| Background:With the increasing reproductive needs of advanced age women in modern society,the number of patients of advanced age with repeated IVF-ET failure also increases.Endometrial receptivity is one of the main causes of repeated IVF-ET failure.At present,there is a lack of clinical research and mechanism discussion on endometrial receptivity in patients of advanced age with repeated IVF-ET failure.Traditional Chinese medicine has certain advantages in improving female endometrial receptivity.Modified Erbu Zhuyu Formula is the optimized formula of Erbu Zhuyu decoction.Erbu Zhuyu decoction is an empirical prescription of Professor Xiao Chengcong,a master of Chinese medicine,in improving endometrial receptivity.The clinical effect of Modified Erbu Zhuyu Formula is remarkable.Based on clinical experience and previous studies,this study explored the clinical efficacy and mechanism of Modified Erbu Zhuyu Formula on endometrial receptivity in advanced age patients with repeated IVF-ET failure.This is to provide a theoretical basis for the clinical application of famous and old TCM empirical prescriptions.Objective:1 To explore the clinical effect of Modified Erbu Zhuyu Formula on endometrial receptivity in women of advanced age with repeated IVF-ET failure.2 The aged mice model was selected.Through the comparative evaluation of three animal models,to confirm that the factors of advanced age could have an important effect on endometrial receptivity.3 To explore the improvement effect and potential mechanism of Modified Erbu Zhuyu Formula on endometrial receptivity in aged mice from the endometrial morphology.4 To explore the molecular mechanism of Modified Erbu Zhuyu Formula regulating endometrial receptivity in aged mice,based on P53/P21 signal pathway mediated by oxidative stress.Methods:1 Clinical efficacy observationUsing the method of clinical control study,67 women of advanced age with repeated IVF-ET failure were collected.According to the treatment scheme,they were divided into two groups:34 cases in the traditional Chinese medicine group and 33 cases in the western medicine group.The traditional Chinese medicine group was treated with Modified Erbu Zhuyu Formula,and the western medicine group was treated with hormone replacement therapy(Estradiol Valerate Tablets+Progesterone Capsules).The general situation,menstrual condition,clinical syndrome of traditional Chinese Medicine(TCM),ultrasonic indexes in the middle luteal phase,basic level of sex hormone in the early follicular phase,serum oxidative stress level in the middle luteal phase and pregnancy outcome were compared and observed between the two groups.To explore the clinical effect of Modified Erbu Zhuyu Formula on endometrial receptivity in women of advanced age with repeated IVF-ET failure.2 Animal experimental researchAged female mice with natural aging were selected and compared with classical embryo implantation disorder mice and normal mice of childbearing age.The models were compared and evaluated with pregnancy rate,average number of embryo implantation sites,endometrial morphology and molecular markers of endometrial receptivity.Aged female mice were used as the research object,in addition to the blank group of childbearing age,the aged mice were randomly divided into aged model group,low,medium and high dose groups of Modified Erbu Zhuyu Formula,progynova group and aspirin group.The pregnancy rate and average number of embryo implantation sites of mice in each group were counted.The expression of microvilli and pinopodes in endometrium were detected by HE staining and scanning electron microscope.The activity of SOD and the content of MDA in serum and uterine tissue were measured by WST-1 method and colorimetry.RT-PCR,WB and immunohistochemistry were used to detect the expression of ERα,PGR,LIF,HOXA10,P53 and P21 in endometrium.To explore the mechanism of Modified Erbu Zhuyu Formula on improving endometrial receptivity in aged mice.Results:1 Clinical observation on improving endometrial receptivity in women of advanced age with repeated IVF-ET failure1.1 Menstrual condition:After treatment,the scores of menstrual volume,menstrual color and blood clot of patients in the traditional Chinese medicine group decreased,and the improvement rate of menstrual color and blood clot in the traditional Chinese medicine group was higher than that in the western medicine group(P<0.05).1.2 Clinical syndrome of TCM:After treatment,the total score of TCM syndrome decreased in both groups(P<0.05).After treatment,the