(English) Effect Of Huluantang On The Endometrial Receptivity Of Controlled Ovarian Hyperstimulation Rats Assisted By GnRHa

ObjectiveResearch the effect of Huluantang on the endometrial receptivity of controlled ovarian hyperstimulation rats assisted by GnRHa. Main observation conclude the effects of Huluantang on rat uterus endometrium cell morphology, the related markers expression of endometrial receptivityMethodsThe first part Research backgroundApplication of computer network technology, searching for a large number of related literatures, doing thorough and systematic collation and analysis, research on IVF-ET, COH, endometrial receptivity, Chinese and Western medicine on the influence factors of endometrial receptivity and the latest developments in this field. A large number of studies have shown that Chinese medicine in the treatment process of IVF-ET with characteristics of good efficacy, safety, no advantage of drug resistance, worthy of further exploration and study.The second part Effects of Huluantang on endometrial tissue morphology of GnRHa hyperstimulation ratsExperiments using SPF level SD rats, divided rats randomly into3groups:model group, Huluantang group, respectively, while setting normal group. Observation on vaginal exfoliated cells in smear, removed uterus, weighed and produced specimens in pregnant2,5days, observed endometrial morphology under light and electron microscopic, mainly observed and tested on uterine maturity index, endometrial thickness, endometrial degree of maturation, glycogen, pinopodes indicators in different groups of pregnant rats.The third part Effects of Huluantang on endometrial receptivity related factors expression of GnRHa hyperstimulation rats Experiments using SPF level SD rats, divided rats randomly into3groups:model group, Huluantang group, respectively, while setting normal group. Taking jugular venous blood, removed uterus, produced specimens inpregnant2,5days, applied Radioimmunoassay method, protein chip detectionmethod for testing endometrial receptivity related factors in different groups of pregnant rats, such as E2,P,P/E2, IFN-γ,TNF-α,IL-4,IL-6,TGF-β1,E-selectin,L-selectin,Leptin,OPN, TIMP-3,ICAM-1, and observed effects of Huluantang on endometrial receptivity related factors expression.ResultsThe first part Research backgroundBy on network database retrieved and large literatures finishing and analysis, we found, important links of general IVF-ET is COH, the application of long programme is most universal; however application of COH drugs brings inevitable problems, effects the endometrial receptivity directly and indirectly, reduces pregnancy rate. Theory of TCM think that endometrial receptivity is closely related to kidney, Qixue, and chongren, kidney essence filling, and Qixue adequate, Zang-Fu, Meridian, and Qixue keep balance then uterine can capacity real; Modern medical think that endometrial receptivity is controlled bymultiple hormones, cytokines, and enzymes. Mentor based on many years of clinical experience, Self-made Huluantang, it respects kidney role, coordinating multiple viscera function, conditioning ChongRen, adjusting Qixue, herbs in Huluantang can control hormones, cytokines, and improve the endometrial receptivity effectively, so,we makes Huluantang as participants.The second part Effects of Huluantang on endometrial tissue morphology of GnRHa hyperstimulation rats1. General conditions: During the animal experiments, rats can be in good spirit; activities freely; bite and sup, defecation and urination will be normal, shiny hair. No adverse reaction during medication.2. Estrous cycle: Vaginal exfoliated cells smears shows that estrous cycle of model group is disappearing after injecting Alarelin5d, estrous cycle recovery after injecting HMG, estrous cycle of Huluantang group and normal group are not disappearing, showing the cells performance, but the cycle variation rate of Huluantang group slows down, cyclical extension.3. The uterus index of rats: Pregnant2d,5d model group, Huluantang group, normal group compared with each other, uterus index were statistically significant (P<0.01); compared with each same group, the uterus index also were statistically significant (P<0.01).4. Rats endometrial thickness and endometrial degree of maturity:Pregnant2d,5d model group, Huluantang group, normal group compared with each other, endometrial thickness were not statistical significant (P>0.05); Compared with each same group, were statistically significant (P<0.01). Pregnant2d model group, Huluantang group, normal group compared with each other, endometrial degree of maturity were not statistical significant (P>0.05), Pregnant5d model group compared with Huluantang group, were statistical significant (P<0.05), compared with normal group, were statistical significant (P<0.01); Huluantang group compared with normal group, was no statistical significant (P>0.05).5. Observation on the morphology of the rats'endometrium under light microscopic: Pregnant2d model group