Serum Micro RNA Studies Of Follicular Maturation-Related Microvesicles In PCOS

Background:Polycystic ovary syndrome(PCOS)is one of the endocrine diseases that seriously affects the physical and mental health of PCOS patients.PCOS often leads to abnormal follicular development and maturation in women of childbearing age and is one of the main causes of female infertility.Microvesicles(Mvs),as new intercellular communication tools,carry miRNA and play an important role in pathophysiology through direct budding of plasma membrane,similar to the function of exosomes formed by plasma membrane invagements.There are a large number of mirnas with significant differential expression in serum exosomes of PCOS patients,which are closely related to PCOS disease and follicular development.Recent studies on PCOS microvesicles have found that the concentration of PCOS in circulating blood tends to increase.However,it is not clear whether mirnas involved in PCOS disease and follicular development exist in the microvesicles of PCOS patients.Circulating blood-derived microvesicles miRNA may be a new idea for the study of PCOS diseases.Objectives:1.To characterize PCOS serum microvesicles and detect their concentration levels.2.To establish PCOS serum microvesicles miRNA differential expression profiles for bioinformatics analysis3.To observe whether the PCOS-like phenotype could be induced in wild-type ICR female rats by tail vein injection of PCOS serum microvesicles.Methods:1.Serum samples from patients admitted to reproductive Center of Affiliated Hospital of Zunyi Medical University from May 2020 to June 2021 were collected.The experimental group consisted of 50 patients diagnosed with PCOS,and the control group consisted of 50 non-PCOS patients with infertility caused by simple tubal factors.2.The microvesicles were extracted by differential centrifugation.The morphology and scale of microvesicles were observed by transmission electron microscopy and nano-particle tracking analysis(NTA).The expression of CD63 was examined by western blotting.3.The miRNA expression profile of serum microvesicles was constructed by miRNAs high-throughput sequencing method,and the miRNAs with significant differential expression were verified by q RT-PCR.R software cluster Profiler was used to analyze GO and KEGG pathway and Reactome pathway of target genes with significantly differentially expressed miRNAs.4.8-9 weeks female wild-type ICR mice were injected with 100μL of serum microvesicle suspension from PCOS patients by tail vein injection.Continuous monitoring of estrous cycles;The ovary was taken 48h after injection,and the ovary was made into HE staining sections to observe the ovarian structure.Immunohistochemical methods were used to detect the expression and localization of NF-κB and WNT5A proteins associated with PCOS inflammation in the ovaries of female ICR mice.Serum microvesicles from PCOS patients were injected into experimental group,serum microvesicles from non-PCOS patients were injected into control group,and 100μL PBS was injected into blank group.Results:1.The serum microvesicles show circular or elliptic bilayer structures of different sizes,which can express the membrane surface antigen molecule CD63.The total concentration of nanoparticles in PCOS group was 1.87 times higher than that in control group(9.58×10~8Particles/m L in PCOS group and 5.12×10~8Particles/m L in control group).2.PCOS group and the control group of serum detected 730 miRNAs in micro vesicles,there are 19 significantly differentially expressed miRNAs micro vesicles(P<0.05,|-log2(Flod Change)|≥1.5),including 8 expression,11 expression down;A total of9 miRNAs with significant difference and 4 miRNAs with no difference in expression were verified by q RT-PCR,and the results showed that,The sequencing results of hsa-mi R-486-3p,hsa-mi R-125b-1-3p,hsa-mi R-885-3p and hsa-mi R-125b-2-3p were the same.3.Nineteen miRNAs with significantly differentially expressed microvesicles were screened out from the prediction database and 195 target genes were identified.These miRNAs may be involved in biological processes such as Wnt signaling pathway,cell cycle,and regulation of cell metabolism.The enrichment pathways include interleukin family,Nuclear factor kappaβ(NF-κB),MAPK signaling pathway,TGF-βsignaling pathway,cholesterol metabolism related pathways,etc.Inflammatory signaling pathway,insulin secretion regulation,cancer miRNA pathway and circadian rhythm and other pathways.4.Injection of serum microvesicles from PCOS patients could induce the disturbance of estrous cycle in female wild-type ICR mice,mainly manifested in prolonged estrous period or prolonged interphase.HE sections of ovary showed normal ovarian structure and normal follicle morphology in each group.NF-κB and WNT5A proteins were mainly located in the cytoplasm of female follicular granulosa cells,and NF-κB protein was positively expressed in the nucleus.WNT5A protein was positively expressed in both large and small ovarian follicles of female mice injected with PCOS serum microvesicles.NF-κB protein was expressed in cytoplasm and nucleus of granulosa cells of female ovarian follicles,and the color of positive signal from dark to light was as follows:PCOS group>control group>blank group.Conclusion:1.The concentration of serum microvesicles in PCOS group showed an upward trend,and there were more microvesicles with smaller diameter.2.There are significantly differentially expressed mirnas in serum microvesicles of PCOS patients,whose target genes are involved in the regulation of signaling pathways including inflammation,lipid metabolism,insulin resistance,circadian rhythm and cancer.These differentially expressed serum microvesicle mirnas may increase the risk of PCOS and may be involved in related genes that regulate follicular development.3.Serum-derived microvesicles from PCOS patients induced a mild PCOs-like phenotype in female wild-type ICR mice.