Analysis Of Gene Expression Profile And Recurrent Implantation Failure Of Polycystic Ovary Syndrome With Different Androgen Levels

ObjectivePolycystic ovary syndrome(PCOS)is the most common reproductive disease bothering women of child-bearing age.Hyperandrogenism,polycystic ovaries,and oligoor an-ovulation are the typical clinical characteristics of PCOS.Androgen excess has been proven to cause the deterioration of insulin resistance and metabolic disorders which are correlated with poor pregnancy outcome including recurrent spontaneous abortion(RSA)and embryo recurrent implantation failure(RIF).Ovarian granulosa cells serving as endocrine cells producing hormones in the ovary is critical for folliculogenesis and oocyte maturation.Hyperandrogenism involves aberrant gene expression and modification processes,inducing ovarian function deficiency.Poor pregnant outcomes of PCOS are closely correlated with hyperandrogenism and ovarian granulosa cell dysfunction.However,molecular mechanisms underlying this correlation have not been fully elucidated.Our study is aimed to explore gene expression profiles of ovarian granulosa cells of PCOS with or without hyperandrogenism,screen and select prognostic biomarkers for pregnancy outcomes of PCOS patients,providing the basis for therapeutic targets and improving the prognosis of PCOS.MethodsUsing Gene Expression Omnibus(GEO)database to download microarray datasets of PCOS and RIF.Differentially expressed genes(DEGs)were analyzed and we performed GO(Gene Ontology)enrichment analysis of DEGs.Co-expressed modules and shared genes of PCOS with hyperandrogenism were selected through Weighted CoExpression Network Analysis(WGCNA)analysis.Shared gene signatures of hyperandrogenic PCOS and RIF were compared with DEGs in PCOS and RIF.Prognostic biomarkers of PCOS with hyperandrogenism were selected.We collected clinical data and samples of ovarian granulosa cells of 13 PCOS patients with hyperandrogenism,12 PCOS patients with non-hyperandrogenism,and 13 healthy controls and validated targeted gene expression.CIBERSORT algorithm analyzed immune cell distribution and immune infiltration of PCOS patients.ResultsThe differential analysis of 271 DEGs in granulosa cells of PCOS with hyperandrogenism was selected by differential analysis.Enrichment analysis showed that DEGs in hyperandrogenic PCOS were related to terms of myeloid leukocyte activation,cytokine production regulation,inflammatory response,and immune receptors activation.720 DEGs in non-hyperandrogenic PCOS were related to terms of extracellular space and mesenchymal cell.WGCNA analysis proved that there were 26 co-expressed genes in hyperandrogenic PCOS and RIF,which enriched in GO terms of bone maturation,immune regulation,and corticosteroid metabolism.In those 26 genes,according to the correlation analysis of genes and enrichment pathways,we found that the five genes which most closely related to the enriched biological functional pathways are ALPL,ANXA3,IDH1,RFLNB,and LRG1.DAPK2.Furtherly,co-expressed genes that were upregulated in both hyperandrogenic PCOS and RIF datasets were screened.DAPK2 was selected and verified by real-time quantitative fluorescence PCR(q RT-PCR).The expression of the DAPK2 gene was negatively correlated with embryo implantation(r=-0.474,P=0.003)and acted as an independent risk factor of embryo implantation through logistic analysis.Immune infiltration analysis showed upregulation of eosinophils and NK cell resting,while downregulation of NK cells activated and T cells CD4 memory resting in hyperandrogenic PCOS versus non-hyperandrogenic PCOS.ConclusionsIn this study,the gene expression features in ovarian granulosa cells of PCOS patients with different androgen levels were analyzed by bioinformatics methods.The differentially expressed genes in ovarian granulosa cells of hyperandrogenic PCOS are mainly involved in the inflammatory immune response pathway,while the differentially expressed genes in non-hyperandrogenic PCOS ovarian granulosa cells are related to cell growth and development.This study identified 26 key genes involved in the occurrence of RIF in hyperandrogenic PCOS patients.The overexpression of the DAPK2 gene was first found closely related to RIF in hyperandrogenic PCOS,providing the basis for improving the prognosis of PCOS patients.