Effects Of Kidney-invigorating And Liver-soothing Treatments On Endometrial Receptivity And The Relationship With Sphk1/S1P Signal Pathway

With the rapid development of modern society, people’s living environment and lifestyle has changed, mental pressure increased and other factors, infertility patients is increasing year by year. Infertility has become a global medical and social problems affecting human health and development, has attaching more and more attention. In vitro fertilization and embryo transfer(IVF-ET) is one of the main means of treatment of infertility at present, has brought the gospel to the majority of infertility patients. But IVF-ET still has low success rate and high abortion rate situation, so how to further improve the clinical pregnancy rate in fertile patients, and reduce the rate of abortion is a hot topic in the research of assisted reproductive technology. According to the research report, the cause of IVF-ET failure, 1/3 owing to the oocyte quality and embryo inherent defect, while 2/3 depends on the uterine endometrium. Endometrial receptivity plays an important role in embryo implantation. In embryo implantation period, endometrial occur a serious of corresponding changes to create a suitable micro environment for embryo implantation. The previous has found that kidney-invigorating and liver-soothing treatments can improve the pregnancy outcome in patient with controlled ovarian hyperstimulation. Therefore, this thesis intends to further observe the effect of kidney-invigorating and liver-soothing treatments on endometrial receptivity and its mechanism, in order to further explore the mechanism of the two treatment in improving the pregnancy outcome, provide the experimental basis for the application of traditional Chinese medicine in the field of assisted reproduction.Part one Expression of Sphk1 in endometrium of normal women’s menstrual cycles and the relationship with estrogen receptor (ER), progesterone receptor(PR), vascular endothelial factor (VEGF)Objective : To study the expression pattern of Sphk1 in uterine endometrium of normal menstrual cycle in women, and to explore the relationship between ER,PR and VEGF.Methods: Forty patients came from the reproductive centers of Shijiazhuang Obstetrics and Gynecology Hospital and Bethune International Peace Hospital for tubal infertility in clinic with regular menstruation were involved in this study. Immunohistochemistry was used to detect the ER, PR, VEGF, Sphk1 expression in normal menstrual cycle endometrium.Results:1 Comparison of general data of patients: between the endometrial staging in patients with age, course of disease, BMI, b FSH analysis has no obvious statistical difference(P>0.05). The study is comparable.2 ER, PR expression in different endometrium periods: Expression of estrogen receptor in the proliferative phase was more than in the secretory phase. Gland express weak in the early proliferation, shows the main expression in mesenchymal cells; mid proliferation gland epithelial nuclei increased expression; up to late proliferation, glandular epithelium and interstitial cell nucleus express was stronger than before(P<0.05); early secretory nucleus and interstitial expression were decreased compared to late proliferation, there was has no statistical significant difference(P>0.05); the expression of mid secretory had decreased, most expressed in cytoplasm; the expression in late secretory had decreased compared with the late proliferation and early secretory expression was statistically significant(P < 0.05). Progesterone receptor in early proliferation mainly stromal staining; mid proliferation gland epithelial cells appeared a few nuclear staining; until to the late proliferation, gland epithelial nuclear staining increased significantly; early secretoty glandular epithelial and stromal expression were increased to peak; mid secretoty intensity decreased, compared with the early secretory was statistically significant(P < 0.05); to the late secretory expression disappeared, comparison with other phases were statistically significant(P<0.05).3 The expression of VEGF in different endometrium periods: In normal menstrual cycle of human, VEGF protein expression in endometrial mainly localized in the glandular epithelial cells and stromal cells and vascular endothelial cells, the expression change of intensity presented regulary with the menstrual cycle. The expression of VEGF is that weak in the proliferative phase and strong in secretory phase. During early and midproliferation, the expression of VEGF