Study Of Exosomal MiRNA Expression Profile In Follicular Fluid Of Premature Ovarian Insufficiency

ObjectivePremature ovarian insufficiency(POI)refers to the ovarian function decline or even failure that appears before the age of 40,leading to infertility,seriously endangers women’s reproductive health.At present,no specific biomarker for diagnosis of POI has been found.Previous studies have found that a large number of miRNAs in female follicular fluid are enriched in exosomes,which are protected by exosome membrane and have good stability,and thus serve as an ideal molecular marker.However,the expression of exosomal miRNAs in follicular fluid of POI patients has not been reported.Therefore,High-Throughput Sequencing technology is applied to explore exosomal miRNAs expression in follicular fluid of POI patients,screening differentially expressed exosomal miRNAs,and understanding of its function,help to elucidate the molecular mechanism of POI disease,research and development of new diagnostic methods,new diagnostic markers and effective method is of great significance for the treatment of POI.Methods1.We recruited 5 patients in POI group and 5 patients in control group and treated with IVF/ICSI-ET,were collected for follicular fluid.Exosomes were isolated from the follicular fluid of the POI group and the control group by hypervelocity centrifugation,and the morphology of exosomes was observed by transmission electron microscopy.Nanoparticle Tracking Analysis(NTA)was used to analyze the particle size of exosomes,and the expression of signature proteins on the surface of exosomes was detected by flow cytometry to identify exosomes.2.The follicular fluid exosomal miRNA of the POI group and the control group were detected by High-Throughput Sequencing technology,and the expression profile of exosomal miRNA in follicular fluid of POI patients was analyzed,and the differentially expressed miRNA was screened out and bioinformatics analyzed.3.Exosomal miR-21-3p,miR-486-3p and miR-6511b-5p were selected from the differentially expressed miRNAs of the two groups,and the sample volume was increased.The expressions of the above three exosomal miRNAs in follicular liquid and plasma were explored and verified by real-time quantitative PCR(qRT-PCR),and the differentially expressed miRNAs were analyzed by receiver operating characteristic(ROC).Results1.Transmission electron microscopy successfully observed that exosomes were isolated from the follicular fluid of POI and control.The diameter of exosomes was within 30-200nm,and the surface marker proteins CD63 and CD 81 of exosomes were positive.Compared with the control group,the morphology,size and density of the exosomes in follicular fluid of POI patients were not significantly different,but the content of exosomal miRNA in POI group was less than that in the control group(16.1%vs 24.3%).At the same time,the types of exosomal miRNAs are also reduced(1142 vs 1269).2.A total of 1410 known miRNAs were detected by High-Throughput Sequencing,among which 26 exosomal miRNAs showed statistical differences.Among them,6 exosomal miRNAs with high expression and 20 exosomal miRNAs with low expression were detected in POI follicular fluid.Bioinformatics analysis of differentially expressed exosomal miRNAs indicated that PI3K-Akt,Hippo,MAPK,Hedgehog,TGF-β and other signaling pathways may be involved in the pathogenesis of POI.3.Real-time quantitative PCR results showed that exosomal miR-21-3p,miR486-3p and miR-6511b-5p were all low expressed in POI follicular liquid,but there was no significant difference in the expression levels of the three exosomal miRNAs in plasma compared with the control group.ROC curve analysis showed that exosomal miR-21-3p,miR-486-3p and miR-6511b-5p in follicular liquid had good sensitivity and specificity for the identification of POI diseases.Conclusions1.Exosomes in the follicular fluid of POI patients have normal shape,size and density,but the content and type of miRNAs carried by exosomes is reduced,which may be related to reduced information communication between cells in follicle.2.Based on High-Throughput Sequencing technology,differential exosomal miRNA analysis was performed in follicular fluid of POI patients,and the exosomal miRNA expression profile in follicular fluid of POI patients was successfully established.3.The low expression of exosomal miR-21-3p,miR-486-3p and miR-6511b-5p in POI disease may be the specific changes in follicular fluid,and the three exosomal miRNAs have the potential to become diagnostic markers of POI disease.Exosomal miR-21-3p may help predict the progression of POI disease.