| Objectives:1 To provide ideas and basis for the clinical diagnosis and treatment of endometrial receptivity,we consulted the domestic and foreign literature on endometrial receptivity,and summarized the research advancement on endometrial receptivity in western medicine and traditional Chinese medicine.2 To observe the effects of Erbuzhuyu Decoction on TCM syndromes,ultrasound parameters in the mid-luteal phase,serum angiogenesis-related factor levels,levels of estrogen and progesterone and pregnancy outcomes in patients with recurrent implantation failure(RIF).Thus to explore the clinical effects of Erbuzhuyu Decoction on endometrial receptivity in patients with RIF.3 To observe the effects of Erbuzhuyu Decoction on autophagy and angiogenesis of endometrial cells during the implantation window of mice with embryo implantation dysfunction,based on the PI3K/Akt/mTOR pathway and the expression levels of LC3B,VEGF,Ang-1 and Ang-2.Thus to further explore its targets and mechanisms for improving endometrial receptivity.Methods:1 Clinical researchThirty-two cases of RIF with low endometrial receptivity and syndrome of kidney deficiency and blood stasis were collected by self-contrast test.The patients were treated with Erbuzhuyu Decoction for 1-3 consecutive treatment courses(1 menstrual cycle was 1 treatment course).The general condition,menstrual blood loss scores,TCM syndrome scores,as well as the ultrasound parameters in the mid-luteal phase,serum angiogenesis-related factor levels,levels of estrogen and progesterone were observed and compared before and after treatment.And the implantation rate and clinical pregnancy rate of the next IVF-ET/ICSI cycle were followed up.2 Experimental researchMifepristone was used to establish embryo implantation dysfunction mouse model.We divided the mice into blank group,model group,low-dose,medium-dose and high-dose group of Erbuzhuyu Decoction,progynova group and aspirin group.After drug intervention and sampling,the uterine morphology of each group was observed and the number of embryo implantation sites was counted with naked eyes.The morphology of endometrial pinopodes of each group was observed with scanning electron microscope;the autophagosomes of endometrial cells were observed with transmission electron microscope.Real-Time PCR,Western blot and immunohistochemistry were used to detect mRNA expression,protein expression and localization of PI3K,Akt,mTOR,LC3B,VEGF,Ang-1,Ang-2.Then analyze the differences of expression and protein localization in each group.Results:1 Clinical study of Erbuzhuyu Decoction on endometrial receptivity in patients with RIF(1)Menstruation and TCM syndromes:Erbuzhuyu Decoction significantly increased menstrual volume in patients with RIF(P<0.01).The color of menstrual blood was brighter,menstruation was smoother and blood clots were reduced than before,but the differences were not statistically significant(P>0.05).And the syndrome of kidney deficiency and blood stasis were significantly improved in patients with RIF(P<0.01).(2)Ultrasound parameters in the mid-luteal phase:After the treatment with Erbuzhuyu Decoction,the thickness and volume of endometrium increased significantly(P<0.01),the number of cases of type A and type B endometrium increased,but there was no statistically difference(P>0.05).The PI,RI,and S/D values of bilateral uterine arteries were significantly lower after treatment(P<0.01).And the Salle scores were significantly increased after treatment(P<0.01).(3)Serum angiogenesis-related factors in the mid-luteal phase:After the treatment with Erbuzhuyu Decoction,the levels of VEGF and Ang-1 in the mid-luteal phase were significantly higher than those before treatment(P<0.01),Ang-2 was also higher than before,but there was no statistically difference(P>0.05).(4)Serum estrogen and progesterone in the mid-luteal phase:After the treatment with Erbuzhuyu Decoction,the level of E2 was significantly higher(P<0.05);P was also higher than that before treatment,but there was no statistically difference(P>0.05).(5)Pregnancy outcome:After the treatment with Erbuzhuyu Decoction,the embryo implantation rate of the next IVF-ET/ICSI cycle was 40.74%,and the clinical pregnancy rate was 37.50%2 Experimental research of the effect of Erbuzhuyu Decoction on the endometrial receptivity of mice model with embryo implantation dysfunction(1)Uterine morphology,pregnancy rate and average embryo implantation sites:The uteri of the model group were white and thinner,the number of average embryo implantation sites was less,and the pregnancy rate was significantly lower than that of the blank group(P<0.01)The uteri of mice in the high-dose group,progynova group,and aspirin group were rosy,with a small amount of hyperemia or swelling at the implantation site.The average number of embryo implantation sites was significantly higher than that of the model group(P<0.05),and the pregnancy rate was significantly higher than that of the model group(P<0.01).Among the Erbuzhuyu Decoction groups,the uterine morphology and pregnancy outcome in