The Role And Mechanism Of IRF9 In The Pathogenesis Of Polycystic Ovary Syndrome And Follicular Fluid Proteomics Study

BACKGROUNDPolycystic ovary syndrome(PCOS)is one of the most common endocrine and metabolic disorders,afflicting 5%-20%of women of childbearing age worldwide.The pathogenesis of PCOS is still unclear.PCOS is one of the leading causes of anovulatory infertility,associated with impaired oocytes,embryos,and endometrial competence,resulting in reduced fertility potential.Patients with PCOS usually exhibit varying degrees of obesity,dyslipidemia,insulin resistance,glucose metabolism abnormalities,and metabolic syndrome,which may interact with each other and harm the follicular environment.The coordinated interaction between granulosa cells(GCs)and follicular fluid is essential for maintaining a healthy follicular environment,which supports oocyte development.Therefore,GCs and follicular fluid are critical determinants of oocyte quality,thus affecting the fertility of patients with PCOS.During folliculogenesis,GCs and oocytes engage in bidirectional nutrient and paracrine signaling.Follicular fluid,as a complex microenvironment for oocyte growth and follicle maturation,is a medium for communication between oocytes and GCs.Therefore,understanding the changes in the function of GCs and protein levels in the follicular fluid of patients with PCOS can improve the understanding of the pathogenesis of PCOS and may provide new ideas for improving fertility in patients with PCOS.Our team performed high-throughput sequencing of GCs from patients with PCOS to screen for differentially expressed genes.Meanwhile,previous studies have suggested that differentially expressed proteins in the follicular fluid of patients with PCOS may be involved in the pathogenesis of PCOS and may serve as diagnostic biomarkers.However,due to the limitations of previous proteomic techniques,relatively few differentially expressed proteins were detected.With the improvement of proteomics technology,such as isobaric mass labeling technology,the throughput of protein identification has been improved and biomarkers can be found more effectively.In addition,the risk factors that affect clinical pregnancy loss in patients with PCOS are not fully understood.Our team had previously conducted a large multicenter randomized trial comparing the live birth of fresh embryo transfer versus elective frozen embryo transfer(FET)in women with PCOS.We found that frozen embryo transfer resulted in a lower risk of clinical pregnancy loss than fresh embryo transfer.However,whether other risk factors associated with clinical pregnancy loss in women with PCOS are still unclear.Therefore,in this study,we explored the pathogenesis of PCOS from the perspectives of GCs and follicular fluid,respectively.We investigated the effect of interferon regulatory factor 9(IRF9),which was selected by high-throughput sequencing screening,on the function of ovarian GCs and possible mechanisms.We also attempted to identify differentially expressed proteins in the follicular fluid of patients with PCOS through tandem mass tag(TMT)-based proteomics technology to explore their roles in the pathogenesis of PCOS.In addition,we also studied the reduced fertility caused by PCOS from a clinical perspective and investigated the risk factors for clinical pregnancy loss after in vitro fertilization in patients with PCOS,thus providing a basis for clinical management.Part 1 Study on the role and mechanism of IRF9 in polycystic ovary syndromeObjective:We found increased expression of IFR9 in ovarian granulosa cells of patients with PCOS through high-throughput sequencing.As a transcription factor,IRF9 can mediate interferon activation and regulate cell growth,differentiation,apoptosis,and modulate immune system activity.Various studies have shown abnormalities in proliferation and apoptosis in ovarian GCs of patients with PCOS,but there is no relevant research on the role of IRF9 in this process.This part of the study aimed to explore the effect of IRF9 on the function of ovarian GCs and its possible mechanism.Methods:A total of 72 patients with PCOS and 74 control patients were included in this part of the study.The mRNA and protein expression of IRF9 in ovarian GCs were detected by real-time quantitative PCR and Western blot.Spearman correlation analysis was used to analyze the correlation