was decreased,while both BAX,Caspase-3 proteins and m RNA expression were increased,the expression of GOLPH3 protein in granulosa cells was correlated positively with the expression of BCL-2 protein,while was negatively with the expression of BAX and Caspase-3 protein.It’s suggested that a decrease in the expression of GOLPH3 level could increase the apoptosis rate of granulosa cells through BCL-2,BAX and Caspase-3 signaling pathways.Its mechanism might be by regulating anti-apoptotic gene BCL-2 and apoptosis gene BAX to promote the apoptosis of granulosa cells and affects the developmental potential of oocytes.3.Higher expression levels of GOLPH3 predicted better pregnancy outcome.The expression of GOLPH3 protein in granulosa cells will valuably predict clinical pregnancy rate.