TCM symptom scores in the traditional Chinese medicine group decreased(P<0.05).After treatment,the total effective rate of TCM syndrome in traditional Chinese medicine group was higher than that in western medicine group(P<0.05).1.3 Ultrasonic indexes during midluteal phase:After treatment,the Salle scores of the two groups were lower than before(P<0.05).After treatment,the endometrial thickness during midluteal phase increased in both groups(P<0.05).The parameters of the uterine artery blood flow:after treatment,PI,RI and S/D of bilateral uterine arteries during midluteal phase of western medicine group were lower than those before,but the difference was not significant(P>0.05).After treatment,the RI of bilateral uterine arteries during midluteal phase of the traditional Chinese medicine group was lower than that before(P>0.05),and the PI and S/D of bilateral uterine arteries during midluteal phase were lower than that before,but the difference was not significant(P>0.05).Endometrial and subendometrial blood flow parameters:after treatment,FI during midluteal phase of western medicine group increased(P<0.05),VI decreased and VFI increased,but the difference was not significant(P>0.05).After treatment,VI,FI and VFI during midluteal phase of the traditional Chinese medicine group were higher than those before,but the difference was not significant(P>0.05).1.4 Basic level of sex hormone during the early follicular phase:After treatment,the serum levels of FSH,LH,E2,P and FSH/LH of the two groups increased,but the difference was not significant(P>0.05).After treatment,the serum levels of FSH,LH,E2,P and FSH/LH of the traditional Chinese medicine group were lower than those in the western medicine group,but the difference was not significant(P>0.05).1.5 Serum oxidative stress level during midluteal phase:After treatment,the activity of serum SOD was higher than that before treatment(P<0.05),and the content of serum MDA was lower than that before treatment(P<0.05).After treatment,the activity of serum SOD in the traditional Chinese medicine group was higher than that in the western medicine group(P<0.05),and the content of serum MDA was lower than that in the western medicine group(P<0.05).1.6 Pregnancy outcome:The rate of embryo implantation in the next cycle in the traditional Chinese medicine group was higher than that in the western medicine group,but the difference was not significant(P>0.05).The clinical pregnancy rate in the next cycle in the traditional Chinese medicine group was higher than that in the western medicine group(P<0.05).2 Experimental study on the mechanism of Modified Erbu Zhuyu Formula on endometrial receptivity in aged mice2.1 Evaluation of endometrial receptivity in aged mice model2.1.1 Pregnancy rate and the average number of embryo implantation sites:The pregnancy rate and the average number of embryo implantation sites in the classical model group were lower than those in the childbearing blank group(P<0.05).The pregnancy rate of mice in the aged model group was lower than that in the childbearing blank group,but the difference was not statistically significant(P>0.05).The average number of embryo implantation sites in the aged model group was lower than that in the childbearing blank group(P<0.05).The pregnancy rate and the average number of embryo implantation sites in the aged model group were higher than those in the classical model group,but the difference was not statistically significant(P>0.05).2.1.2 Morphology:Under the light microscope,the endometrial blood vessels and glands decreased in the classical model group and the aged model group.Under scanning electron microscope,the microvilli on the surface of endometrium in the classical model group and the aged model group were sparse,and the score of pinopodes was lower than that in the childbearing blank group(P<0.05).2.1.3 Molecular markers related to endometrial receptivity:The results of RT-PCR,WB and immunohistochemistry showed that the expression of ERα、PGR in endometrium of classical model group was lower than that of the childbearing blank group(P<0.05).The expression of ERα、PGR in the endometrium of the aged model group was higher than that of the childbearing blank group(P<0.05).The expressions of LIF and HOXA10 in endometrium of the classical model group and the aged model group were lower than those of the childbearing blank group(P<0.05).2.2 Effect of Modified Erbu Zhuyu Formula on endometrial morphology of aged mice2.2.1 Pregnancy rate and the average number of embryo implantation