| Background and ObjectivePolycystic ovary syndrome?PCOS?is a common reproductive endocrine disorder in women of childbearing age.There are many clinical symptoms,involving in the menstrual sparseness,abnormal ovulation,polycystic ovarian,hyperandrogenism,obesity and insulin resistance,accompanied by many long-term complications,such as diabetes,hypertension,hyperlipidemia,cardiovascular diseases and so on,which seriously endanger women's health.At present,the pathogenesis of PCOS is not clear.Studies have shown that the pathogenesis of PCOS is related to heredity,environment,race,lifestyle and chronic low-grade inflammatory reaction.Exploring the possible pathogenesis of PCOS and finding an effective method to treat PCOS and delay long-term complications has become a hot topic in the field of reproductive medicine.MicroRNAs?microRNAs?are short-chain non-coding RNAs that regulate gene expression at the pre-translational level.MicroRNAs participate in various life activities and regulate cell proliferation and apoptosis.Abnormal expression level of microRNAs can cause disorders in the body's life activities,leading to cancer,immunity,endocrine and other diseases.Studies have shown that microRNAs may be associated with insulin resistance,follicular dysplasia,granulosa cell proliferation and apoptosis,and follicular hormone synthesis,and play a certain role in the occurrence and development of PCOS.Recently,exosomes have become a new research hotspot,and more and more studies on exosomes have been carried out.The exosome is a membranous vesicle with a diameter of 30-100 nm secreted by cells through endocytosis.It has a lipid bilayer membrane structure.It has a cup-shaped structure under the microscope.It contains many bioactive substances such as microRNAs,RNA,lncRNAs,lipids and proteins.It is widely distributed in serum,plasma,follicular fluid,ascites,saliva,urine and other body fluids.Exosomes,as a new intercellular communication medium,play an important role in cell communication by transferring proteins,microRNAs,and RNA to target cells.Therefore,the research on the role of exogenous microRNAs has become a new direction to explore the possible molecular mechanism of exogenous microRNAs in the occurrence and development of various diseases.In this study,we will start with the serum and follicular fluid exosome microRNAs of PCOS patients and screen out the differentially expressed serum exosome-derived microRNAs from PCOS patients through high-throughput sequencing and bioinformatics analysis,which will provide a theoretical basis for the diagnosis and pathogenesis of PCOS.At the same time,we will screen out the differentially expressed exosome-derived microRNAs from PCOS patients.MicroRNAs,a specific exosome of follicular fluid,provide a basis for exploring the molecular mechanism of embryo quality decline and abnormal ovulation in PCOS patients.Materials and methodsPatients diagnosed as PCOS according to Rotterdam criteria were selected as the experimental group.Patients with infertility due to simple fallopian tube factor were selected as the control group.With the approval of Henan Provincial People's Hospital Ethics Committee,patients who signed the informed consent form were selected to receive hormone therapy within six months.Peripheral blood was collected on the 2nd to 4th day of menstruation.Peripheral blood and the first tube of clear and non-blood-contaminated follicular fluid were collected at the same time.The collected samples were centrifuged at 3000 rpm for 20 minutes.The supernatant was taken and frozen in the refrigerator at-80 C for reserve.Subsequently,exosomes in serum and follicular fluid were extracted by Exoquick TM kit,and the size and morphology of the extracts were observed by transmission electron microscopy.The expression of exosome-specific protein markers CD63 and CD9 was detected by Western-Blot.The exosome microRNAs were extracted and purified by Exoquick TMM kit,and the microRNAs in serum and follicular fluid exosomes of PCOS group and normal group were analyzed by high throughput sequencing and bioinformatics.Differential expression of as was confirmed by qRT-PCR.SPSS22.0 software was used to input data,and t-test analysis was carried out.The measurement data in the experimental data were expressed as x+s?mean+standard deviation?.The difference between the two groups was statistically significant?P<0.05?.Results1.The extract isolated from serum and follicular fluid was found to be exosomes by transmission electron microscopy and Western-blot.2.High-throughput sequencing and bioinformatics analysis showed that there were 243 differentially expressed exo-miRNAs in serum,of which 115 were up-regulated and 128 were down-regulated.The results of qRT-PCR showed that the expression levels of miR-146a-5p,25-5p,27a-3p,93-5p,23a-3p,223-5p,25-3p,483-5p,383-5p were significantly higher in the PCOS group than in the control group?P<0.05?;while the expression level of miR-141-3p in the PCOS group was lower than that in the normal group,but the difference between the groups was not statistically significant?P>0.05?;3.The results of qRT-PCR showed that the expression levels of 10 miRNAs in the PCOS group were significantly higher than those in the control group in follicular fluid?P<0.05?.4.The results of sequencing of follicular fluid and the sequencing of blood samples on the day of egg retrieval showed that there were 58 differentially expressed miRNAs expressed in follicular fluid,of which 25 were up-regulated and 33 were down-regulated.Conclusion1.Compared with NON-PCOS,there are differentially expressed exosome miRNAs in the serum of PCOS,miR-146a-5p,93-5p,27a-3p,-23a,483-5p may participate in insulin signaling pathway and regulate granulosa cells proliferation and apoptosis in PCOS.2.Exo-miR-16-5p,15b,486-5p in follicular fluid may be associated with ovulation disorders and insulin resistance in PCOS patients.Follicular fluid-specific expression of exosome miRNA is present in PCOS. |
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