Investigation Of The Mechanism Of IL-1β In Regulating Embryo Implantation In Mice

Objective:To construct an exvivo model of mice embryo implantation that allows intervention of IL-1β (interleukin-1beta) at different concentrations. To find the optimal dose of1L-1β for embryo implantation by examining the development of mice embryos, and the expression levels of MMP-9(matrix metalloproteinase-9),1CAM-1(intercellular adhesion molecule-1) in endom-etrial cells and in embryo-endometrial epithelial cell in vitro culture. To provide some theoretical basis for the clinical application of IL-1β in human embryo implantation to improve human pregnancy rate.Method:1. Primary culture of endometrial epithelial cells during mice embryo implantation, Endometrium tissue was obtained from white female mice of KM strain, and was shredded and filtered from stainless filters of100and400meshes, followed by digestion using0.25%I collagenase, and culturing in the DMEM/F12culture medium with10%FCS.2. Examination of endometrial epithelial cells:Cells of exuberant growth were collected, and examined with an optical microscope and an inverted microscope, respectively, for the morphology of endometrial epithelial cells. Immunohistochemistry was used to test the expression of cell cytokeratin.3. Cell Immunohistochemistry was applied to investigate the effect on IL-1β on the expression of MMP-9and ICAM-1in the endometrial epithelial cells. Embryos were obtained from pregnant D4mice, and cultured for24hrs in the endometrial epithelial cell primary culture. IL-1β of different concentrations was then added to the culture. Positive cells were counted and the expression levels of MMP-9and ICAM-1in the endometrial epithelial cells were measured and compared to the control group. The images were analyzed using the IPP6.0high-definition imaging analysis system. Five object plates were randomly selected in each treatment group. The positive cells were counted automatically from five different angles in the10x20times visual field, the average of which was taken as the final count measurement. The optical density values were also measured and averaged in a similar manner.4. Morphological examination of the effect of IL-1β of different concentrations on the embryo development in vitro:the2-cell embryos from the pregnant mice were collected and then treated for96hrs with IL-1β of different concentrations and compared to then control group on the blastocyst and hatched blastocyst rates at72hrs and96hrs.5. ELISA was used to test the effect of IL-1β and IL-IRa of different concentrations on the expression level of MMP-9and ICAM-1in the primary culture of embryos and endometrial epithelium. Before the in vitro culturing of endometrial epithelial cells, the8-cell embryos from the pregnant mice were collected to set up the co-culturing model. The culture was treated for48hrs with IL-1β and IL-1β+IL-IRa of different concentrations. The liquid supernatant was collected from the culture to measure the expression of MMP-9and ICAM-1.Results:1. The endometrial epithelial cells after digestion appeared to be clustered under the inverted phase contrast microscope. Adherence was observed after24hrs since inoculation. The cells stretched into irregular shapes and fused into a single layer after48hrs culturing, closely aligned with relatively large cell nucleus in the center of the cytoplasm.2. Immunohistochemistry and cytokeratin in endometrial epithelial cells:The Cytokeratin-18antibody appeared positive with cytoplasm colored brown while cell nucleus colored blue with the dye. The positive rate was about96%. There was no positive reaction in cytoplasm in the control group.3. The results from the cell Immunohistochemistry demonstrate that cytoplasm displayed brownish yellow particles of different shades due to the various expressions of MMP-9and ICAM-1with treatment of IL-1β of different concentrations. Compared to the control group, the expressions of MMP-9and ICAM-1both increased statistically significantly (P<0.05) in a dose-response manner.4. Observation of embryo morphology suggests the following. The blastocyst rate was72.6%s71.4%,69.9%, respectively with72-hr treatment of1L-1β of lng/ml。10ng/ml%、100ng/ml, which were all statistically significantly higher than the control group (P<0.01).The rate of hatched blastocysts was58.0％、58.4%, respectively with96-hr treatment of IL-1β of lng/ml、10ng/ml, which were both statistically significantly higher than the control group (P<0.05);there was no statistically significant difference (P>0.05) between the100ng/ml and the control groups in the96-hr blastocyst rate (42.9%in the100ng/mL group). 5. The ELISA test results suggest that:1) there was an increase in the expression of MMP-9and ICAM-1in the exvivo cultured embryo with lng/mL and10ng/mL IL-1β; Compared to the control group, the increase was statistically significant (P<0.05).2) There was no statistically significant increase in the expression of MMP-9and ICAM-1with intervention of100ng/mL IL-1β compare to the control group (P>0.05).3) The expression of both MMP-9and ICAM-1dropped with treatment of10ng/ml IL-1β+10ug/mI IL-1Ra in a statistically significant manner as compared to the control group (P<0.05).4) Treatment of lng/ml IL-1β+10ug/ml IL-1Ra and of100ng/ml IL-1β+10ug/ml IL-lRa on the cultured embryo did not appear to affect the expression of MMP-9and ICAM-1in a statistically significant manner (P>0.05).Conclusion:1.IL-1β improves the endometrial receptivity during embryo implantation in mice.2.IL-1β improves the blastocyst-forming rate and blastocyst-hatching rate of in cultured mice2ce)l embryos in vivo.3. IL-1β of1to10ng/mL is most appropriate for culturing the mouse embryo in vivo.4.The proper ratio of IL-1β vs. IL-1Ra can affect the expression of implantation-related proteins.5. It is postulated that IL-β regulates embryo implantation via its effect on the expression levels of MMP-9ICAM-1which are the proteins related to the employ development and implantation.