基于线粒体功能揭示《黄帝内经》“七七”理论内涵及高龄IVF-ET患者的中药干预机制

Mitochondrial Function Reveals Connotation of "Seven-Seven" Theory of Huangdi's Internal Classic and Mechanism of Herbal Intervention for Elderly Women Undergoing IVF-ET

目的:研究不同年龄阶段女性卵巢颗粒细胞线粒体功能的变化及补肾中药二至天癸方对高龄妇女体外受精-胚胎移植技术(IVF-ET)结局的影响,从而验证《黄帝内经》“七七”理论的科学内涵。方法:收集山东中医药大学附属医院中西医结合生殖与遗传中心行辅助生殖技术的不孕症患者150例,按照《黄帝内经》“七七”理论分为三七、四七组(30例),五七组(60例)和六七组(60例),五七组和六七组采用随机数字+信封法分为对照组和中药组各30例,中药组自促排卵周期启动日给予二至天癸方至扳机日。观察各组之间的IVF结局,使用蛋白免疫印迹法(Western blot)测定各组卵巢颗粒细胞中线粒体融合蛋白1(Mfn1)、线粒体融合蛋白2(Mfn2),线粒体分裂蛋白1(Drp1)的含量。结果:与三七、四七组比较,五七对照组和六七对照组患者获卵数、双原核(2PN)数、可用胚胎数、优质胚胎数、临床妊娠率和活产率均明显下降(P<0.05),六七对照组鲜胚移植率明显下降(P<0.05)。与五七对照组比较,六七对照组患者患者获卵数、2PN数、可用胚胎数、优质胚胎数和临床妊娠率均明显下降(P<0.05)。与五七对照组比较,五七中药组患者获卵数、2PN数、可用胚胎数明显升高(P<0.05);与六七对照组比较,六七中药组患者获卵数明显升高(P<0.05)。与三七、四七组比较,五七和六七对照组患者卵巢颗粒细胞中Mfn1和Mfn2蛋白表达明显降低,Drp1蛋白表达升高(P<0.05);与五七对照组比较,六七对照组患者卵巢颗粒细胞中Mfn1和Mfn2蛋白表达明显降低,Drp1蛋白表达升高(P<0.05)。与五七对照组比较,五七中药组患者卵巢颗粒细胞中Mfn2蛋白表达升高,Drp1蛋白表达降低(P<0.05);与六七对照组比较,六七中药组患者卵巢颗粒细胞中Mfn1和Mfn2蛋白表达升高,Drp1蛋白表达降低(P<0.05)。结论:随着年龄的增长,女性在“五七”后IVF结局预后变差,卵巢颗粒细胞中线粒体融合能力下降,而裂变增多,从线粒体功能验证了《黄帝内经》“七七”理论的内涵。二至天癸方可以调节高龄女性的卵巢颗粒细胞线粒体功能,提高Mfn1、Mfn2表达水平,促进线粒体融合,抑制Drp1而减少线粒体分裂,缓解年龄带来的卵巢功能减退,提高卵母细胞发育潜能,改善高龄女性的IVF结局,但对于“六七”之年的超高龄女性临床效果欠佳。

Objective:To study the changes of mitochondrial function of ovarian granulosa cells in women of different ages and the effect of Erzhi-Tiangui prescription on in vitro fertilization-embryo transfer(IVF-ET) outcomes for elderly women,so as to verify the connotation of the "Seven-Seven" theory in the Huangdi's Internal Classic (《黄帝内经》).Method:A total of 150 infertility patients undergoing IVF-ET at the Reproductive and Genetic Center of Integrative Medicine,Affiliated Hospital of Shandong University of Traditional Chinese Medicine were recruited and assigned into "hree-Seven/Four-Seven(30 cases),Five-Seven(60 cases),and Six-Seven(60 cases) groups according to the "Seven-Seven" theory.The Five-Seven and SixSeven groups were further assigned into control and Chinese medicine subgroups using the random number plus envelope method,and the Chinese medicine group was administrated with Erzhi Tiangui prescription from the start day of controlled ovulation stimulation cycle to the trigger day.The IVF outcome was observed,and Western blot was employed to determine the levels of mitofusin 1(MFN1),mitofusin 2(MFN2),and dynaminrelated protein 1(Drp1) in the ovarian granulosa cells.Result:Compared with the Three-Seven/Four-Seven group,the control subgroups of the Five-Seven and Six-Seven groups showed decreased retrieved oocytes,two pronuclear(2PN) embryos,available embryos,high-quality embryos,clinical pregnancy rate,and live birth rate(P<0.05).Moreover,the control subgroup of the Six-Seven group showed decreased fresh embryo transfer rate(P<0.05).Compared with the control subgroup of the Five-Seven group,that of the Six-Seven group showed reduced retrieved oocytes,2PN embryos,available embryos,high-quality embryos,and clinical pregnancy rate(P<0.05).The Chinese medicine subgroup had more retrieved oocytes,2PN oocytes,and available embryos than the control subgroup in the Five-Seven groups(P<0.05).The Chinese medicine subgroup had more retrieved oocytes,than the control subgroup in the Six-Seven groups(P<0.05).The control subgroup of the SixSeven group showed lower expression levels of Mfn1 and Mfn2 and higher level of Drp1 than the control subgroup of the Five-Seven group(P<0.05),which indicated that the levels of Mfn1 and Mfn2 in ovarian granulosa cells were down-regulated while the expression of Drp1 was up-regulated with aging(P<0.05).The Chinese medicine subgroup had higher Mfn2 level and lower Drp1 level than the control subgroup in the FiveSeven group(P<0.05),and the Chinese medicine subgroup had higher Mfn1 and Mfn2 levels and lower Drp1level than then control subgroup in the Six-Seven group(P<0.05).Conclusions:The prognosis of IVF in women after "Five-Seven" became worse with aging,and the mitochondria in ovarian granulosa cells showed decreased fusion ability and increased fission,which verified the connotation of the "Seven-Seven" theory from the mitochondrial function.Erzhi Tiangui prescription can regulate the mitochondrial function of ovarian granulosa cells in elderly women,up-regulate the expression levels of Mfn1 and Mfn2 to promote mitochondrial fusion,and down-regulate the expression of Drp1 to reduce mitochondrial fission,thus alleviating the ovarian hypofunction caused by aging,improve the development potential of oocytes,and improve the IVF outcomes of elderly women.However,this prescription has limited efficacy for the elderly women in the age range of "SixSeven".