基于表观遗传学探讨补肾中药改善IVF治疗结局的机制研究
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摘要
目的:从表观遗传学角度探讨补肾中药改善体外受精-胚胎移植(IVF-ET)患者治疗结局的机制。
方法:临床研究:将66例行IVF-ET治疗的肾阴虚证不孕患者按照随机数字表法分为治疗组33例(二至天癸颗粒联合促性腺激素(Gn),33个周期)和对照组33例(单用Gn,33个周期)。治疗组在IVF前3个月卵泡期服用二至天癸颗粒,对照组不服药,两组均在IVF前一个月经周期的黄体中期获取子宫内膜行DNMT1检测。观察两组患者黄体中期子宫内膜DNMT1表达,肾虚证候积分变化情况,Gn用量及用药天数,子宫内膜厚度及分型,子宫动脉、子宫内膜PI和RI,取卵数、优质卵率、受精率、优质胚胎率及临床妊娠率。实验研究:将105只8-9周龄、健康昆明种雌性小鼠按照随机数字表法分为空白对照组、肾阴虚模型组及肾阴虚治疗组,每组各35只。造模成功后,进行超排卵,每组30只小鼠取卵行卵细胞印迹基因Snrpn和Pegl/Mest甲基化测定,另5只雌雄合笼后取受精卵,观察卵裂率、成胚率。
结果:临床研究:治疗组患者肾虚症状得到明显改善,治疗组Gn用量及用药天数低于对照组,单个卵雌激素(E2/卵泡)水平高于对照组,优质卵率、优质胚胎率及临床妊娠率均高于对照组,差异均有统计学意义(P<0.05);治疗组着床期子宫内膜DNMT1表达量高于对照组,子宫内膜分型优于对照组,子宫动脉、子宫内膜PI和RI均低于对照组,差异均有统计学意义(P<0.05)。实验研究:肾阴虚模型小鼠卵细胞Snrpn和Pegl/Mest基因均发生了不同程度的去甲基化(印迹丢失),而该去甲基化在肾阴虚治疗组得到部分纠正;各组小鼠受精卵数目、卵裂率及成胚率比较,肾阴虚模型组低于空白对照组,肾阴虚治疗组高于肾阴虚模型组,差异有统计学意义(P<0.05)。
结论:1.在IVF-ET治疗周期,应用补肾中药可明显改善患者肾虚症状,减少Gn用量,提高优质卵率、优质胚胎率,进而改善临床妊娠率。2.补肾中药可提高着床期子宫内膜DNMT1表达量,改善子宫内膜分型,增加子宫动脉及子宫内膜血流灌注,以改善子宫内膜容受性利于胚胎着床,这可能是补肾中药改善IVF结局的机制之一。3.补肾中药可在一定程度上防止肾阴虚模型小鼠卵细胞印迹基因Snrpn和Pegl/Mest的异常去甲基化(印迹丢失),并提高其受精卵卵裂及成胚能力;推论DNA异常去甲基化可能是肾阴虚不孕的表观遗传学机制之一,补肾中药防止异常去甲基化的发生可能是其治疗不孕症的机制之一。
Objective: In view of epigenetics, to explore the mechanism of reinforcing Kidney Chinese herbs to improve the treatment outcome of in vitro fertilization
Method: In clinical study, 66 infertile women of kidney yin defficiency undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomized into the test group, treated with ETG and Gn(33 cases), and the control group, treated with Gn only(33 cases). The test group had taken reinforcing Kidney Chinese herbs for 3 months, while the control group hadn’t. Just one month before IVF, all patients of both groups were scraped the endometrium during the midluteal phase to evaluate the DNMT1. The long term protocol was adopted. The syndrome scores of TCM, administration time and number of Gn, thickness and type of endometrium, PI/RI of uterous artery and endometrial blood stream, number of retrieved oocytes, fertilization rate, high quality embryo rate and pregnancy rate were evaluated. In experimental study, 105 Kunming female mice of 8～9 weeks old were randomized into the kidney yin defficiency model group, the kidney yin deficiency test group, and the control group. After the model was established successfully, all the mice were treated with COH. The occtyes of thirty mice were retrieved to evaluate the methylation the imprinted gene of Snrpn and Pegl/Mest. After cohbaiting,the fertilized ovum of the other five mice were retrieved to evaluate the ability of merogenesis and forming the embryo.
