| Objective: This study was to observe the effect of endometrial receptivity of kidney-invigorating and liver-soothing treatments on patients of embryo implantation repeated failures in IVF-ET before and after,to explore the mechanism with two methods on improving the endometrial receptivity,increase embryo implantation rate and clinical pregnancy rate and compare their similarities and differences.Methods: Based on the inclusion criteria and exclusion criteria,117IVF-ET patients with embryo implantation failure were made as experimental group.According to the syndrome differentiation of traditional Chinese medicine,they were divided into different groups of bushen and shugan,bushen group 62 cases(35 cases of proliferative phase,27 cases of secretory phase),shugan group 55 cases(33 cases of proliferative phase,22 cases of secretory phase).Another cases of normal subjects were 58 cases of the control group(32 cases at proliferative phase and 26 cases at secretory phase),which were caused by tubal factor IVF-ET preparation period and reproductive hormone and endometrial biopsy.Patients of bushen group and shugan group respectively,at the time of graft failure after menstruation the5 th day taking Bushen Zhuyun decoction,Xiaoyao Pill three menstrual cycles.After traditional Chinese medicine in the treatment of 3 cycles,underwent in vitro fertilization and embryo transfer(IVF-ET)in the conventional scheme or embryo transplantation.Patients of bushen group and shugan group before and after treatment with traditional Chinese medicine in proliferative phase(menstrual clean the3-5 days)and secretion(6-8 days after ultrasonic detection of ovulation)extracted 3ml in venous blood,separation of serum set-20℃ for preservationof the refrigerator until after used for a full automatic chemiluminescence detection.The same Bushen group and Shugan group patients in traditional Chinese medicine before the treatment,in its last transplant failure underwent hysteroscope or diagnostic curettage operation(endometrial time with the venous blood samples were obtained on),endometrial tissue samples were collected,traditional chinese medicine in the treatment of the three cycles after collected corresponding patients with proliferative or secretory phase endometrium.In control group,the venous blood and endometrial time,method and experimental group were collected,and the results were compared with the experimental group.After Chinese medicine treatment and the control group collecting specimen,the next cycle underwent IVF-ET treatment or embryo transfer.After taken each fresh endometrial tissue,first with saline will be cleaning the attachment on the surface of blood,mucus,such as clean,and then paraffin embedded and sectioned for HE staining and immunohistochemistry tissue fixed in 4% poly formaldehyde fixative,will be used for scanning electron microscopic observation of tissue fixed in 2.5%glutaraldehyde solution,more than endometrial tissue were kept at 4℃refrigerator,set aside;all remaining tissue were put into the liquid nitrogen to freeze,after 24 hours,then moved in the-80℃the refrigerator to save until ELISA enzyme-linked method and real-time fluorescence quantitative PCR detection were used.The content of E2 and P in serum was detected by fully automatic chemiluminescence assay.The morphology of different stages of endometrial tissue was observed HE staining.Expression and localization of ER,PR,VEGF,VEGFR-2,PCNA,CyclinD1,MMP-9 and e NOS protein in Uterine endometrium was detected by immunohistochemistry.Pinopodes process with planting window period was observed by scanning electron microscope(SEM).Expression VEGF,VEGFR-2,PCNA,CyclinD1,MMP-9 and eNOS protein in endometrial tissue was detected by ELISA.Expression VEGF,VEGFR-2,PCNA,CyclinD1,MMP-9 and eNOS mRNA in uterine endometrium was detected by real-time fluorescence quantitative PCR.Resluts:1 Comparison of clinical pregnancy rate and intrauterine pregnancy rateBushen proliferation treatment group 17 cases of pregnancy,pregnancy rate was 48.6%,intrauterine pregnancy rate was 42.9%,Bushen secretion treatment group 15 cases of pregnancy,pregnancy rate was 55.6%,intrauterine pregnancy rate was 51.9%.Shugan proliferation treatment group pregnancy in15 cases,pregnancy rate was 45.5%,intrauterine pregnancy rate was 42.4%and Shugan secretion treatment group pregnancy in11 cases,the pregnancy rate was 50.0%,intrauterine pregnancy rate was 50.0%.Proliferation control group pregnancy in 13 cases,the pregnancy rate was 40.6%,intrauterine pregnancy rate was 37.5%.Secretory control group 10 cases of pregnancy,pregnancy rate was 38.5%,intrauterine pregnancy rate was 30.8%.Compared with control group,the clinical pregnancy rate and intrauterine pregnancy rate in Bushen groups and Shugan groups were higher obviously,and the differences were statistically significant(P<0.05).The clinical pregnancy rate in Bushen proliferation treatment group was higer than Shugan proliferation treatment group,and the differences were statistically significant(P<0.05).The clinical pregnancy rate and intrauterine pregnancy rate in Bushen secretion treatment group were higher than Bushen proliferation treatment group and Shugan secretion treatment group,and the differences were statistically significant(P<0.05).The clinical pregnancy rate and intrauterine pregnancy rate in Shugan secretion treatment group were higher than Shugan proliferation treatment group,but the differences were not