between the expression level of IRF9 and clinical characteristics.To identify the role of IRF9 in the function of ovarian GCs,we constructed IRF9 overexpression and knockdown human ovarian granulosa cell lines by overexpression lentivirus infection and specific siRNA transfection of KGN and SVOG cells,respectively.CCK-8 and EdU staining assays were employed to detect the role of IRF9 on the cell proliferation of GCs.Flow cytometry was performed to analyze the effect of IRF9 on the cell cycle and apoptosis of GCs.We conducted RNA sequencing(RNA-seq)and chromatin immunoprecipitation followed by high-throughput sequencing(ChIP-seq)after overexpressing IRF9 in KGN cells using lentiviral to screen for downstream target genes directly bound by IRF9.Changes in the expression of downstream genes were verified after overexpression and knockdown of IRF9 in KGN and SVOG cells.Dual-luciferase reporter gene assay was performed to detect the regulation effect of IRF9 on the promoter region of the downstream target gene.At the same time,the ChIPPCR experiment was used to detect the enrichment of IRF9 in the promoter region of the downstream target gene.We examined the effects of the target gene on the function of proliferation,cell cycle distribution,and apoptosis in ovarian GCs after overexpression and knockdown of the target gene in KGN and SVOG cells.Rescue experiments were performed by knocking down the target gene in IRF9 lentiviral-transfected KGN cells to explore whether IRF9 affects the function of GCs by mediating the target gene.Protein expression of CCND2 and BCL2 downstream of the IRF9-target gene pathway was examined by Western blot.The effect of dihydrotestosterone(DHT)on the expression of IRF9 and target gene was detected in KGN cells and IRF9 siRNA-transfected KGN cells by qRT-PCR and Western blot.Results:We found that the mRNA and protein expression levels of IRF9 were significantly higher in ovarian GCs of patients with PCOS than in the control group.The relative expression level of IRF9 mRNA was positively correlated with serum AMH、T and DHE-s.We found that overexpression of IRF9 could promote the G1/S phase transition of the cell cycle,thereby promoting granulosa cell proliferation and inhibiting apoptosis,while the knockdown of IRF9 blocked the G1/S phase progression of the cell cycle,inhibited granulosa cell proliferation,and promoted apoptosis.Combined with RNA-seq and ChIP-seq analysis,GATA4 binding protein 4(GATA4)was screened as a direct downstream target gene of IRF9 binding to its promoter region.Overexpression of IRF9 in KGN and SVOG cells led to increased expression of GATA4,while knockdown of IRF9 expression resulted in decreased GATA4 expression.ChIP-PCR and dual-luciferase reporter assays confirmed that IRF9 could bind to the GATA4 promoter region and promotes its transcription.These findings demonstrated that GATA4 was a direct target of IRF9.Overexpression of GATA4 in KGN and SVOG cells was found to promote granulosa cell proliferation,promote G1/S phase transition,and inhibit apoptosis,while knockdown of GATA4 suppressed cell proliferation by arresting Gl/S phase progression and induced cell apoptosis.In KGN cells with stable overexpression of IRF9,the knockdown of GATA4 partially rescued the increase in cell proliferation and decrease in apoptosis induced by IRF9 overexpression.We also found that the IRF9-GATA4 pathway can promote the expression of CCND2 and BCL2.In addition,DHT treatment significantly increased the expression of IRF9 and GATA4 in KGN cells,and the upregulation of GATA4 expression induced by DHT was partially mediated by IRF9.Taken together,these findings demonstrated that IRF9 might affect the function of granulosa cells by regulating GATA4,indicating its potential role in the pathogenesis of PCOS.Conclusion:The expression level of IRF9 was significantly increased in ovarian GCs of patients with PCOS.Abnormal expression of IRF9 is mainly associated with characteristic hormonal changes of PCOS.Overexpression of IRF9 promoted the proliferation and inhibited apoptosis of ovarian GCs.The possible mechanism was that IRF9 directly binds to the GATA4 promoter region and promotes the expression of GATA4,which in turn promotes the G1/S cell cycle process and inhibits apoptosis.Part 2 Quantitative proteomics analysis of follicular fluid from patients with polycystic ovary syndromeObjectives:The