in glandular epithelial expression is weak, the majority expressed in mesenchymal cells; to the late proliferation, glandular epithelial expression increased, compared with early metaphase has statistical difference(P<0.05), increasing the expression of stromal cells, no significant difference(P>0.05). Late proliferative and secretory phase VEGF expression increased gradually, the expression of VEGF in the mid secretory glands and stroma were significantly higher than that in other various periods(P<0.05), the expression of VEGF were significantly in the menstrual period glandular epithelium.4 Sphk1 expreses in different endometrium periods: Sphk1 mainly expressed in endometrial vascular endothelial cells, a small amount of expression in the glandular epithelial cells and stromal cells. The Sphk1 protein expression in proliferative stage were lower than these secretory phase(P<0.05), in the mid and late secretory phase Sphk1 expression was higher than proliferative mid and late phase that in endometrium(P<0.05), appeared to the strongest expression for cycle.5 Correlation analysis of Sphk1 and ER, PR expressionin in normal menstrual cycle endometrium: Correlation analysis showed that, ER, PR and Sphk1 showed a positive correlation between the expression of proliferative phase(r=0.839, P<0.01; r=0.877, P<0.01), the secretion of ER, PR and Sphk1 in early and midsecretory expression presented negative correlation(r=-0.887, P<0.05; r=-0.858, P<0.01). The late secretory ER, PR expression to a minimum, while the expression of Sphk1 is lower than the secretion of mid, higher than proliferative phase.6 Linear correlation analysis of Sphk1 and VEGF expression in normal menstrual cycle endometrium: Endometrial glandular epithelium of VEGF protein and Sphk1 protein expression was significantly correlated(r=0.938, P< 0.01).Conclusion: 1 The expression of Sphk1 protein was always detected in the endometrial tissues in normal women throughout the menstrual cycle; 2 Sphk1 mainly expressed in endometrial vascular endothelial cells, a small amount of expression in the glandular epithelial cells and stromal cells; 3 Sphk1 expression in human endometrium has periodicity, during the peri-implantation period expression reached its peak, the change of Sphk1 may be involved in the establishment of endometrial receptivity, as one of the molecular markers to predict endometrial receptivity; 4 Sphk1 expression is regulated by ER, PR and relates to VEGF expression, to jointly participate in the angiogenesis in endometrium.Part two The Study on the influence of kidney-invigorating and liver-soothing treatments on the pregnancy outcome and hemodynamics of uterine endometrium with the people under IVF-ETObjective: To research the effect of kidney-invigorating and liver-soothing treatments on the pregnancy outcome and hemodynamics of uterine endometrium with the people under IVF-ET.Methods: One hundred and thirteen infertile patients met criteria for inclusion and exclusion criteria from Bethune International Peace Hospital undergoing the treatment of IVF-ET were randomly divided into liver-soothing group which has 25 people(Xiaoyao San+Gn RHa/FSH/h CG) and liver-soothing control group(Gn RHa/FSH/h CG) with 30 people, kidney-invigorating group(Bushentiaojing Recipe+Gn RHa/FSH/h CG) which has 30 patients and kidney-invigorating control group(Gn RHa/FSH/h CG)with 28 patients. Observe the content of days and dose of the used gonadotropin, the hormone levels of E2, LH, P on h CG day, ultrasonic detection of h CG endometrial thickness, type; uterine artery blood flow index(RI, S/D, PSV, EDV); endometrial blood parameters,pregnancy rate and complications.Results:1 Foundation data of age, BMI, AFC, basic FSH has no statistical significance between each groups(P>0.05).2 Comparison of the hormone levels of E2, LH, P on h CG day among each group: The content of E2 was significantly higher in kidney-invigorating and liver-soothing groups than in control group(P<0.05). The level of LH, P was no statistical significance between each groups(P>0.05).3 Comparison of days and dose of the used gonadotropin and endometrial thickness and pattern between each group :The time of gonadotropin were no statistical significance between each group(P>0.05) while the dose of gonadotropin were significantly lower in liver-soothing group than in liver-soothing group control group(P<0.05). The dose of gonadotropin were lower in kidney-invigorating group than in kidney-invigorating group control group(P>0.05).4 Comparison of types and thickness of endometrium: The