目的 基于INSR基因甲基化以及胰岛素代谢信号通路探讨电针改善痰湿型多囊卵巢综合征(polycystic ovary syndrome,PCOS)患者胰岛素抵抗及体外受精-胚胎移植(in vitro fertilization and embryo transfer,IVF-ET)妊娠结局的作用机制.方法 采用前瞻性随机对照研究,纳入2020年1月至2022年12月期间于山东中医药大学附属医院生殖与遗传科就诊拟行IVF-ET助孕的痰湿型PCOS患者100例,采用随机数字表法均分为电针治疗(试验组)50例,伪针刺激(对照组)50例,采用固定拮抗剂方案促排卵,两组患者分别于取卵前1个月经周期经净始予电针治疗和伪针刺激,每周2次,直至扳机日.收集颗粒细胞.观察比较两组患者痰湿证候积分、胰岛素抵抗指数(homeostasis model assessment-insulin resistance,HOMA-IR)、促性腺激素(gonadotropin,Gn)的用量及使用时间、获卵数、受精率、优质胚胎数、临床妊娠率、流产率和活产率.采用亚硫酸氢盐扩增子测序测定颗粒细胞中INSR基因启动子区甲基化水平的变化;实时荧光定量PCR和Western blotting测定INSR、PI3K、GLUT4 mRNA和蛋白的含量.结果 试验组治疗后痰湿证候积分(15.23±1.57)、HOMA-IR(2.82±0.39)较治疗前(21.65±3.61、3.34±0.56)明显降低,差异均具有统计学意义(P<0.001,P=0.014).试验组治疗前后证候积分差值(-5.76±2.86)、HOMA-IR差值(-2.67±0.06)的绝对值明显大于对照组(-1.64±0.84、-0.11±0.04),差异均具有统计学意义(P<0.001,P=0.021).试验组患者Gn用量[(2 119.53±338.28)U]及使用时间[(10.16±1.25)d]较对照组[(2 405.65±434.20)U、(10.94±1.46)d]明显减少(P=0.005,P=0.026),优质胚胎数[(3.54±1.04)枚]较对照组[(2.66±1.87)枚]明显增多(P=0.014),受精率[66.91%(552/825)]、临床妊娠率[63.27%(31/49)]和活产率[51.02%(25/49)]较对照组[60.20%(475/789)、41.67%(20/48)、31.25%(15/48)]明显升高,差异均具有统计学意义(P=0.005,P=0.033,P=0.048);两组患者的获卵数、早期流产率差异均无统计学意义(均P>0.05).试验组患者颗粒细胞中INSR基因启动子区38、47、56、59、94、143位点甲基化水平较对照组明显降低,差异均具有统计学意义(均P<0.05),两组患者71、74、154、156、162位点甲基化水平差异均无统计学意义(均P>0.05).与对照组相比,试验组患者卵巢颗粒细胞中INSR、PI3K、GLUT4基因mRNA和蛋白表达均上调,差异均有统计学意义(均P<0.05).结论 电针可能是通过降低痰湿型PCOS患者INSR基因启动子区甲基化水平,使INSR、PI3K、GLUT4基因表达上调,从而改善患者胰岛素抵抗,提高其胚胎的质量,最终改善妊娠结局.
Mechanism of electroacupuncture on improving insulin resistance and IVF-ET pregnancy outcome in polycystic ovary syndrome patients with phlegm-damp syndrome based on DNA methylation
Objective To study the mechanism of electroacupuncture(EA)on improving insulin resistance and in vitro fertilization and embryo transfer(IVF-ET)pregnancy outcome in polycystic ovary syndrome(PCOS)patients with phlegm-damp syndrome based on DNA methylation and insulin metabolism signaling pathway.Methods In this prospective randomized controlled study,100 PCOS patients with phlegm-damp syndrome who underwent IVF-ET in Department of Reproduction and Genetics in Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2020 to December 2022 were enrolled and divided into a treatment group(EA therapy)and control group(placebo needling)by random number table,50 patients in each group.Fixed antagonist regimen was used to promote ovulation in the two groups.Patients received EA therapy and placebo needling respectively twice a week from the menstrual cycle before oocyte retrieval till human chorionic gonadotrophin injection day.The granulosa cells were collected.The improvement of phlegm and dampness syndrome,homeostasis model assessment insulin resistance(HOMA-IR),dosage and number of days of gonadotropins(Gn)used,number of oocytes retrieved and high-quality embryos,fertilization rate,clinical pregnancy rate,early miscarriage rate and live birth rate of patients in the two groups were compared.Bisulfite amplicon sequencing was used to evaluate the methylation levels of the INSR gene promoter region in ovarian granulosa cells of patients in the two groups.Real-time polymerase chain reaction and Western blotting technology were used to detect the expression of INSR,PI3K and GLUT4.Results The phlegm-dampness score(15.23±1.57)and HOMA-IR(2.82±0.39)of the experimental group after treatment were significantly lower than those before treatment(21.65±3.61 and 3.34±0.56),and the differences were statistically significant(P<0.001,P=0.014).The differences of the phlegm-dampness score(-5.76±2.86)and HOMA-IR(-2.67±0.06)before and after treatment in the experimental group were significantly greater than those in control group(-1.64±0.84,-0.11±0.04),and the differences were all statistically significant(P<0.001,P=0.021).In the experimental group,the dosage of Gn used by patients[(2 119.53±338.28)U]and the duration of Gn used[(10.16±1.25)d]were significantly reduced compared with control group[(2 405.65±434.20)U,P=0.005;(10.94±1.46)d,P= 0.026],and the number of high-quality embryos(3.54±1.04)was significantly increased compared with control group(2.66±1.87,P=0.014).Fertilization rate[66.91%(552/825)],clinical pregnancy rate[63.27%(31/49)]and live birth rate[51.02%(25/49)]were significantly higher in the experimental group than in control group[60.20%(475/789),41.67%(20/48),31.25%(15/48)],and the differences were all statistically significant(P=0.005,P=0.033,P=0.048).There were no significant differences in the number of oocytes retrieved and early abortion rate between the two groups(all P>0.05).The methylation levels of INSR gene promoter sites 38,47,56,59,94 and 143 in the experimental group were significantly lower than those in control group(all P<0.05),but there were no significant differences in methylation levels of sites 71,74,154,156 and 162 between the two groups(all P>0.05).Compared with control group,the mRNA and protein expression of INSR,PI3K and GLUT4 was up-regulated in the experimental group(all P<0.05).Conclusion EA may improve IR by down-regulating INSR methylation levels and up-regulating INSR,PI3K and GLUT4 expression,thereby enhancing the quality of embryos and improving the pregnancy outcome in PCOS patients.
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