The Role Of Activin A And Related Growth Factors In Pathogenesis And Clinical Treatment Of Endometriosis

BackgroudEndometriosis is a common and frequently-occurring disease in women of childbearing age, whose incidence trend has increased significantly in recent years, reaching10%-15%. The main manifestations are infertility, dysmenorrhea, dyspareunia, and chronic pelvic pain, seriously affecting women’s health and quality of life. Although it is a benign disease, it has extensive lesions, morphological diversity and malignant biological behavior with implantation, invasion, proliferation, metastasis and recurrence.Pathogenesis of endometriosis remains unclear. Sampson propounded the theory of retrograde menstruation in1921which was proved in many clinical and experimental data, but it is common and even physiological phenomenon that menstrual blood reflux to the pelvis, and most of people did not suffer from endometriosis. In recent years, chinese scholars have proposed "eutopic endometrium determinism" that the biological characteristics of the eutopic endometrium is the determing factor of endometriosis. Under the cyclical regulation of ovarian hormones, a lot of cytokines produced by the endometrium cyclically change. Such as Activin A, its related growth factors (Follistatin、Cripto), and etc. In normal physiological conditions, under the regulation of ovarian hormones, when the endometrium converts from the proliferative phase to the secretory, Activin A gradually increases to the highest level at the late secretory phase, Cripto increases at the proliferative phase, while Follistatin remains relatively low. New research shows the expression of Activin A and its related growth factors completely differs between the endometriotic eutopic endometrium and the one with normal menstrual cycle. The mRNA expression of Activin A in endometriotic eutopic endometrium at the proliferative phase is significantly higher than the normal, but it is not higher at the secretory phase. The mRNA expression of Follistatin was significantly higher at the secretory phase than that at the proliferative phase and the nomal endometrium.The mRNA expression of Cripto in endometriotic eutopic endometrium is significantly lower than that in the normal endometrium at the proliferative phase, while it is relatively high at the secretory phase. Thus, we concluded there would be some kind of dynamic equilibrium between Activin A and its related growth factor in the endometrial tissue, which would control the Activin signal pathway to express the needed physiological effect. The change of the dynamic equilibrium may strengthen the capacity of proliferation, adhesion, invasion and release cytokines which promote the occurrence and development of endometriosis. But the reason that influence the expression and secretion of Activin A, Follistatin, Cripto in the eutopic endometrium is still not clear at present.As is known to all, endometriosis is a kind of hormone-dependent diseases and inflammatory disease. A large number of abnormal inflammatory factors expression, interleukin-1β is one of the important molecules among them.So, this study was to investigate the effects of interleukin-1β and estrogen on Activin A, Follistatin, Cripto and other factors in endometriotic eutopic endometrium.It has been more than a century since Von Recklinghausen first proposed and named endometriosis in1885. Even though the clinical method of therapy has been initially established in endometriosis, we still face problems of pain, infertility, high recurrence rate after surgery and drug withdrawl, and treatment of deep lesion.The novel treatment have been the research focus. Promoting decidualization of the secretory phase membrane,promoting apoptosis, anti-adhesion, anti-invasive, and anti-angiogenic have gradually become new strategies for endometriosis. The mRNA expression of Activin A in endometriotic eutopic and ectopic endometrium reduces in the secretory phase,but the mRNA expression of Follistatin and Cripto,antagonist of Activin A, increases.Thus we speculat that this makes the function of Activin A decrease in secretory phase, loss the opportunity of decidualization. And Cripto can promot apoptosis, adhesion, invasive, and angiogenic,which also promot the development of endometriosis.Therefore, our study investigated the effect of Cripto monoclonal antibodies on secretory phase endometriotic ectopic endometrial stromal and epithelial cells in vitro, and Preliminarily explored possible therapeutic effect of Cripto monoclonal antibody on endometriosis at the cellular level.ObjectiveIn this study, endometriotic eutopic endometrium was primarily cultured, which was identified as endometrial stromal cells. The culture was intervened with different concentrations of interleukin-1β and estrogen. Reverse transcription real-time PCR and ELIS A techniques were used to detect the change of Activin A, Follistatin, Cripto at the mRNA and protein levels.At the same time endometrial stromal and epithelial cells in ovarian endometriotic cyst wall were isolated,cultured and identifiedj,which was devided to treatment group with Cripto monoclonal neutralizing antibody and control group. Annexin V-FITC/PI flow cytometry was used to test apoptosis, crystal violet staining was used to test the capacity of cell adhesion and Transwell chamber was used to test the capacity of cell invasion.Our research explored the role of interleukin-1β to the expression and protein excretion of Activin A、Follistatin、Cripto in the endometriotic eutopic endometrial stromal cells. Inhibition of Cripto neutralizing antibodies against endometriotic ectopic endometrial cells was also investigated. It provided