Study On Expression Of Gonadotropin-releasing Hormone And Its Receptors In Endometriosis

Background: Endometriosis (EM), defined histologically as a pathological entity of the presence of endometrium-like glands and stroma outside the uterus, with a morbidity as high as 10-15%. It seems that the condition shows a trend with gradual increase among the population. It shares features with malignant tumors (metastasis and planting) but is not neoplastic. Despite of the fact that endometriosis is one of the most frequent gynecological diseases, the mechanism of its happening is still under detailed scrutiny with some controversy and it is in lack of effective cure methods. In recent years, Gonadotropin-releasing hormone analogs(GnRH-a),together with laparoscopy, has been used in treatment of endometriosis and confirmed effective.The recognized mechanism of this kind of drugs is that it binds to the GnRH receptors(GnRH-R) on the surface of gonadotropin cells in pituitary and makes eutopic and ectopic endometrium atrophy through down-regulating the pituitary-gonad axis. Since 1970's there is growing evidence that auto-paracrine gonadotropin-releasing hormone (GnRH) system exists in human extrapituitary tissues. In human normal or tumor tissues controversial data have been obtained relating to the presence and function of GnRH-R and its reaction to different GnRH-a. Based on this we inspected the expression and significance of GnRH system in endometriosis ,it would provide some clues leading to effective diagnosis and treatment strategies for endometriosis. Part I Protein Expression of GnRH and GnRH-R in Different Kinds of Endometrial Tissues Objective: To investigate and compare the protein expression of GnRH and GnRH-R in endometriosis ,endometrial hyperplasia ,endometrial carcinoma and control endometrium, to further discuss the function of GnRH system in different kinds of endometrial tissues. Materials and Methods: 1. Endometrial samples ,obtained from women undergoing surgery in hospital , were classified according to pathological diagnosis into five groups:eutopic endometrium of EM(n=28), ectopic endometrium of EM(n=24) ,endometrial hyperplasia(n=13),endometrial carcinoma(n=17) and control(n=22). 2. Immunohistochemistry(IHC) assay was used to detect the protein expression levels of GnRH and GnRH-R in these specimen. Results: 1. GnRH and GnRH-R were expressed at the protein level in all eutopic endometrium of EM and control group, majorly localized in the plasma of glandular epithelium cells and some stroma cells,with the most intense staining during the luteal phase. 2. In approximately 50% ectopic endometrium of EM GnRH and GnRH-R protein were found,lower than control group(P<0.01).They were localized majorly in the plasma of ectopic glandular epithelium cells . 3. GnRH and GnRH-R protein exist in 92.3%endometrial hyperplasia, in endometrial carcinoma their positive rate are 58.8%* and 82.4% respectively. Only one of them (with*)has statistic significance compared with control(P<0.01).They majorly localized in the plasma of hyperplastic or carcinomatous changed glandular epithelium cells ,stroma cells were no or faint stained. Conclusion: 1. GnRH and its receptor protein exist in all eutopic endometrium,with the most intense staining in luteal phase. 2. GnRH and its receptor protein were expressed in approximately 50% ectopic endometrial tissue of EM,it maybe because that about half of the ectopic endometrium samples have no distinctive glandular epithelium cells. 3. The positive expression of GnRH and GnRH-R protein in endometrial hyperplasia and endometrial carcinoma is lower than control ,the reason of this is not clear but GnRH-R still can be diagnosis or therapy target for its high positive rate . Part ⅡmRNA Expression of GnRH and GnRH-R in Endometriosis Objective: Study on the mRNA expression of GnRH and GnRH-R in endometriosis to confirm their auto-paracrine function in endometriosis.Materials and Methods: 1. Thirty-six endometrial sampls , preserved in liquid nitrogen immediately after operation, were classified according to pathological diagnosis and laparoscopy into three groups:eutopic and ectopic endometrium of EM, control without EM. 2. The extraction of RNA from the tissue sample was carried out according to TRIZOL one step assay.For each mRNA to be detected ,RT reaction was completed as described in reagent box. 3. Quantitative competitive PCR(QC-PCR) technique was used to examine GnRH gene expression in samples of endometriosis and control. 4. Nested PCR assay was used to examine GnRH-R gene expression in eutopic and ectopic endometrium and control group without endometriosis. Results: 1. GnRH mRNA existed in all eutopic endometrium come from women with or without endometriosis ,and its level was higher in the luteal phase than in the follicular phase(P<0.05). 2. GnRH -R mRNA was also present in all eutopic endometrium. 3. GnRH and its receptor mRNA were present in 6/10 and 5/10 of ectopic endometrium,lower than eutopic endometrium(P<0.01). Conclusion: The results of RT-PCR is consistent with that of IHC,the auto/paracrine of GnRH and GnRH -R in eutopic and ectopic endometrium of endometriosis were approved. Part ⅢCell Culture of Eutopic Endometrium of EM Objective: To create cell culture of eutopic endometrium of EM and to further detect the expression of GnRH and its receptors at cell level。Materials and Methods: 1. Seventeen eutopic endometrial sampls of EM and control were cultured with enzyme digest method. 2. Mouse-anti-human keratin and vimentin were used as first antibody to evaluate the cultured cells. 3. GnRH and GnRH-R were detected in cultured cells using immunocytochemistry(ICC)assay. Results: 1. Fifteen eutopic endometrial samples were successfully cultured,two were bacteria contaminated. 2. Mixed culture of endometrial cells contains glandular epithelium and stroma cells,after 3 to 4 times of transfer of culture more than 90% of them were stroma cells. 3.GnRH and GnRH-R protein were found in cultured glandular epithelium and stroma cells too, after 2 to 3 times of transfer of culture GnRH expression wes no more exist. Conclusion: Mixde culture of eutopic endometrium by enzyme digest method is convenient and can be used as research model of EM;ICC confirm that GnRH and GnRH-R protein exist in cultured glandular epithelium and stroma cells.