statistically significant(P>0.05).The intrauterine pregnancy rate in Bushen proliferation treatment group was higher than Shugan proliferation treatment group,but the differences was not statistically significant(P>0.05).2 The content of E2 and P in serumCompared with the proliferating control group,Bushen proliferation before treatment group and Shugan proliferation before treatment group,thecontent of E2 and P was higher in Bushen proliferation after treatment group and Shugan proliferation after treatment group,but the differences was not statistically significant(P>0.05).The content of E2 and P was obviously higher in Bushen proliferation after treatment group than in Shugan proliferation after treatment group,but the differences was not statistically significant(P>0.05).The content of E2 and P was no statistical significance between Bushen proliferation before treatment group and Shugan proliferation before treatment group(P>0.05).Compared with secretory control group,Bushen secretion before treatment group and Shugan secretion before treatment group,the levels of E2、P in Bushen secretion after treatment group and Shugan secretion after treatment group,and the diferece was statistically significant(P<0.05).The content of E2、P in Bushen secretion after treatment group was higher Shugan secretion after treatment group,and the diferece was statistically significant(P<0.05).Compare with secretory control group,the levels of E2、P in Bushen secretion before treatment group and Shugan secretion before treatment group were no statistical significance(P>0.05).3 The results of human endometrial tissue by hematoxylin and eosin(HE)staining3.1 Proliferation before treatment group(Bushen proliferation before treatment group,Shugan proliferation before treatment group)and the proliferation control group HE staining showed: less glandular tissue,most of the short and the main oval,occasionally slender curved shape,glandular epithelial low columnar,small nuclei,nuclear division less,almost no secretion phenomenon and interstitial cells arranged closely.3.2 Secretory before treatment group(Bushen secretory before treatment group,Shugan secretory before treatment group)and the secretory control group HE staining showed: more gland organization and more curved dilatation,glandular epithelial cell nuclei of vacuoles and release,covering a single layer of columnar epithelial cells within the gland,large nuclei,light color,interstitial loose.3.3 Proliferation after treatment group(Bushen proliferation after treatment group,Shugan proliferation after treatment group)HE staining showed: more glandular tissue,most of slender curved shape,occasionally short,glandular epithelium with pseudostratified and epithelial cells,and accumulation tendency,nuclear division more,release,stromal cell arranged loosely,edema.3.4 Secretory after treatment group(Bushen secretion after treatment group,Shugan secretion after treatment group)HE staining showed: more glandular tissue and significantly curved expansion,glandular epithelium for cuboidal,nucleus located in the central and large,light color,transparent cytoplasm,secreted phenomenon obvious,interstitial cell osteoporosis and edema significantly.4 The results of the human endometrial pinpode during the implantation window by scanning electron microscope4.1 The Bushen secretion before treatment group: many microvilli appeared edema and protruding into the uterine cavity,a few short microvilli fusion,apical cell appeared smooth and slender protrusions of the plasma membrane(developing pinopodes).4.2 The Bushen secretion after treatment group: almost no microvilli,a large number of smooth surface of the plasma membrane protrusions,the same size,as "pattern" swelling(fully developed pinopodes).4.3 The Shugan secretion before treatment group: a lot of short microvilli,mutual integration,at the top of the whole cell appeared smooth and slender protrusions of the plasma membrane(developing pinopodes);occasionally a cobblestone appearance of plasma membrane protrusions,like a mushroom(fully developed pinopodes).4.4 The Shugan secretion after treatment group: less microvilli,the emergence of a large number of intimal surface protrusions of the plasma membrane,projections of smooth surface,as mushrooms(fully developed pinopodes).4.5 The secretory control group: a small number of plasma membraneprotrusions and creaseing,some protrusions of the plasma membrane covering the top of the microvilli,cell volume increase(recession of pinopodes);almost no the "pattern" swelling of the plasma membrane protrusions.5 The result of the expression of ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS protein in human endometrium by immunohistochemical method.5.1 The position expression of ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1,eNOS protein in human endometrium by immunohistochemical method.ER and PR located in uterine luminal epithelium,glandular epithelium and interstitial nucleus.ER mainly located in glandular epithelial cell nuclei and expression of proliferation phase was significantly higher than secretory phase(P<0.05).PR mainly located in the interstitial nucleus and expression of proliferation phase was significantly higher than secretory phase(P<0.05).VEGF,VEGFR-2 located in the cytoplasm of luminal epithelium,glandular epithelium,stromal cells and vascular endothelial cells,and the expression of the cytoplasm of epithelial cells were most obvious,the expression