the valuable scientific data to further explore the pathogenesis of endometriosis and new ideas for treatment of endometriosis to relieve the suffering of patients,reduce the recurrence rate and cancerization, and improve the capacity of fertility and clinical outcomes.Methods1. The endometriotic eutopic endometrium were taken as samples.Human endometrial cells were isolated enzymatically from endometrium. Stromal cells were seperated by filters with200mesh and identified by vimentin and keratin.2. The endometrial stromal cells at the third generation of the logarithmic phase were intervened with different concentrations of interleukin-1β and estrogen.3. RT-qPCR technique was applied to detect different mRNA expression of Activin A, follistatin and crypto, intervened by defferent concentrations of interleukin-1β and estrogen.4. ELISA was used to detect changes of protein excretion of Activin A、 Follistatin and crypto, intervened by defferent concentrations of interleukin-1β and estrogen.5. Ovarian endometriotic cyst wall were taken as samples.The stromal cells and epithelial cells were isolated by which were identified by vimentin and keratin.6. The third generation of cultured stromal cells and the second generation of epithelial cells both in logarithmic growth phase were respectively devided to treatment group with Cripto monoclonal neutralizing antibody (concentration to5μg/mL)and control group (only complete medium).7. Annexin V-FITC/PI Flow cytometry was applied to detect apoptosis both in the treatment group and the control group.8. Crystal violet staining was used to test the capacity of cell adhesion both in the treatment group and the control group.9. Transwell chamber was applied to test the capacity of invasion of cells both in the treatment group and the control group.10. Statistical analysis: all of the experimental quantitative data was described with the arithmetic mean±standard deviation (x±SD).The differences of means in groups was compared by One-way ANOVA. Bonferroni or Dunnett T3test was used for multiple comparison. Independent samples t-test was used to compare the differences between the treatment group and control group after homogeneity of variance was test, P<0.05was considered statistically significant.Results1.The primary culture, observation and identification of eutopic endometrial stromal cells.Before culture flasks were completely covered by cells, they were flat shaped and separated from each other,whose outline and nuclear were not clear. When the cells overgrow culture flasks, they were slim, shuttle-shaped, packed tightly and arranged in parallel. They were positive for vimentin but negative for cytokeratin by Immunohistochemical staining.2. mRNA expression of Activin A and Cripto have a dose dependent increase to Interleukin1β and estrogen. mRNA expression of follistatin have a dose dependent increase to Interleukin1β. The difference was statistically significant. But estrogen did not change the mRNA expression of follistatin.3.Secresion of protain of Activin A has a dose dependent increase to Interleukin1β and estrogen. Secresion of protain of follistatin has a dose dependent increase to Interleukin1β. The difference was statistically significant. But estrogen did not change the secresion of protain of follistatin. When concentration of interleukin-1β was500pg/ml, content of Cripto was much higher than the control group, and the difference was statistically significant.The content of Cripto had a slight increase compared with the control group when concentration of interleukin-1β was not500pg/ml, the difference was not statistically significance. When concentration of estrogen was10-6M and10-5M, content of Cripto was much higher than the control group, and the difference was statistically significant.4. Culture, observation and identification of ectopic endometrial cells.Before culture flasks were covered completely by stromal cells, they grow dispersed and stretched, whose outline and nuclear were not clear. When the cells overgrow culture flasks, they were slim shuttle-shaped, packed tightly and arranged in parallel.They were positive for vimentin but negative for cytokeratin by Immunohistochemical staining. Epithelial cells formed dense colonies, which insert each other during growth.They were polygonal or tadpole-shaped, whose outline were clear and nuclear were significantly larger.They were swirling-packed or group-packed. They were negative for vimentin but positive for cytokeratin by Immunohistochemical staining. 5. Cripto monoclonal neutralizing antibodies had an role on endometriotic ectopic endometrial stromal cells and epithelial cells,which increased apoptotic cells compared with the control group, the difference was statistically significant.6. Cripto monoclonal neutralizing antibodies had an role on endometriotic ectopic endometrial stromal cells and epithelial cells,which inhibited cell adhesion compared with the control group, the difference was statistically significant.7. Cripto monoclonal neutralizing antibodies had an role on endometriotic ectopic endometrial stromal cells and epithelial cells,which decreased the amount of invasive cells compared with the control group, the difference was statistically significant.Conclusion1. Interleukin-1βand estrogen affect the secretion and expression of Activin A, follistatin and crypto in endometriotic eutopic endometrium, suggest that it may participate in the control of Activin A and related factors.2. Cripto monoclonal neutralizing antibodies can induce apoptosis of stromal and epithelial cells in endometriotic ectopic endometrium and inhibit its adhesion, invasion, suggest that it may inhibit the development of endometriosis.