| Object:To study the whole-genome DNA methylation profile and methylation differentially expressed genes of ovarian granulosa cells in phlegm-dampness type polycystic ovary syndrome and normal women by the Reduced representation bisulfite sequencing technology(RRBS).To observe the effect of Cang-Fu-Dao-Tan-Wan on the outcome of phlegm dampness type polycystic ovary syndrome patients on clinical symptoms and the pregnancy rate.Objectively evaluate the clinical efficacy of Cang-FuDao-Tan-Wan in the treatment of phlegm-dampness type polycystic ovary syndrome and the nature of its epigenetic syndromes,providing an objective basis for TCM syndrome differentiation and treatment.Method:(1)A total of 60 patients with polycystic ovary syndrome of phlegmdampness type were selected and randomly divided into experimental group and control group 1,and 60 patients with non-phlegm-dampness type as control group 2.In addition,60patients who underwent IVF due to male factors were selected as the control group.3 groups of patients were planned to undergo IVF treatment,and ovarian granulosa cells were extracted as the research object.Six patients in each group were randomly selected for RRBs methylation sequencing,through GO and KEGG analysis,to obtain the target gene pathway of phlegm-damp polycystic ovary syndrome.(2)A total of 120 women with phlegm-dampness type polycystic ovary syndrome who were planned for IVF treatment were selected,and they were divided into treatment group,normal group and control group according to the random number table method.60 women who were planned for IVF treatment with non-phlegm-dampness type polycystic ovary syndrome were selected as the normal group.The treatment group was given CangFu-Dao-Tan-Wan for the first two menstrual cycles,and the three groups of patients were treated with conventional IVF to promote drainage,and cryopreserved granular cells were separated.Observe the changes in phlegm-dampness syndrome scores of patients before and after treatment,the number of eggs obtained,2PN fertilization rate,IVF transferable embryo rate,high-quality embryo rate and clinical pregnancy rate,and finally use RT-PCR technology detect the mRNA expression level of differential gene to verify the candidates of ovarian granulosa cells in each group of patients.Results:(1)We used DMR analysis on the whole genome DNA methylation sequencing data of AC,CD,DA,and BA groups.Draw a clustering heat map of the average methylation level of the differentially methylated regions,and take the intersection of each group of genelists to obtain 171 candidate genes.According to the gene coexpression network,we found that PKP3 is at the core of the network.According to the results of DMR analysis,we found that the PKP3 gene was hypermethylation after treatment with Cang-Fu-Dao-Tan-Wan.The KEGG analysis results of DMR indicated that these candidate genes are mainly involved in cancer-related pathways and five major biological processes including endocytosis,biological metabolic pathway,calcium ion signaling pathway and mTOR signaling pathway.This study focused on the hypermethylation of PKP3,which is involved in the inhibition of the PKP3/p38 MAPK/ERCC1 signaling pathway.The regulation of these DNA methylation genes may be closely related to the phlegm-damp polycystic ovary syndrome.(2)The analysis of clinical data showed:Group A(phlegm-damp PCOS placebo group)2PN fertilization rate(52.1%vs 69.79%),transferable embryo rate(57.43%vs 64.95%),high-quality embryo rate(20.4%vs 37.01%)and The clinical pregnancy rate(32.76%vs 53.45%)was significantly lower than that of group D(normal group),and the difference was statistically significant(p<0.05).The number of eggs obtained in group A(15.15±4.93vs15.50±4.38)was slightly lower than that of group D,The difference was not statistically significant(p>0.05);Group B(phlegm-damp PCOS medication group)2PN fertilization rate(69.95%vs 52.1%),transferable embryo rate(66.61%vs 57.43%),highquality embryo rate(44.77)%vs 20.4%),clinical pregnancy rate(55.17%vs 32.76%)compared with group A(phlegm-damp PCOS placebo group),significantly improved,the difference was statistically significant(p<0.05),the number of eggs obtained in group B(15.56±4.05vs15.15±4.93)slightly higher than group A,the difference was not statistically significant(p>0.05).After treatment with Cangfudaotan granules,the scores of phlegmdampness TCM syndromes in group B(32.64±4.34 v.s.24.84±3.92)were significantly reduced,and the clinical symptoms were significantly improved.(3)The target signal pathway was verified by real-time quantitative fluorescent PCR and Western blot:the mRNA expression of PKP3 and ERCC1 in group A(phlegm-damp PCOS placebo group)was significantly higher than that in group D(normal group),and group B(phlegm-damp PCOS medication group)compared with group A(phlegm-damp PCOS medication group),the mRNA expression of PKP3 and ERCC1 was significantly reduced.The expression of ERCC1,phosphorylated ERK1/2 and phosphorylated p38MAPK protein in ovarian granulosa cells in group A(phlegm-damp PCOS placebo group)was significantly increased compared to group D(normal group);group B(phlegmdamp PCOS medication group)The protein expression of p-p38 MAPK,p-ERK1/2 and ERCC1 decreased significantly,but there was no significant difference from the D group(normal group).Conclusion:Abnormal methylation of the PKP3 gene in ovarian granulosa cells may be one of the important reasons for the decline of oocyte quality in patients with phlegmdamp polycystic ovary syndrome.This study confirmed that Cang-Fu-Dao-Tan-Wan can inhibit PKP3/p38 MAPK through PKP3 hypermethylation/ERCC1 expression to play a critical role in the treatment of polycystic ovary syndrome. |
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