intensity from proliferative to secretory phase decreased slightly.PCNA located in endometrial epithelial and stromal cell nucleus and the expression of secretory phase higher than proliferation phase(P<0.05).MMP-9 located in endometrial luminal epithelium and glandular epithelial cells,the expression of glandular epithelium was higher than luminal epithelium,cytoplasm of stromal cells has a small amount of expression,and the expression of secretion phase was higher than proliferation phase(P<0.05).CyclinD1 was mainly expressed endometrial glandular epithelial nuclei,interstitial cells weakly,and the expression of secretory phase was higher than that proliferative phase(P<0.05).eNOS was expressed endometrial glandular epithelium,luminal epithelium and vascular endothelial cell plasma,and the expression of cytoplasm of interstitial cells weak and the expression of secretory phase was higher than proliferation phase(P<0.05).5.2 Comparison of the expression of ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1,eNOS protein in human endometrium among each group by immunohistochemical method.Compared with proliferation control group,Bushen proliferation before treatment group and Shugan proliferative before treatment group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS protein in Bushen proliferation after treatment group and Shugan proliferative after treatment group was higher,and the difference was statistically significant(P<0.05).Compare with proliferation control group,Bushen proliferation before treatment group and Shugan proliferative before treatment group,the expression of ER and PR protein in Bushen proliferation after treatment group and Shugan proliferation after treatment group was lower,and the difference was statistically significant(P<0.05).The expression of PCNA,MMP-9,CyclinD1 and eNOS protein in Bushen proliferation after treatment group was higher Shugan proliferation after treatment group,and the difference was statistically significant(P<0.05).The expression of ER and PR protein in Bushen proliferation after treatment group was lower Shugan proliferation after treatment group,and the difference was statistically significant(P<0.05).The expression of VEGF and VEGFR-2 protein in Bushen proliferation after treatment group was lower Shugan proliferation after treatment group,and the difference was not statistically significant(P>0.05).Compare with proliferation control group,the expression of ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS protein in Bushen proliferation before treatment group and Shugan proliferative before treatment group was not tatistically significant(P>0.05).Compared with secretory control group,Bushen secretion before treatment group and Shugan secretion before treatment group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS protein in Bushen secretion after treatment group and Shugan secretion after treatment group was higher,and the difference was statistically significant(P<0.05).Compare with secretory control group,Bushen secretion before treatment group and Shugan secretion before treatment group,the expression of ER and PR proteinin Bushen secretion after treatment group and Shugan secretion after treatment group was lower,and the difference was statistically significant(P<0.05).The expression of PCNA,MMP-9,CyclinD1 and eNOS protein in Bushen secretion after treatment group was higher than Shugan secretion after treatment group,and the difference was statistically significant(P<0.05).The expression of ER and PR protein in Bushen secretion after treatment group was lower than Shugan secretion after treatment group,and the difference was statistically significant(P<0.05).The expression of VEGF and VEGFR-2protein in Bushen secretion after treatment group was lower than Shugan secretion after treatment group,and the difference was not statistically significant(P>0.05).Compare the expression of ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS protein among secretory control group,Bushen secretion before treatment group and Shugan secretion before treatment group was not statistically significant(P>0.05).6 Comparison of the expression of ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1,eNOS protein in human endometrium among each group by ELISA.Compared with proliferation control group,Bushen proliferation before treatment group and Shugan proliferative before treatment group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS protein in Bushen proliferation after treatment group and Shugan proliferative after treatment group was higher,and the difference was statistically significant(P<0.05).Compare with Bushen proliferation after treatment group,the expression of PCNA,MMP-9,CyclinD1 and eNOS protein in Shugan proliferation after treatment group was lower,and the difference was statistically significant(P<0.05).The expression of VEGF and VEGFR-2protein in Bushen proliferation after treatment group was lower than Shugan proliferation after treatment group,and the difference was not statistically significant(P>0.05).Compare with proliferation control group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and e NOS protein in Bushen proliferation before treatment group and Shugan proliferative before treatmentgroup was not tatistically significant(P>0.05).Compared with secretion control group,Bushen secretion before treatment group and Shugan secretion before treatment group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and