good status of endometrium in liver-soothing group group is higher than liver-soothing group control group(P<0.05); while kidney-invigorating group compared with kidney-invigorating control group, no statistical significance(P>0.05). The endometrial thickness of kidney-invigorating group, liver-soothing group were higher than those of the control group, there is statistical significance(P<0.01; P<0.05).5 Comparison of uterine artery hemodynamics parameters: The expression of PSV of uterine artery in kidney-invigorating group on h CG day is higher than that of kidney-invigorating control group; the expression of RI of uterine artery in liver-soothing group on h CG day is lower than that of liver-soothing control group, with significant difference(P<0.05). kidney-invigorating group and liver-soothing group S/D ratio are lower than the control group, there were no statistically significant(P> 0.05).6 Comparison of uterine endometrial blood parameters: The endometrial VI was higher than that of the control group, the difference was statistically significant(P<0.01); soothing liver group endometrial VI was higher than that of the control group, the difference was statistically significant(P<0.05). Bushen, Shugan group two The value of VFI in kidney-invigorating group and liver-soothing group were higher than the control group, the difference was not statistically significant(P>0.05). The group of tonifying kidney, soothing liver group was not statistically significant(P>0.05).7 Comparison of endometrial volume: the endometrial volume of kidney-invigorating group and liver-soothing group were increased compared with their respective control group, but with no statistical difference(P>0.05).8 Comparison of pregnancy number and pregnancy rate: kidney-invigorating group 20 cases of pregnancy, the pregnancy rate was 66.67%; the kidney-invigorating control group 13 cases of pregnancy, the pregnancy rate was 46.43%, pregnancy rate were significantly difference between the two groups, with statistical significance(P<0.05); liver-soothing group 18 cases of pregnancy, the pregnancy rate was 72%; liver-soothing control group 14 cases of pregnancy, pregnancy rate of 46.67%, two groups were significant difference in the rate of pregnancy, with statistical significance(P<0.05).9 Comparison of complication: one cases biochemical pregnancy in kidney-invigorating group; one cases of ectopic pregnancy and one embryo damage in kidney-invigorating control group; one cases of ectopic pregnancy in liver-soothing group, and one cases of biochemical pregnancy, one cases of OHSS in liver-soothing control group. The incidence of complications between the groups had no statistical significance(P>0.05).Conclusions: 1 Xiaoyao San and Bushentiaojing Recipe combined with controlled ovarian hypersimulation can reduce the amount of Gn, which can lighten economic burden of IVF-ET patients; 2 Xiaoyao San and Bushentiaojing Recipe can improve pregnancy rate who is undergoing IVF-ET; 3 Xiaoyao San and Bushentiaojing Recipe can improve the endometrial blood supply to better the pregnancy outcome. 4 Bushentiaojing recipe, Xiaoyao san both can increase the level of serum E2 in patients on h CG day, increase of the thickness of endometrium. In improving the ultrasound endometrial morphology liver-soothing group is better than kidney-invigorating group; as for the uterine artery blood and endometrial flows, the expression of PSV increased in the kidney-invigorating group, while soothing liver group focused on reducing RI, the two party can improve the endometrial blood supply, creating a suitable microenvironment of embryo implantation. Part three Effects of kidney-invigorating and liver-soothing treatments on endometrial receptivity in Gn RHa controlled ovarian hyperstimulation ratObjective: To observe the effect of kidney-invigorating and liver-soothing treatments on endometrial tissue morphology and angiogenesis in Gn RHa controlled ovarian hyperstimulation rat.Methods: Totally 80 female sprague-dawley rats with normal estrous period were randomly divided into normal group, model group, kidney-invigorating group and liver-soothing group with 20 rats in each. Except the normal group, all the rats were made Gn RHa controlled ovarian hyperstimulation model(Gn RHa+HMG+h CG). First day of molding, kidney-invigorating group rats were given Bushen Tiaojing recipe, liver-soothing group rats were given Xiaoyao Pill suspension, 1ml/200g/d intragastric administration, control group and normal group rats were given the same dose of distilled water, consecutive