pathogenesis of PCOS is still unclear.Although novel proteins and pathways potentially involved in the pathogenesis of PCOS have been found in recent years,it is still a challenge to identify the proteins that may contribute to the risk of developing PCOS.In the present study,we performed TMT-based quantitative proteomics technology and bioinformatics analysis to investigate the proteomic changes in follicular fluid of patients with or without PCOS to identify differentially expressed proteins(DEPs)that may be associated with the pathogenesis of PCOS.Methods:Follicular fluid samples were collected from infertile patients with(n=9)or without(n=9)PCOS.Total protein was extracted,quantitatively labeled with a tandem mass tag(TMT),and analyzed using liquid chromatography-mass spectrometry(LC-MS).TMTbased proteomics and bioinformatics analysis were used to determine the differentially expressed proteins and understand the protein networks.Selected differentially expressed proteins were confirmed by ELISA,and correlation analysis was performed between these DEPs and the clinical characteristics.In addition,the effects of platelet factor 4(PF4)on the function of GCs were examined using CCK-8,EdU assay,flow cytometry and immunofluorescence staining.Results:In this study,we have identified 1216 proteins,including 70 DEPs(32 upregulated proteins,38 downregulated proteins).Bioinformatics analysis revealed that the inflammatory response,complement and coagulation cascades,activation of the immune response,lipid transport,and regulation of protein metabolic processes were co-enriched in patients with PCOS.ELISA results demonstrated that the expression of PF4 was increased,while insulin-like growth factor binding protein 1(IGFBP1)and apolipoprotein C2(APOC2)expression levels were decreased in the follicular fluid of patients with PCOS.Treatment of KGN cells with different concentrations of PF4 revealed that PF4 did not affect the proliferation,apoptosis and DNA damage of KGN cells.Conclusions:Our study identified DEPs in the follicular fluid of patients with PCOS.We verified elevated levels of PF4 and decreased levels of IGFBP1 and APOC2 in the follicular fluid of patients with PCOS.Inflammatory response,complement and coagulation cascades,activation of the immune response,lipid transport,and regulation of protein metabolic process were dysregulated in PCOS,which may play essential roles in the pathogenesis of PCOS.Part 3 Clinical study on the risk factors for clinical pregnancy loss after in vitro fertilization in women with polycystic ovary syndromeObjective:To investigate which factors were associated with the risk of clinical pregnancy loss in women with PCOS who underwent IVF and whether the risk factors differ between fresh embryo transfer cycles and frozen embryo transfer cycles.Methods:A case-control study nested in a multicentre randomized trial comparing live birth rates between fresh and frozen embryo transfer(FET)in women with PCOS.Women with the outcome of clinical pregnancy loss were selected as the case group,those with live birth as the control group.Parameters before IVF treatment and variables during ovarian stimulation and embryo transfer were compared.Results:Women with clinical pregnancy loss had greater maternal body mass index(BMI),more antral follicle count(AFC),higher level of 2-h glucose level after 75g oral glucose tolerance test(OGTT),and a higher proportion of fresh embryo transfer.Moreover,there were significant interactions between the types of transfer and AFC,2-h glucose level after OGTT on clinical pregnancy loss in PCOS,which indicated these factors may have different effects on pregnancy loss after fresh vs.frozen embryo transfer.When the multivariable logistic regression analysis was stratified by the fresh or frozen embryo transfer,AFC was a risk factor for clinical pregnancy loss after fresh embryo transfer,while 2-hour glucose level after OGTT was associated with clinical pregnancy loss in FET cycles.Conclusions:In women with PCOS who underwent IVF,fresh embryo transfer,higher BMI,AFC,and 2-h glucose level after OGTT were risk factors for clinical pregnancy loss.FET may be a better choice to decrease the risk of clinical pregnancy loss,especially for those with more AFC.During FET,2-h glucose after OGTT seemed to be associated with clinical pregnancy loss and warranted close monitoring.