Establishment Of A Novel In Vitro Model-human Endometriosis Histologic Model And Investigation Of The Attachment Characteristic Of The Eutopic Endometrium From Endometriosis And Non-endometriosis Women

Establishment of a novel in vitro model—human Endometriosis histologic model and investigation of the attachment characteristic of the eutopic endometrium from endometriosis and non-endometriosis women.BackgroundEndometriosis is a common disease of the reproductive women that induces menorrhalgia, chronic pelvic pain and infertility and affects their health as well as life quality. The pathogenesis has not been clearly clarified and treatment options are limited. Among the theories of endometriosis, the implantation theory of Sampson is widely accepted. However, it can't explain that up to 90％of women may experience some degree of retrograde menstruation yet only a few develop endometriosis and how endometriosis can form outside the peritoneal cavity. The latest research has revealed some differences of eutopic endometrium between endomertiosis patients and normal women, as far as the endometrial ultramicrostructure, gene, expression product of DNA and functional status are concerned.Models are the important tool for studying endometriosis. Some kinds of endometriosis models have been established, but they have insufficiency. Such as cell models, quite different from the real physiological status in human being, are unable to reflect the comprehensive and complicated interactions of cells in vivo. Some animal models, such as rabbit model and rat model, are regarded by some scholars not suitable and lack a menstrual cycle. Ideal models of macaque or baboon are rare and too expensive so lacks of feasibility for research.Endometriosis histo]ogic model is a novel model in recent 10 years, established by inoculation of in-vitro-separated endometrium fragments on peritoneum, It has the following advantages:①good imitation: It reflects direct interaction of endometrium and peritoneum and imitates pathogensis in early stage.②It presents a visibly dynamic and progressive development for understanding the adhesion, aggressiveness and agiogenesis of eutopic endometrium to peritonaeum and provides direct histologic proof.③suitable for comparison of endometrium and peritoneum in patients and normal women.④aboundant in resources and feasible in practice. But the suitable culture condition about the model is not found out and no research is involved in quantitation up to now.Objective1. To investigate culture environment for endometrium and the adhesion of endometrium to peritoneum.2. To analyze the cellular and biomolecular features in adhesion of endometrium to peritoneum.3. To compare the adhesion features of eutopic endometrium from patients with endometriosis and the control.Methods1. Observe and compare the effect on endometrium and adhesion of endometrium to peritoneum using gas-liquid surface culture and in-medium culture.2. Collect specimens at different times for observation under light microscope and electron microscope to learn the timing of adhesion and the cellular features at adhesion sites. Detect secretion of sICAM-1, MMP-2 and VEGF in cultures using ELISA technique.3. Identify the cells at adhesion sites using immunohiotochemistry and immunofluorescence techniques.4. Observe and compare the endometrium as well as peritoneum from patients with endometriosis and the control under electron microscope.5. Observe and compare the adhesion characteristic after auto inoculation and cross inoculation of endometrium to peritoneum using vibration test, light microscope and electron microscope.6. Culture endometrium of patients with endometriosis and the control for detection of sICAM-1 secretion by ELISA. Results1. Gas-liquid surface culture is superior to in medium culture in maintaining tissue structure and survival.2. Faster adhesion and invasion of endometrium to peritoneum is observed in gas-liquid surface culture.3. Peritoneal mesothelium defects were found after adhesion by gland cells and stromal cells of endometrium and disappeared gradually as the cultivation continued.4. Apparent invasion of endometrium into peritoneum was observed in culture 3 days after inoculation. There was a seemingly trend that the endometrium of patients with endometriosis had an earlier invasion than that of the control.5. Active secretion of sICAM-1, MMP-2 and VEGF was detected in endometrium culture and peritoneum culture with inoculation of endometrium, sICAM-1 was expressed in peritoneum with little MMP-2 and VEGF.6. Ultrastructural discrepancy was found between endometrium of patients with endometriosis and that of the control, the former demonstrating more active functional units.7. There was little difference in adhesion ability in vitro between endometrium of patients with endometriosis and that of the control.8. There was an higher increase in sICAM-1 secretion in cultured endometrium of patients with endometriosis than that of the control.Conclusion1. Gas-liquid surface culture is suitable for human endometriosis histologic model.2. Adhesion of endometrium to peritoneum progresses very quickly as evidenced by formation of firm adhesion only one hour after inoculation and apparent invasion into the fibrous layer of peritoneum 3 days later.3. Some differences were observed in the eutopic endometrial ultrastructure between endometriosis and non-endometriosis women indicating the former has an active function, with suggests eutopic endometrium plays an important role in the pathogenesis of endometriosis. 4. There was little difference in adhesion ability in vitro between endometrium of patients with endometriosis and that of the control, however, there might be some differences in the clearance of the adhesion and subsequent invision.5. The mesothelial layer defect may be due to the multiple actions of cellular factors in the abdominal cavity.6. The sICAM-1 levels increase significantly more quickly in cultured endometrium of patients with endometriosis than that in the control, indicating easier adhesion of endometrium fragments with little clearance.