endometrial glands maldevelopment, form does not rules, not more, glands cavity mild expand and bent, mesenchyme loose edema, vascular less; Huluantang group glands have good development, part form does not rules, not more, glands cavity slightly increases, secretion real slightly increased, mesenchyme loose and edema does not obvious, less vascular hyperplasia, filling; normal group glands have very good development, increased does not obvious, glands cavity slightly increases, mesenchyme loose and density, majority cells are shuttle shaped, vascular less. Pregnant5d model group less glands, glands epithelial cell hyperplasia, glands cavity narrow, bent is not obvious, mesenchyme density, majority are shuttle shaped cell, contains large matrix cell gathered, and has giant peritoneal macrophages cell infiltration, vascular less; Huluantang group glands development obvious, more, glands cavity obvious increases, secretion real obvious increased, the matrix cell in mesenchyme obvious hyperplasia, mesenchyme loose and edema, more round, less shuttle shaped, vascular hyperplasia; normal group glands more, development good, cavity large and filled with rich glandular secretions, apparent proliferation of mesenchymal stromal cells, loose, cells more round, hyperplasia of vascular clearly.6. Observation on glycogen of ratsendometrium under light microscope: Pregnant2d,5d model group compared with Huluantang group, normal group were statistical significant in the number of rats endometrial glycogen (P<0.01), and Huluantang group compared with normal group was not statistical significant (P>0.05). Compared with each same group, were statistically significant (P<0.01). Pregnant2d model group's glycogen was more than Huluantang group and normal group, Pregnant5d model group's glycogen was significantly less than that Huluantang group and normal group.7. Observation on the rat endometrium pinopodes under the electron microscope: Pregnant2d model group endometrial surface form is hot uniform, has less pinopodes expression, form size varies, development does not synchronization, most surface is covered by thick micro-fluff, no shaped bumps, cell boundaries is clear; Huluantang group surface form structured, no pinopodes expression, epithelial cell distribution is uniform, form size is relative consistent, development basic synchronization, its top is covered by long micro-fluff, cell boundaries is clearly, micro-fluff mutual fusion and constitute small bumps; Normal group surface form structured, no pinopodes expression, epithelial cell distribution, size and form is consistent basically, development synchronization, the top is covered by relatively short micro-fluff, cell boundaries is lack of clarity, micro-fluff mutual fusion and constitute small bumps. Pregnant5d model group surface form flat, but not uniform, pinopodes are full disappeared, epithelial cell form size varies, distribution does not uniform, its top is covered by length varies of micro-fluff, cell boundaries is not clearly, endometrium is advanced secretion; Huluantang group surface has large of shaped bumps, pinopodes expression are clear, its surface is not very smooth, distribution relative uniform, form size relative consistent, cell boundaries is basic clearly, development is basic synchronization, bumps surface covers more short and fine micro-fluff; Normal group has a large number of pinopodes, micro-fluff disappeared, surface is smooth, bumps are significantly, distribution, form and size are consistent, synchronized development, mutual boundaries is clear, for the full development of pinopodes.The third part Effects of Huluantang on endometrial receptivity related factors expression of GnRHa hyperstimulation rats1. Comparison of E2,P,P/E2of pregnant rats: Pregnancy2d E2of model group, Huluantang group are higher than normal group, were statistically significant (P<0.01), model group compared with Huluantang group, was not statistically significant (P>0.05); pregnant5d model groups, Huluantang group are also higher than normal group, were statistically significant (P<0.05, P<0.01), model group compared with Huluantang group, was not statistically significant (P>0.05). Pregnant2d,5d compared between same groups, statistically significant in model group and Huluantang group, normal group was not statistically significant.Pregnant2d P of model group, Huluantang group is higher than normal group, was statistically significant (P<0.01), model group is higher than Huluantang group, was statistical significant (P<0.01); pregnant5d model group, Huluantang group is higher than normal group, was statistically significant (P<0.05), model group is lower than Huluantang group, was statistically significant (P<0.05). Pregnant2d,5d compared between same groups, model group has no statistical significant (P>0.05), Huluantang group and normal group were statistically significant (P<0.01).Pregnancy2d model group. Huluantang group P/E2are lower than normal group, were statistically significant (P<0.01), model group is slightly higher than Huluantang group, but was not statistically significant (P>0.05). Pregnant5d Huluantang group and normal group P/E2are higher than model group, were statistically significant (P<0.05). Huluantang group compared with normal group, was not statistically significant (P>0.05).2. Endometrial Thl,Th2cytokine expression of pregnant rats: Pregnant2d,5d IFN-γ, and TNF-a of model group are higher than normal group, and expression is raised than1.5times, Huluantang group are lower than model group, and expression is declined than1.5times; pregnant2d Huluantang group IL-4, IL-6are higher than model group and normal group, but raised less, model group is declined, but not clear; pregnant5d model group IL-4, IL-6are lower than Huluantang group and normal group, and expression is declined than1.5times, Huluantang group compared with model group expression is raised than1.5times. Model groupThl/Th2is raised; Huluantang group Th1/Th2is declined.3. Endometrial receptivity related factors expression of pregnant rats: Pregnant2d, compared with normal group, model group TGF-β1, E-selectin, L-selectin and Leptin expression is reduced more than1.5times, OPN, TIMP-3, ICAM-lis declined slightly. Compared with model group, Huluantang group TGF-β1, TGF-β3, E-selectin, L-selectin, Leptin and TIMP-3is raised more than1.5times, OPN and ICAM-1are raised slightly; pregnant5d, compared with normal group, model group TGF-β1, TGF-(33, E-selectin, L-selectin, Leptin TIMP-3and ICAM-1expression is declined more than1.5times, OPN also declined slightly. Compared with model group, Huluantang group TGF-(31, TGF-β3, E-selectin, L-selectin, Leptin, TIMP-3expression is raised more than1.5times, OPN and ICAM-1are also raised slightly.Conclusions:1. By on the collation, analysis, summary of ancient literatures of traditional Chinese medicine and modern medical advances, we found that bad endometrial receptivity is the main reason for the failure of IVF-ET, which caused low pregnancy rate.In recent years, modern research shows traditional Chinese medicine may improve endometrial receptivity, and increase pregnancy rate. TCM believes that deficiency of the kidney, deficiency of the kidney essence is of poor endometrial receptivity main pathogenesis, Qixue weak, ChongRen disharmony is synergistic pathogenesis. Mentor respects coordination of the kidney, liver, heart, spleen, dual position on time frames and Zang-Fu, pay attention to the coordination of the Qixue and ChongRen, Primary and secondary treatment, self-made Huluantang to improve endometrial receptivity, and increase pregnancy rate.2. GnRHa hyperstimulation model rats implantation window period ahead of open and close, uterine index and endometrial degree of maturation are lower; endometrial bad developed, development of glands and mesenchyme does not synchronize; less glycogen; pinopodes appears ahead, expression does not uniform; E2, P increased obviously, and P rised ahead; IFN-y and TNF-alpha are raised, IL-4, and IL-6are declined, Th1/Th2is imbalances, important molecular markers of embryo implantation:TGF-β1, TGF-β3, E-selectin, L-selectin, Leptin, TIMP-3, ICAM-1are reduced, it shows that COH drugs has direct and indirect impact on the above factors, reduced endometrial receptivity, led to the failure of implantation, declined the pregnancy rate.3. Huluantang can delay the open and close of implantation window, increase uterine index and endometrial degree of maturity; led glands and mesenchyme development synchronized, increased glycogen; change expression time range of pinopodes, makes it uniform; coordinated E2, P and P/E2ratio; reduced IFN-γ, TNF-α expression, increased IL-4, IL-6expression, makes Th1/Th2cytokines ratio be balanced, overall regulation of immune imbalance; increased TGF-β1, TGF-β3, E-selectin, L-selectin, Leptin, TIMP-3expression, and ultimately achieve the goal of improve endometrial receptivity, embryo implantation, and improve pregnancy rate.4. In this study, the innovation lies in:(1) Using various experimental methods to discuss the effects of Huluantang on endometrial receptivity of GnRHa hyperstimulation rats on multi-level protection. Provide the scientific basis for the clinical treatment of IVF-ET.(2) Discussion on the mechanism of improving endometrial receptivity by Huluantang then provides experimental basis for further research and development of new traditional Chinese medicine.(3) Provided a preliminary idea for treatment of IVF-ET by traditional Chinese medicine, deeper research of mechanism of traditional Chinese medicine on endometrial receptivity, timely start implantation of auxiliary therapeutic effect and mechanism, further improvement of IVF-ET pregnancy rates safely and effectively.5. Ideas for future research:improving research design, on the basis of existing research, through further research of cytokine signaling pathway and endometrial gene expression, in order to discuss on the mechanism and links of Huluantang in deep level.