| Objective:In this study,the ovarian low response(POR)was used as the disease vector in in vitro fertilization embryo transfer(IVF)technology.The random controlled trial(RCT)was used to observe the effect of acupuncture on the quality of IVF derived oocytes in por patients.Meanwhile,the whole genome DNA methylation sequencing technology was used to analyze the follicular fluid and peripheral blood of POR patients.The effects of acupuncture on DNA methylation of POR patients were explored,which provided epigenetic ideas and methods for further clarifying the mechanism of acupuncture effect on POR.Method:1.clinical researchPOR patients were randomly divided into acupuncture plus IVF group and IVF group.The study period was 20 weeks,including baseline period of 1 week,acupuncture treatment for 8 weeks,IVF cycle for 3 weeks and an 8-week follow-up.Among them,acupuncture was performed in the acupuncture plus IVF group from the two menstrual cycles before IVF ovulation promotion,except for three times a week,except for the menstrual period.Until the h CG injection day of IVF promoting cycle,the only IVF group was not treated by acupuncture;both groups received the IVF micro stimulation ovulation promotion program.The main evaluation indexes are mature oocytes and high quality embryo rate.The secondary evaluation indexes include the number of eggs taken,the number of fertilized eggs,the rate of fertilization,the rate of high-quality embryos,the number of available embryos,the available embryo rate,the amount of GN,the number of GN days,etc.the above evaluation indexes were evaluated on the third day of embryo culture after the egg taking.2.Analysis of DNA methylationThe cases were from the first part of randomized controlled trial.The follicle fluid and peripheral blood samples were taken from IVF of two groups.8 subjects were randomly selected from each group and Illumina 850k chip was used for sequenced for the whole genome DNA methylation,and the selected significant heterotopic sites and regions of DNA methylation were enriched.Gene Ontology(GO)function analysis and Kyoto Encyclopedia of Gene and Genomics(KEGG)were performed for genes and genomes,The results showed that the potential biological function and signal pathway related to acupuncture on IVF derived oocytes quality in por patients were selected.Result:1.clinical researchThe baseline of two groups were comparable.The main outcomes:the number of mature oocytes in the two groups was similar,and the difference was not statistically significant(P>0.05);the high quality embryo rate of acupuncture plus IVF group was significantly higher than that of IVF group,and the difference was statistically significant(P<0.01).Secondary outcomes:The number of high-quality embryos and the rate of available embryos in the acupuncture+IVF group were significantly higher than those in the simple IVF group(P<0.05);the number of retrieved eggs,the number of fertilized embryos,the rate of fertilized embryos,the number of available embryos,the amount of GN and the days of GN in the two groups were similar(P>0.05);the basic FSH,LH and E2 levels in the two groups were similar(P>0.05);the baseline SAS and SDS scores of the two groups were similar,and the SDS scores of the two groups were similar on the day of h CG injection(P>0.05)The SAS score of the acupuncture+IVF group was significantly lower than that of the simple IVF group(P<0.01);meanwhile,the cycle cancellation rate of the acupuncture+IVF group was lower than that of the simple IVF group,but the difference was not statistically significant(P>0.05).2.Analysis of DNA methylation(1)The differential methylation sites,regions and genesThere were 2950 significant heterotopic sites of DNA methylation in the follicle fluid of the two groups,corresponding to 1081 genes,of which 558 methylation levels were up regulated and 523 were down regulated;636 were significantly different in methylation in peripheral blood,corresponding to 279 genes,of which 206 methylation levels were up regulated and 73 were down regulated;follicle fluid and peripheral blood had the same methylation difference characteristics There were 61 loci.There were 435 genes in the different areas of methylation in follicular fluid of the two groups,of which 200 genes were up regulated and 235 down regulated;149 genes were enriched in the different methylation regions in peripheral blood,109 were up regulated and 40 were down regulated.(2)GO analysisIn the analysis of go enrichment,the highest concentration of different methylation sites in the follicle fluid of the two groups included negative regulation of cholesterol storage,isolation of extracellular ligands and receptors,positive regulation of NFAT signal cascade of calmodulineurin,fine cell response stimulated by follicle stimulating hormone and voltage gated chloride channel activity;the enrichment process of differentiated methylation region in follicle fluid The highest biological processes include smooth muscle tissue development,catecholamine stimulated cell response,eye development of embryo camera,gene imprinting and catecholamine reaction.The biological processes with the highest concentration of heterotopic points in peripheral blood methylation include cytoplasmic isolation of proteins,regulation of virus entering host cells,negative regulation of transcription factors into nucleus,differentiation of adenoepithelial cells and calcium dependent phospholipid binding,etc.;the biological processes with the highest concentration in the different methylation areas include the regulation of phospholipase activity,protein phosphatase regulating activity The results showed that phosphatase regulated activity,mi RNA binding in gene silencing after transcription and virus entering host cells.(3)KEGG pathway analysisAmong the 30 signal pathways with the highest concentration of different methylation sites in the follicular fluid of the two groups,including small cell lung cancer,long-term depression and bladder cancer,among them,ovarian steroid generation and GnRH signaling pathway were related to the pathogenesis of reproductive medicine memory por;30 channels with the highest concentration in the different methylation regions included cholesterol metabolism,mannose-o-glycan biosynthesis,lipid metabolism,lipid metabolism The regulation of lipolysis of fat cells,among which estrogen signaling pathway,ovarian steroid production and GnRH signaling pathway may be related to the pathogenesis of POR.Among the 30 pathways with the highest concentration of heterotopic points in the peripheral blood DNA methylation difference between the two groups,including trypanosomiasis,propionic acid metabolism and cell aging,no por related pathway was found;30 pathways with the highest concentration in the different regions of DNA methylation in the follicle fluid of the two groups included niacin and niacinamide metabolism,cancer central carbon metabolism,p53 pathway,estrogen signaling pathway and Hippo signaling pathway may be related to the pathogenesis of POR.After analyzing the signal pathway with high concentration in follicular fluid and peripheral blood,it was found that the methylation difference of GNAS/AC/PKA/CREB pathway also involved GnRH signal pathway,ovarian steroid generation,estrogen signal pathway,Ca2+signal pathway,etc.Conclusion1.Acupuncture can improve the quality of IVF derived oocytes in por patients,mainly by improving the potential of embryo development.2.The effect of acupuncture on improving the quality of IVF oocytes from por patients may be realized by improving the DNA methylation level in the growth environment of oocytes,which is mainly detected in the follicular fluid.3.The DNA methylation mechanism of acupuncture in improving the quality of IVF oocytes from por patients may be closely related to improving the methylation levels of Hox,DGAT1,Sox2 and CASP8 genes in follicular fluid,influencing the biological processes of gonadotropin response and follicle stimulating hormone cells,and regulating the methylation levels of GNAS/AC/PKA/CREB pathway and Wnt/Ca2+pathway. |
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