Results: Symptoms of kidney asthenia of the test group was significantly improved. The level of E2 per occtye, the ratio of high quality occtye and embryo, the clinical pregnancy rate were higher than those of the control group. The difference were statistically significant (P<0.05). The expression of DNMT1 of the endometrium of the test group was higher than that of the control group, the type of endometrium was better than that of the control group, PI and RI of uterous artery and endometrial blood stream of the control group were lower than those of the control group. The difference was statistically significant (P<0.05). The DNA methylation of Snrpn and Pegl/Mest of the kidney yin defficiency model mice occtye were lost. The abnormality was prevented partly in the kidney yin deficiency test group. The number, ability of merogenesis and forming the embryo of fertilized ovum of the kidney yin defficiency model mice were lower than those of the blank mice. The difference was statistically significant (P<0.05).
Conclusions: Reinforcing Kidney Chinese herbs combined with western medicine can decrease the dosage of Gn obviously and improve symptoms of kidney asthenia, the quality of oocyte, the ratio of high quality occtye and embryo, the ratio of pregnancy. Reinforcing Kidney Chinese herbs can improve the thickness and type of endometrium, PI and RI of uterous artery, endometrial blood stream, DNMT1 expression of the endometrium. The kidney yin defficiency can make the methylation the imprinted gene of Snrpn and Pegl/Mest lost. It indicates that the abnormality of DNA methylation may be the epigenetic mechanism of the infertility of kidney yin defficiency. Reinforcing Kidney Chinese herbs can prevent the abnormality partly and improve the ability of merogenesis and forming the embryo of the fertilized ovum.That maybe the mechanism of reinforcing Kidney Chinese herbs to cure the infertility.
方法:临床研究:将66例行IVF-ET治疗的肾阴虚证不孕患者按照随机数字表法分为治疗组33例(二至天癸颗粒联合促性腺激素(Gn),33个周期)和对照组33例(单用Gn,33个周期)。治疗组在IVF前3个月卵泡期服用二至天癸颗粒,对照组不服药,两组均在IVF前一个月经周期的黄体中期获取子宫内膜行DNMT1检测。观察两组患者黄体中期子宫内膜DNMT1表达,肾虚证候积分变化情况,Gn用量及用药天数,子宫内膜厚度及分型,子宫动脉、子宫内膜PI和RI,取卵数、优质卵率、受精率、优质胚胎率及临床妊娠率。实验研究:将105只8-9周龄、健康昆明种雌性小鼠按照随机数字表法分为空白对照组、肾阴虚模型组及肾阴虚治疗组,每组各35只。造模成功后,进行超排卵,每组30只小鼠取卵行卵细胞印迹基因Snrpn和Pegl/Mest甲基化测定,另5只雌雄合笼后取受精卵,观察卵裂率、成胚率。
结果:临床研究:治疗组患者肾虚症状得到明显改善,治疗组Gn用量及用药天数低于对照组,单个卵雌激素(E2/卵泡)水平高于对照组,优质卵率、优质胚胎率及临床妊娠率均高于对照组,差异均有统计学意义(P<0.05);治疗组着床期子宫内膜DNMT1表达量高于对照组,子宫内膜分型优于对照组,子宫动脉、子宫内膜PI和RI均低于对照组,差异均有统计学意义(P<0.05)。实验研究:肾阴虚模型小鼠卵细胞Snrpn和Pegl/Mest基因均发生了不同程度的去甲基化(印迹丢失),而该去甲基化在肾阴虚治疗组得到部分纠正;各组小鼠受精卵数目、卵裂率及成胚率比较,肾阴虚模型组低于空白对照组,肾阴虚治疗组高于肾阴虚模型组,差异有统计学意义(P<0.05)。
结论:1.在IVF-ET治疗周期,应用补肾中药可明显改善患者肾虚症状,减少Gn用量,提高优质卵率、优质胚胎率,进而改善临床妊娠率。2.补肾中药可提高着床期子宫内膜DNMT1表达量,改善子宫内膜分型,增加子宫动脉及子宫内膜血流灌注,以改善子宫内膜容受性利于胚胎着床,这可能是补肾中药改善IVF结局的机制之一。3.补肾中药可在一定程度上防止肾阴虚模型小鼠卵细胞印迹基因Snrpn和Pegl/Mest的异常去甲基化(印迹丢失),并提高其受精卵卵裂及成胚能力;推论DNA异常去甲基化可能是肾阴虚不孕的表观遗传学机制之一,补肾中药防止异常去甲基化的发生可能是其治疗不孕症的机制之一。
Objective: In view of epigenetics, to explore the mechanism of reinforcing Kidney Chinese herbs to improve the treatment outcome of in vitro fertilization
