Influence Of Growth Hormone On Oxidative Stress Of Patients With Polycystic Ovary Syndrome Undergoing In Vitro Fertilization And Embryo Transfer

Objective:Polycystic ovary syndrome(PCOS)is a common cause of infertility for women of childbearing-age,and some of them may seek in vitro fertilization and embryo transfer(IVF-ET)treatment.However,the outcomes of IVF-ET for patients with PCOS are restrained by lower oocyte development potential and pregnancy rate,and higher miscarriage rate despite more oocytes retrieved.Previous studies have discovered oxidative stress(OS)in patients with PCOS,which may affect the quality of oocyte and IVF-ET outcomes.Growth hormone(GH)can function as an antioxidant to alleviate mitochondrial dysfunction and improve oocyte quality and IVFET outcomes for poor ovarian responders.However,the ability of GH to alleviate OS,and improve oocyte quality and IVF-ET outcomes in patients with PCOS has not been assessed in detail.The phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway is dysregulated in patients with PCOS.And GH can suppress OSinduced apoptosis in certain types of cells by activating the PI3K/Akt signaling pathway.As the effects of GH on the PI3K/Akt signaling of GCs in patients with PCOS have not been studied,this study was set to explore whether GH can improve OS and IVF-ET outcomes and the possible mechanism.Materials and Methods:A prospective randomized clinical trial enrolling 117 patients with PCOS whom underwent IVF-ET treatment was conducted at the Department of Reproductive Medicine,West China Second University Hospital,Sichuan University,between November 2018 and May 2020.The patients were randomly assigned to undergo treatment with(PCOS-GH,n = 57)and without(PCOS-C,n = 60)GH.Patients with tubal infertility who underwent IVF-ET treatment during the same peroid were settelled as the normal control group(non-PCOS,n = 59).The controlled ovarian stimulation(COS)protocol for all participants was Gn RH antagonist protocol.The PCOS-GH group was injected with an additional 4 IU/day of GH until the trigger day during the COS.Blood samples were collected before the COS and on the trigger day,and follicular fluid(FF)and granulosa cells(GCs)were collected during oocyte retrieval.OS markers in serum and FF were measured,which included malondialdehyde(MDA),superoxide dismutase(SOD),total antioxidant capacity(TAC),and total oxidant status(TOS).Reactive oxygen species(ROS)generation in GCs was estimated by fluorescence microscopy and spectrophotometry.GC apoptosis and mitochondrial membrane potential(MMP)were detected by flow cytometry following Annexin V-FITC/PI double-staining and JC-1 staining,respectively.The expression of apoptosis-related genes and proteins of PI3K/Akt signaling were determined by using quantitative reverse-transcription polymerase chain reaction(qRT-PCR)and western blotting(WB).The concentrations of active caspase-9 and active caspase-3 in GCs lysate were determined by using enzyme-linked immunosorbent assay(ELISA).The COS and IVF-ET outcomes,OS markers in serum and FF,ROS generation,mitochondrial function,and expression of apoptosis-related genes and proteins of PI3K/Akt signaling in GCs were determined and compared.Results:1.Compared with the non-PCOS group,oocyte maturation rate,normal fertilization rate,day 3(D3)embryo development rate,higher-quality D3 embryo rate,blastocyst development rate and higher-quality blastocyst rate were significantly lower in PCOS-C group(P < 0.05).Compared with the PCOS-C group,the oocyte maturation rate,normal fertilization rate,higher-quality D3 embryo rate,blastocyst development rate,higher-quality blastocyst rate,and the number of mature oocytes,normal fertilized oocytes,D3 embryos and higher-quality D3 embryos were significantly higher in the PCOS-GH group(P < 0.05).The implantation rate,clinical pregnancy rate,miscarriage rate,and live birth rate per fresh or thawed embryo transfer cycle did not differ significantly among the three groups(P > 0.05).2.Basal serum MDA,TOS,and OSI levels were higher in patients with PCOS,compared with those without PCOS(P < 0.05).MDA on human chorionic gonadotropin(HCG)day was significantly higher in the PCOS-C compared with the non-PCOS group(P < 0.05).OSI was significantly higher in the PCOS-C group compared with the non-PCOS and PCOS-GH groups(P < 0.05).During COS,serum levels of MDA,SOD,TOS,and OSI on the HCG day were increased,whereas TAC was decreased in three groups(P < 0.05).The changes of serum TAC,TOS,and OSI were significantly samller in the PCOS-GH group compared with the PCOS-C and non-PCOS groups(P < 0.05).3.The basal levels of serum MDA,TOS and OSI in patients with PCOS were positively correlated with that of FINS,HOMA-IR and TT(P < 0.05);and negatively correlated with that of SHBG(P < 0.05).No correlation was found between SOD and TAC levels with various metabolic and androgen parameters(P > 0.05).4.FF MDA,TOS and OSI were higher in the PCOS-C group compared with the PCOS-GH and non-PCOS groups(P < 0.05),while no significant difference was found between the PCOS-GH and non-PCOS group(P > 0.05).FF TAC was higher in the PCOS-GH group compared with the PCOS-C and non-PCOS groups(P <0.05),but no significant difference was found between the PCOS-C and non-PCOS groups(P > 0.05).No signigicant difference was found in FF SOD among the three groups(P > 0.05).5.Compared with the PCOS-C group,lower ROS levels and apoptotic rate,and higher MMP was found in the GCs of the non-PCOS and PCOS-GH groups(P <0.05);while no significant difference was found between the PCOS-GH and nonPCOS groups(P > 0.05).6.FF TOS,OSI and MDA were positively correlated with the ROS level and apoptosis(P < 0.05),while FF TOS and SOI were negatively correlated with MMP in the GCs(P < 0.05).FF TAC was negatively correlated with late apoptosis in GCs(P < 0.05).7.Compared with the PCOS-C group,the mRNA levels of FOXO1,Bax,caspase-9,and caspase-3 mRNA were significantly decreased,whereas the level of Bcl-2 was increased in the GCs of the PCOS-GH group(P < 0.05).p-PI3K/PI3 K,p-Akt/Akt,p-FOXO1/FOXO1 and Bcl-2 protein were significantly increased,whereas cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3 protein,and concentrations of active caspse-9 and active caspase-3 were decreased(P < 0.05).Conclusion:This study revealed systemic and ovarian OS state,GCs mitochondrial dysfunction,increased GC apoptosis and dysregulated PI3K/Akt signaling pathway,and decreased oocyte quality in patients with PCOS undergoing IVF-ET.GH can alleviate systemic and ovarian OS,GC mitochondrial dysfunction and apoptosis,activate PI3K/Akt signaling,and improve the quality of oocyte in these patients.