Differential Proteomics Studies Of Plasma-derived Exosomes From Patients With Polycystic Ovary Syndrome

Part 1BackroundPolycystic ovary syndrome(PCOS)is a common cause of anovulatory infertility in women of childbearing age,which seriously affects the patient’s reproductive status,quality of life and long-term health.According to statistics,the global incidence rate is 6% to 10%,showing a trend of increasing year by year.The high heterogeneity of the etiology and clinical manifestations of PCOS has led to many controversies in its clinical diagnosis and treatment options.In the past,most comparative PCOS proteomics studies were based on ovarian tissue or plasma.Exosomes are the medium for the exchange of material and energy information between cells,and play an important role in various pathophysiological processes of the body.However,there were no relevant reports of PCOS-related differential proteomics study with plasma exosomes.ObjectivesTo screen the differential proteome of plasma exosomes associated with of PCOS,and to deepen the understanding of the molecular mechanism of PCOS from the perspective of plasma exosomes.MethodsPlasma samples were collected from PCOS with hyperandrogenemia(PCOSHA),PCOS with non-hyperhomogenemia(PCOS-NHA)and healthy controls(Control).Plasma exosomes were extracted by differential centrifugation,and differential proteomics analysis was performed by Label-free comparative proteomics platform to screen differential proteomes that may be related to the molecular mechanism of PCOS,furthermore,bioinformatics analyses of differential proteins were performed.ResultsPlasma exosomes were successfully extracted,and 51 differential proteins were screened in PCOS-HA/Contol,additionly,39 and 59 were detected in PCOSNHA/Control and PCOS-NHA/PCOS-HA.After Wayne’s analysis,it was found that8 specific differential proteins of PCOS-HA/Contol,6 in PCOS-NHA/Contol and 11 in PCOS-NHA/PCOS-HA.GO functional annotation analysis found that differential proteins between the three groups are mainly involved in biological processes such as biological regulation,cellular processes and stress responses,distributed in subcellular organs or extracellular regions,and have molecular functions such as binding activity,molecular regulation activity and catalytic activity..GO clustering enrichment analysis showed that differential proteins of PCOS-HA/Control were mainly enriched in biological processes such as cell processes,tissue organization and regeneration processes,and immune system processes;differential proteins of PCOSNHA/Control were mainly enriched in regulation process,cellular processes and metabolic processes according to the biological process,and are mainly enriched in binding activity according to molecular function;differential proteins of PCOSNHA/PCOS-HA are mainly enriched in the immune system according to biological processes,and are mainly enriched in catalytic activity according to molecular functions.For KEGG annotation of differential proteins of PCOS-HA/Control and PCOS-NHA/Control are mainly enriched in cell signaling pathways of human papillomavirus(HPV)-infected;the varied proteins of PCOS-NHA/PCOS-HA are mainly enriched in the PI3K-Akt cell signaling pathway.The prediction of String interactions revealed that most of the differential proteins may have direct or indirect interactions.Furthermore,our study has screened three proteins that may play important regulatory functions in the pathophysiology of PCOS,including extracellular matrix protein 1(ECM1),C-reaction protein(CRP),and fibronectins(FN1).However,further experimental evidence is still needed.Conclusions1.Our study have successfully screened the differentially expressed proteins of plasma exosomes associated with the pathogenesis of PCOS-HA/PCOS-NHA,and these changed proteins were mainly enriched in the PI3K-Akt cell signaling pathway,providing experimental data for further study of the pathogenesis of PCOS;2.This study also screened out the differential expressed proteins specific to PCOS-NHA or PCOS-HA.Whether these differential proteins can be used as biomarkers for differential diagnosis of PCOS still requires more experimental verification.Part 2BackroundDue to the extremely long time course of proteomics analysis,including a large number of interference factors affecting the experimental results,it is very easy to cause false positive or false negative phenomenon.Therefore,after comparing the differential proteins selected by proteomic experiments,other techniques are still needed to validate the protein quantification to confirm the reliability and repeatability of proteomic experiments.Enzyme-linked immunosorbent assay(ELISA)is the most commonly used technique for protein quantification of high-throughput samples of tissue fluid.ObjectivesTo assess the reliability of proteomic experimental results with other protein quantification techniques.MethodsTwo differential plasma exosomal proteins of PCOS-HA/Control-specific,namely Zinc-alpha-2-glycoprotein(AZGP1)and Thyroxine-binding globulin(SERPINA7),were selected and verified by ELISA experiments.ResultsCompared with Control group,ELISA experiments showed that the expression levels of AZGP1 and SERPINA7 in plasma exosomes of PCOS-HA and PCOS-NHA groups were consistent with the proteomic results.Conclusions1.The results of the ELISA experiment confirmed the reliability of the proteomic experiment results;2.The expression levels of AZGP1 and SERPINA7 in plasma exosomes of patients with PCOS-HA are significantly increased,and these differential proteins may become specific plasma exosome molecular markers for differential diagnosis of PCOS patients.