Characteristics And Methylation Profile Of Cell-free DNA In Premature Ovarian Failure Patients' Serum

Premature ovarian failure(POF)is the final stage of the development of primary ovarian insufficiency(POI),and the original follicles of these patients are in a depleted state and the depletion state is irreversible.The clinical features of POF are amenorrhea,infertility,osteoporosis,sweating and other menopausal symptoms.POF is not rare in clinical practice,a survey shows that the incidence of POF within 30 years of age is about 0.1%,and 1% before the age of 40.The etiology of POF is complex,the current study shows that genetic factors,immune factors,metabolic factors,iatrogenic injury,viral infection and other environmental factors may lead to premature ovarian failure.However,so far,the exact pathogenesis of premature ovarian failure has not yet fully elucidated.It has been found that several genes that may be associated with the pathogenesis of POF are associated with the apoptotic process.Such as MLH3 gene mutations lead to abnormal meiosis and abnormal oocytes,and then end up with rapid apoptosis;PGRMC1 gene missense mutation will damage the development of progesterone in ovarian anti-apoptotic effect,making the premature loss of follicles,and ultimately lead to POF.According to the method of electrophoresis,cell-free DNA(cfDNA)was consistent with the band formed by apoptotic cells,and the active release of active DNA of T lymphocytes also formed similar bands.It has been shown that cfDNA may be mainly derived from the active release of apoptosis and the synthesis of newly synthesized DNA of active cells.Therefore,we use cfDNA as the starting point to explore whether the impact of apoptosis on the incidence of POF.In addition,DNA methylation is an indispensable epigenetic regulation of the various enzymes involved in the organism.Higher eukaryotes can regulate the extent to which genes are expressed and expressed by changes in DNA methylation status.Studies have shown that the prevalence of DNA methylation in a variety of diseases is prevalent,especially in tumors,which may be the earliest changes in the pathogenesis of tumors.CfDNA as a genetic material different from the intracellular DNA,has its unique source and biological characteristics.The cfDNA methylation characteristic may become a diagnostic target for more related diseases.A number of studies have shown that the methylation status of the cfDNA-related genes has changed in a variety of diseases and has begun to explore the possibility of applying it as a specific diagnostic indicator to clinical practice.Therefore,it is important to explore the pathogenesis of POF from the perspective of methylation of specific gene of cfDNA,which is of great significance in early diagnosis,early treatment and prognosis of premature ovarian failure.ObjectiveTo investigating the characteristics of plasma cfDNA content and integrity and the characteristics of cfDNA methylation profile,and to provides a new perspective on the pathogenesis of POF.MethodsThe study was conducted in this study.The patients were diagnosed as premature ovarian insufficiency(POF).The samples were plasma cells free DNA and normal ovarian function.The content of free DNA was detected by real time-qPCR(RT-qPCR),and the integrity index of free DNA was obtained by calculating the ratio of free DNA in different fragments.The methylation status of plasma cfDNA was detected by methylation technique,and the clustering information of the corresponding methylation site was explored by GO analysis.ResultsThe cfDNA concentration calculated from the CT value obtained by Real-time PCR with the primers ALU115 represented the total cfDNA content in the plasma and the concentrstion obtained by the primers ALU247 represents the concent of larger fragments of plasma cfDNA.The plasma cfDNA concentration in the POF group was higher than that in the control group(9.76 ng / ?l vs.3.73 ng / ?l,p<0.001),and the integrity of plasma cfDNA of POF group was lower than that of the control group(8.37% vs.22.41%,p<0.001).The serum cfDNA of the POF group and the control group was transformed with bisulfite and analyzed by methylation analysis using the BeadChip Array Inf Methyl 8 Sample Beadchip methylation chip.It was found that the methylation level of 127724 sites was significant difference.The proportion of differential methylation sites in the POF group and the control group was 39.52% and 43.68%,respectively,in the high density region of CpG and CpG.Analysis of genomic functional regions revealed that the differential methylation sites of the promoter region(TSS1500,TSS200 and 1st Exon)accounted for 36.66%.GO analysis of the gene corresponding to the differential methylation site in the POF group and the control group was performed on the DAVID website,including cell components,molecular functions and biological processes.The genes of the differential methylation sites are mainly located in the cytoplasm,plasma membrane and other cell components;and have protein binding,ATP binding and other molecular functions;and can regulate GTPase activity,protein phosphorylation and forward regulation of apoptosis and other biological processes.The signal pathway of the gene where the differential methylation site was involved was analyzed by KEGG.The results showed that the genes of differential methylation sites were mainly involved in MAPK signal pathway,endocytosis and estrogen signaling pathway,progesterone-mediated oocyte maturation and GnRH signaling pathway in POF group compared with control group.ConclusionPOF patients' plasma cfDNA content increased,while the the decreased integrity may be due to increased cell apoptosis.The different methylation site of plasma cfDNA in POF are enriched in the KEGG pathways,such as apoptosis pathway,estrogen signaling pathway,progesterone-mediated oocyte maturation and GnRH signaling pathway,and follicular development Closely related signal pathways.