Method: In clinical study, 66 infertile women of kidney yin defficiency undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomized into the test group, treated with ETG and Gn(33 cases), and the control group, treated with Gn only(33 cases). The test group had taken reinforcing Kidney Chinese herbs for 3 months, while the control group hadn’t. Just one month before IVF, all patients of both groups were scraped the endometrium during the midluteal phase to evaluate the DNMT1. The long term protocol was adopted. The syndrome scores of TCM, administration time and number of Gn, thickness and type of endometrium, PI/RI of uterous artery and endometrial blood stream, number of retrieved oocytes, fertilization rate, high quality embryo rate and pregnancy rate were evaluated. In experimental study, 105 Kunming female mice of 8～9 weeks old were randomized into the kidney yin defficiency model group, the kidney yin deficiency test group, and the control group. After the model was established successfully, all the mice were treated with COH. The occtyes of thirty mice were retrieved to evaluate the methylation the imprinted gene of Snrpn and Pegl/Mest. After cohbaiting,the fertilized ovum of the other five mice were retrieved to evaluate the ability of merogenesis and forming the embryo.
Results: Symptoms of kidney asthenia of the test group was significantly improved. The level of E2 per occtye, the ratio of high quality occtye and embryo, the clinical pregnancy rate were higher than those of the control group. The difference were statistically significant (P<0.05). The expression of DNMT1 of the endometrium of the test group was higher than that of the control group, the type of endometrium was better than that of the control group, PI and RI of uterous artery and endometrial blood stream of the control group were lower than those of the control group. The difference was statistically significant (P<0.05). The DNA methylation of Snrpn and Pegl/Mest of the kidney yin defficiency model mice occtye were lost. The abnormality was prevented partly in the kidney yin deficiency test group. The number, ability of merogenesis and forming the embryo of fertilized ovum of the kidney yin defficiency model mice were lower than those of the blank mice. The difference was statistically significant (P<0.05).
Conclusions: Reinforcing Kidney Chinese herbs combined with western medicine can decrease the dosage of Gn obviously and improve symptoms of kidney asthenia, the quality of oocyte, the ratio of high quality occtye and embryo, the ratio of pregnancy. Reinforcing Kidney Chinese herbs can improve the thickness and type of endometrium, PI and RI of uterous artery, endometrial blood stream, DNMT1 expression of the endometrium. The kidney yin defficiency can make the methylation the imprinted gene of Snrpn and Pegl/Mest lost. It indicates that the abnormality of DNA methylation may be the epigenetic mechanism of the infertility of kidney yin defficiency. Reinforcing Kidney Chinese herbs can prevent the abnormality partly and improve the ability of merogenesis and forming the embryo of the fertilized ovum.That maybe the mechanism of reinforcing Kidney Chinese herbs to cure the infertility.
引文
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