for 11 days. The day of inject h CG, female and male rats cage in the proportion of 2:1, the next day observe the vaginal plug at 9:00a.m, see bolt is denoted as G1 day. Until G5 day, detected rats’ serum estradiol(E2), progesterone(P) content by radio immunoassay; to observe the morphological changes of endometrium using HE staining; tested endometrium estrogen receptor, progesterone receptor using immuohistochemicalmethod, electron microscopy observed endometrial pinopodes expression; statistical comparison of the rats pessary number, embryo implantation number and pregnancy rate. Results:1 Changes of general condition of rats in each group: in the course of the experiment, the normal and the treated rats in each group development in good condition, white coat color with good gloss, mental condition, weight increased significantly. The rats in the model group compared with normal group appeared less activity, little hair loss, weight gain compared with the other groups slowly. No obvious adversereaction during the treatment.2 The changes of estrous cycle in each group: normal estrous cycle of rats for 4-5 days, exiting the regularity. Model group and the treatment group rats after continuous injection 5 days after Gn RHa vaginal smears is the large number of white cells, suggesting that the change of estrous cycle disappeared, were in diestrus; estrous cycle changes of rats recovered to normal after injection of h MG, h CG on rat vaginal secretions increase, vaginal smear under display a large number of keratinocytes, in estrus. Cage visible after pessary, visible sperm smear.3 Detection of sex hormone and its receptor levels in each group:Compared with the normal group, model group rats serum P 、P/E2 value decreased;compared with the control group, serum P, P/E2 expression of kidney-invigorating and liver-soothing groups are increased(P<0.05). Among the groups of E2 did not change significantly, the difference was not statistically significant(P>0.05). Expression of model group rats, ER、PR reduced compared to normal control group; kidney-invigorating and liver-soothing groups increased as compared with model group, there were statistically significant(P<0.05).4 Uterus index detection: in the model group, the uterus index was obviously lower than that of normal group, kidney-invigorating and liver-soothing groups with significant difference(P<0.01); kidneyinvigorating and liver-soothing groups’ uterus index lower than that of normal group(P<0.05); between kidney-invigorating and liver-soothing groups were no statistically significant(P>0.05).5 Changes in endometrial surface pinopodes: the surface of normal group rats’ uterine endometrium uniformly distributed on a large number of clear boundary membrane protuberance, uniform distribution and appeared mushroom shaped, as fully developed pinopodes; in model group, the number of endometrial pinopodes lower than the normal group, the surface of which pinopodes shrinkaged, showing degradation performance; kidney-invigorating and liver-soothing groups’ pinopodes number than that in model group increased, the surface is full, relatively uniform distribution.6 The endometrial thickness, morphology, endometrial maturity and the number of uterine spiral artery: endometrial thickness in the rats of model group lower than normal group, each treatment group endometrial thickness increased. Endometrial maturity of model group is low than the the normal group and the treatment group(P<0.05). Compared kidney-invigorating with liver-soothing groups, the difference was not statistically different(P<0.05). The number of uterine spiral artery of model group rats less than the normal group, with statistically significant difference(P<0.01). Increased expression of each treatment groups compared with model group(P<0.05). Compared kidney-invigorating with liver-soothing groups, the difference was not statistically different(P>0.05).7 The rat vaginal plug rate, pregnancy rate: compared with the normal group, model group rats in the vaginal plug number and number of pregnancy were decreased, and the decreased number of embryo implantation; compared with the model group, the treatment group the vaginal plug number increased, no obvious statistical difference(P>0.05),the number of implantation and pregnancy increases the number of, with statistically significant difference(P<0.05); as for kidney-invigorating group, number of pregnancy and embryo implantation number of liver-soothing group is increased, has no significant differences(P>0.05).Conclusion: The therapies of