e NOS protein in Bushen secretion after treatment group and Shugan secretion after treatment group was higher,and the difference was statistically significant(P<0.05).Compare with Bushen secretion after treatment group,the expression of PCNA,MMP-9,CyclinD1 and eNOS protein in Shugan proliferation after treatment group was lower,and the difference was statistically significant(P<0.05).The expression of VEGF and VEGFR-2 protein in Bushen secretion after treatment group was lower than Shugan secretion after treatment group,and the difference was not statistically significant(P>0.05).Compare with secretion control group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS protein in Bushen secretion before treatment group and Shugan secretion before treatment group was not tatistically significant(P>0.05).7 Comparison of the expression of ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1,eNOS mRNA in human endometrium among each group by RT-PCR.Compared with proliferation control group,Bushen proliferation before treatment group and Shugan proliferative before treatment group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS mRNA in Bushen proliferation after treatment group and Shugan proliferative after treatment group was higher,and the difference was statistically significant(P<0.05).Compare with Bushen proliferation after treatment group,the expression of PCNA,MMP-9,CyclinD1 and eNOS mRNA in Shugan proliferation after treatment group was lower,and the difference was statistically significant(P<0.05).The expression of VEGF and VEGFR-2mRNA in Bushen proliferation after treatment group was lower than Shugan proliferation after treatment group,and the difference was not statistically significant(P>0.05).Compare with proliferation control group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and e NOS mRNA in Bushenproliferation before treatment group and Shugan proliferative before treatment group was not tatistically significant(P>0.05).Compared with secretion control group,Bushen secretion before treatment group and Shugan secretion before treatment group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and e NOS mRNA in Bushen secretion after treatment group and Shugan secretion after treatment group was higher,and the difference was statistically significant(P<0.05).Compare with Bushen secretion after treatment group,the expression of PCNA,MMP-9,CyclinD1 and eNOS mRNA in Shugan proliferation after treatment group was lower,and the difference was statistically significant(P<0.05).The expression of VEGF and VEGFR-2 mRNA in Bushen secretion after treatment group was lower than Shugan secretion after treatment group,and the difference was not statistically significant(P>0.05).Compare with secretion control group,the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS mRNA in Bushen secretion before treatment group and Shugan secretion before treatment group was not tatistically significant(P>0.05).8 Correlation analysis of E2,P,ER,PR,VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOSER,PR,VEGF,eNOS and E2 showed positive correlation(r=0.859,P=0.000;r=0.704,P=0.013;r=0.825,P=0.009;r=0.913,P=0.005);ER,PR and P showed negative correlation(r=-0.681,P=0.006;r=-0.735,P=0.017);P,CyclinD1,MMP-9,eNOS and VEGF showed positive correlation(r=0.847,P=0.000;r=0.951,P=0.000;r=0.906,P=0.014;r=0.950,P=0.000);PCNA,MMP-9 and CyclinD1 showed positive correlation(r=0.949,P=0.000;r=0.884,P=0.007);MMP-9 and eNOS showed positive correlation(r=0.829,P=0.022).(P<0.05)Conclusions:1 Bushen and Shugan treatments can significantly improve the clinical pregnancy rate and intrauterine pregnancy rate in IVF-ET patients with embryo implantation repeated failure.The Bushen treatment was better than Shugan treatment on effect of the therapy.Secretory phase was better thanproliferative stage.2 Bushen and Shugan treatments can increase serum E2 and P in IVF-ET patients with embryo implantation repeated failure,reduce the expression of ER and PR in endometrial tissue,improve the expression of VEGF,VEGFR-2,PCNA,MMP-9,CyclinD1 and eNOS in endometrial tissue,promote cell proliferation,increase endometrial angiogenesis and vascular permeability,so as to improve the endometrial receptivity for embryo implantation.3 Bushen and Shugan treatments can improve the IVF-ET patients with repeated failure of embryo implantation in endometrial morphology,promote the formation of pinopodes and development,promote the synchronization of the endometrial stroma and glands.In the end,the endometrium in the implantation window reach the best receptive state.4 Assessment of endometrial receptivity mainly in the secretory phase,Bushen and Shugan in secretory phase endometrial receptivity was better than that in proliferative stage.5 Bushen treatment increased serum E2 and P in patients of IVF-ET embryo implantation repeated failure,reduced the expression of ER and PR in endometrial tissue,improved the expression of PCNA,MMP-9,CyclinD1 and eNOS in endometrial tissue,improved endometrial morphology,promoted the formation of pinopodes and development,promoted the synchronization of the endometrial stroma and glands.These were better than Shugan treatment.6 Shugan treatment to improve the expression of VEGF and VEGFR-2 in endometrial tissue is higher than Bushen treatment.So Shugan treatment to increased endometrial blood supply is better than Bushen treatment. |
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