sites:The pregnancy rate in each treatment group was higher than that in the aged model group(P<0.05),and the average number of embryo implantation sites in each dose group of Modified Erbu Zhuyu Formula was higher than that in the aged model group(P<0.05).2.2.2 Morphology:Under the light microscope,the endometrial blood vessels and glands increased in each treatment group.Under scanning electron microscope,the expression of microvilli and pinopodes in each treatment group were improved,and the improvement effect of low-dose Modified Erbu Zhuyu Formula group was the best(P<0.05).2.3 Study on the molecular mechanism of Modified Erbu Zhuyu Formula regulating endometrial receptivity based on P53/P21 signaling pathway mediated by oxidative stress2.3.1 Expression of molecular markers of endometrial receptivity in endometrial tissue:Compared with the aged model group,the expression of ERa protein and mRNA in each dose group of Modified Erbu Zhuyu Formula decreased(P<0.05).The expression of PGR protein and mRNA decreased in mid-dose and high-dose Modified Erbu Zhuyu Formula groups,progynova group and aspirin group(P<0.05).The expression of LIF protein and mRNA increased in mid-dose and high-dose Modified Erbu Zhuyu Formula groups and aspirin groups(P<0.05),the expression of HOXA10 protein and mRNA increased in each treatment group(P<0.05).2.3.2 The level of oxidative stress:The activity of serum SOD in the high-dose Modified Erbu Zhuyu Formula group and aspirin group were higher than that in the aged model group(P<0.05).The SOD activity of uterine tissue in each dose group of Modified Erbu Zhuyu Formula and progynova group were higher than that in the aged model group(P<0.05).The content of serum MDA in each dose group of Modified Erbu Zhuyu Formula and progynova group were lower than that in the aged model group(P<0.05).The content of MDA in uterine tissue in mid-dose and high-dose Modified Erbu Zhuyu Formula groups were lower than that in the aged model group(P<0.05).2.3.3 Aging markers:Compared with the aged model group,the expression of P53 and P21 protein and mRNA in endometrium of mice in each treatment group increased in varying degrees,and the expression of P53 and P21 in the high-dose Modified Erbu Zhuyu Formula group increased most significantly(P<0.05).Conclusion:1 The results of clinical control study:Modified Erbu Zhuyu Formula can significantly improve menstrual condition,alleviate the syndrome of kidney deficiency and blood stasis,increase the thickness of endometrium,reduce the blood flow resistance of endometrium,and increase the blood perfusion of endometrium in the midluteal phase,thereby improving the endometrial receptivity and the outcome of pregnancy in women of advanced age with repeated IVF-ET failure.Its mechanism may be relate to increasing the activity level of serum SOD,reducing the content of serum MDA,improving the antioxidant capacity and improving the level of serum oxidative stress in the midluteal phase of women of advanced age with repeated IVF-ET failure.2 The results of animal experiments:2.1 The experiment confirmed that the endometrial receptivity of aged mice decreased and there was embryo implantation disorder.It is confirmed that advanced age is an important factor affecting endometrial receptivity.Aged mice could be used as an animal model to study the decrease of endometrial receptivity caused by aging.2.2 Modified Erbu Zhuyu Formula can repair the damage of endometrial epithelial cells in aged mice,promote the development of endometrial glands and blood vessels,promote the growth of endometrial microvilli,enrich the expression of pinopodes,and make corresponding morphological preparations for embryo implantation in the window of implantation,so as to improve endometrial receptivity.2.3 Modified Erbu Zhuyu Formula can regulate the expression level of molecular markers related to endometrial receptivity during the window of implantation(reduce the expression of ERα and PGR,and increase the expression of LIF and HOXA10).Modified Erbu Zhuyu Formula can improve the level of oxidative stress in serum and uterus of aged mice during the window of implantation(increase the level of SOD activity and reduce the content of MDA).Modified Erbu Zhuyu Formula can activate P53/P21 signaling pathway,increase the expression of P53 and P21 in endometrium,and induce endometrial cell senescence during the window of implantation,so as to improve endometrial receptivity and facilitate embryo implantation. |
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