kidney-invigorating, liver-soothing can increase the rat pregnancy rate and embryo implantation number, its mechanism may improve and regulate the level of sex hormone and ER, PR to improve the endometrial morphology and pinopodes expression on Gn RHa controlled ovarian hyperstimulation rat. Part four Effects of kidney-invigorating and liver-soothing treatments on endometrial vascular permeability and angiogenesis in Gn RHa controlled ovarian hyperstimulation ratObjective: To observe the effect of kidney-invigorating and liver-soothing treatments on endometrial vascular permeability and angiogenesis in Gn RHa controlled ovarian hyperstimulation rat.Methods: Animal grouping and modeling same as the third part. The expression of VEGF, Sphk1 protein expression using immunohistochemical and Western blot methods; VEGF m RNA, Sphk1 m RNA expression were detected by RT-PCR; expression of NO, e NOS detected by nitrate reductase method from the rat serum and uterine tissues.Results:1 The location and expression of VEGF m RNA, protein in endometrium: VEGF protein expression was mainly located in endometrial glandular epithelial cells, stromal cells and endothelial cell cytoplasm, brownish yellow granules. In model group, VEGF expression of uterus was lower than that of normal group(P<0.05, P<0.01), detected by immunohistochemistry,Western blot and RT-PCR; In kidney-invigorating and liver-soothing groups VEGF expression increased compared with those in the model group(P<0.05), close to the normal group(P>0.05).Statistical significance is not obvious between kidney-invigorating and liver-soothing groups.2 The location and expression of Sphk1 m RNA, protein in endometrial: Sphk1 protein expression was mainly located in endometrial vascular endothelial cells nuclear and cytoplasmic, a small amount of expression in the glandular epithelium, stromal cells, brownish yellow granules. In model group, Sphk1 expression of uterus was lower than that of normal group, detected by immunohistochemistry, Western blot and RT-PCR(P<0.05); in kidney-invigorating and liver-soothing groups Sphk1 expression increased than those in the model group(P<0.05).3 Comparison of NO in rat serum and uterus homogenate between each group: compared with the normal group, the expression of NO in the serum and uterus homogenate of the model group decreased. Compared with the model group, the content of NO in the serum and uterus homogenate in the kidney-invigorating and liver-soothing groups significantly increased, with significant difference(P<0.05). Compared with the kidney-invigorating group the content of NO in the serum and uterus homogenate in the liver-soothing group significantly increased, with significant difference(P< 0.05).4 Comparison of e NOS in rat serum and uterus between each group: compared with the normal group, the expression of e NOS in the serum and uterus of the model group decreased, with significant difference(P<0.05). Compared with the model group, the content of e NOS in the serum and uterus homogenate in the kidney-invigorating and liver-soothing groups significantly increased, with significant difference(P<0.05). Between group, rat serum and uteruse e NOS of kidney-invigorating and liver-soothing groups had no statistical significance(P>0.05).5 Correlation analysis of Sphk1 and VEGF: Sphk1 and VEGF content of two variables is a linear trend, correlation analysis showed: the correlation coefficient Sphk1 and the detection of VEGF is 0.865, P=0.000, with statistical significance.Conclusion: 1 VEGF, Sphk1 m RNA and protein expression decreased in the controlled ovarian hyperstimulation rat endometrial, leading poor angiogenic and declined permeability. 2 Controlled ovarian hyperstimulation rats endometrium vasodilation factor NO and e NOS expression decreased, endometrium permeability decreased. 3 Sphk1 and VEGF expression has relativity, jointly participate in endometrial angiogenic. 