the high-dose group were the best,and there was no significant difference from the two western medicine groups(P>0.05)(2)PI3K/Akt/mTOR signaling pathway,LC3B:① Gene expression:There was no significant difference in the expression of PI3K mRNA among all groups(P>0.05).The expression of Akt mRNA and mTOR mRNA in the model group was significantly higher than those in the blank group(P<0.01),and the expression of LC3B mRNA was significantly lower than that of the blank group(P<0.01).The expression of PI3K mRNA,Akt mRNA and mTOR mRNA in the middle-dose and high-dose groups of Erbuzhuyu Decoction showed a downward trend compared with the model group,and the expression of LC3B mRNA showed an upward trend compared with the model group,but the differences were not significant(P>0.05).②Protein expression:The expression of PI3K,Akt and mTOR protein in the model group was significantly higher than that of the blank group(P<0.01),the expression of LC3B protein was significantly lower than that of the blank group(P<0.01).Compared with the model group,the expression of PI3K,AKT and mTOR protein in middle-dose and high-dose groups of Erbuzhuyu Decoction groups and aspirin groups was significantly lower(P<0.05),while the expression of LC3B protein were significantly higher(P<0.05).③Protein localization:The PI3K,Akt and mTOR proteins in the model group showed obvious expression in endometrial luminal epithelial cells and glandular epithelial cells,and weak expression in stromal cells and vascular epithelial cells.The LC3B protein in the model group showed very little expression in endometrial luminal epithelial cells and glandular epithelial cells,and no expression in stromal cells and myometrial cells.However,compared to the model group,the Akt and mTOR proteins in the middle-dose group,high-dose group,progynova group and aspirin group showed less expression in endometrial luminal epithelial cells and glandular epithelial cells,the LC3B protein showed obvious expression in endometrial luminal epithelial cells and glandular epithelial cells(3)Autophagosomes in endometrial cells:there were no obvious autophagosomes and autophagolysosomes were found in the endometrial cells of the model group;while there were relatively more autophagolysosomes in the endometrial cells of the middle-dose,high-dose group and aspirin group(4)VEGF,Ang-1,Ang-2:①Gene expression:The expression of VEGF,Ang-1 and Ang-2 mRNA in the model group was significantly lower than that in the blank group(P<0.01).After drug interventions,the expression of VEGF,Ang-1,and Ang-2 mRNA showed an upward trend.Among them,the expression of VEGF mRNA was significantly higher in the middle-dose,high-dose group,progynova group and aspirin group(P<0.05).The expression of Ang-1 mRNA in all groups were significantly higher than that in the model group(P<0.05).The expression of Ang-2 mRNA was significantly higher in the high-dose group,progynova group and aspirin group(P<0.01).②Protein expression:The expression of VEGF,Ang-1 and Ang-2 protein in the model group was significantly lower than those in the blank group(P<0.01).After drug interventions,the expression of VEGF,Ang-1,and Ang-2 protein showed an upward trend.Among them,The expression of VEGF protein in all groups were significantly higher than that in the model group(P<0.05).The expression of Ang-1 protein was significantly higher in the middle-dose,high-dose group,progynova group and aspirin group(P<0.01).The expression of Ang-2 protein was significantly higher in the high-dose group,progynova group and aspirin group(P<0.05).③Protein localization:The VEGF,Ang-1 and Ang-2 proteins in the model group were negatively or weakly expressed in endometrial luminal epithelial cells and glandular epithelial cells,but not significantly expressed in vascular epithelial cells.However,compared to the model group,the VEGF,Ang-1 and Ang-2 proteins in the high-dose group,progynova group and aspirin group showed weakly positive to positive expression in endometrial luminal epithelial cells,glandular epithelial cells and vascular epithelial cells.Conclusions:1 Clinical research showed that Erbuzhuyu Decoction can significantly improve the syndrome of kidney deficiency and blood stasis in patients with RIF,increase the thickness and volume of the endometrium in the mid-luteal phase,improve the blood stream perfusion of uterine artery,endometrial and subendometrial blood stream perfusion,increase the serum angiogenesis-related factor levels.In addition,it can improve the function of luteum.Thereby the Erbuzhuyu Decoction can improve the endometrial receptivity and pregnancy outcomes.2 Experimental research showed that Erbuzhuyu Decoction can inhibit the PI3K/Akt/mTOR pathway,up-regulate the expression of LC3B,increase the expression of VEGF,Ang-1 and Ang-2 in the endometrium of the embryo implantation dysfunction mouse model,thereby enhancing the endometrial autophagy and angiogenesis to improve endometrial receptivity,which is conductive to embryo implantation. |
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