4 kidney-invigorating and liver-soothing methods can through the regulation of endometrial VEGF, Sphk1, NO, e NOS expression, prompting the two methods can increase endometrial angiogenesis and permeability changes to improve the endometrial receptivity in COH rats.Between the two methods, the expression of kidney-invigorating group through the regulation of NO of serum and uterus homogenate enhanced than kidney-invigorating group, prompting kidney-invigorating method is superior to liver-soothing method in improving endometrial receptivity. Part five Influence of kidney-invigorating and liver-soothing treatments on the regulation of Sphk1/S1 P signal pathway in Gn RHacontrolled ovarian hyperstimulation rat endometriumObjective: Preliminary studies have confirmed that kidney-invigorating and liver-soothing treatments can enhance the expression of Sphk1 to improve the endometrial receptivity, therefore this study was designed to further investigate the related mechanism of action.Methods: Animal experiment and specimen collection were the same as the third part. Testing index and method for the expression: Sphk1, S1P1, Akt, p-Akt, Cyclin D1 protein expression were detected by western blot; Sphk1, S1P1, Akt, p-Akt, Cyclin D1 m RNA were detected by RT-PCR.Results:1 Sphk1 m RNA and protein expression: compared with normal group, Sphk1 m RNA expression of COH model group decreased, with significant difference(P<0.05). Compared with the model group, the expression of Sphk1 m RNA in kidney-invigorating and liver-soothing groups enhanced, with significant difference(P<0.05). The expression of Sphk1 m RNA in kidneyinvigorating and liver-soothing groups has decreased than the normal control group, no significant difference(P>0.05). There is no significant difference between kidney-invigorating group and liver-soothing group(P>0.05).2 S1P1 m RNA and protein expression: compared with normal group, S1P1 m RNA expression of COH model group decreased, with significant difference(P<0.01). Compared with the model group, the expression of S1P1 m RNA in kidney-invigorating and liver-soothing groups enhanced, with significant difference(P<0.05). The expression of S1P1 m RNA in kidney-invigorating and liver-soothing groups has decreased than the normal control group, has significant difference(P<0.05). The expression of S1P1 m RNA in kidney-invigorating group enhanced than liver-soothing group, with significant difference(P<0.05).3 Akt m RNA and protein expression: compared with normal group, Akt m RNA expression of COH model group decreased, with significant difference(P<0.05). Compared with the model group, the expression of Akt m RNA in kidney-invigorating and liver-soothing groups enhanced, with no significant difference(P>0.05). The expression of Akt m RNA in kidney-invigorating and liver-soothing groups has decreased than the normal control group, no significant difference(P>0.05). The expression of Akt m RNA in kidney-invigorating group enhanced than the liver-soothing group, with no significant difference(P>0.05).4 p-Akt protein expression: compared with normal group, p-Akt protein expression of COH model group decreased, with significant difference(P<0.05). Compared with the model group, the expression of p-Akt protein in kidney-invigorating and liver-soothing groups enhanced, with significant difference(P<0.05). There is no significant difference between kidney-invigorating group and liver-soothing group, the expression of p-Akt protein in kidney-invigorating group enhanced than liver-soothing group(P>0.05).5 p-Akt/Akt expression: compared with normal group, p-Akt/Akt expression of COH model group decreased, with significant difference(P<0.01). Compared with model group, the expression of p-Akt/Akt in kidney-invigorating and liver-soothing groups enhanced, with significant difference(P<0.05). Compared with normal group, p-Akt/Akt expression of kidney-invigorating and liver-soothing groups decreased, with significant difference(P<0.05). Compared with kidney-invigorating group, the expression of p-Akt/Akt in kidney-invigorating group enhanced, with no significant difference(P>0.05).6 Cyclin D1 m RNA and protein expression: compared with normal group, Cyclin D1 m RNA expression of COH model group decreased, with significant difference(P<0.05). Compared with the model group, the expression of Cyclin D1 m RNA in kidney-invigorating and liver-soothing groups enhanced, with significant difference(P<0.05). The expression of Cyclin D1 m RNA in kidney-invigorating and liver-soothing groups has decreased than the normal control group, with significant difference(P<0.05). The expression of Cyclin D1 m RNA in kidney-invigorating group has decreased than the liver-soothing group, with significant difference(P<0.05).Conclusion: Kidney-invigorating and liver-soothing treatments can increase the expression of Sphk1 in endometrium, active PI3K/Akt signal transduction pathway, up regulation of Cyclin D1 expression, so as to play the role of promoting cell proliferation, as play its role to improve the endometrial receptivity. Kidney-invigorating treatment is superior to liver-soothing treatment in improving S1 P receptor expression and cell proliferation Cyclin D1 aspects.