Tim-3-Gal-9-Mediated Immunity Imbalance During Unexplained Recurrent Spontaneous Abortion

【Objective】To detect the expression of Tim-3and Gal-9at the maternal-fetal interface of spontaneous abortion models and normal pregnant models, for the sake of exploring the relationship between Tim-3-Gal-9pathway and unexplained recurrent spontaneous abortion (URSA).[Methods]1. Female CBA/J mice were mated with male DBA/2mice or BALB/c mice respectively to construct spontaneous abortion models and normal pregnant models.2. The Western blot/Real Time-PCR/FACS assay were used to detect the expression of Tim-3and Gal-9in placenta tissues of spontaneous abortion models and normal pregnant models, and then analyze them by statistics.3. The immunohistochemistry/Western blot/Real Time-PCR/FACS assay were used to measure the expression of Tim-3and Gal-9in the decidual tissues of spontaneous abortion models and normal pregnant models, and then analyze them by statistics.[Results]1. The spontaneous abortion and normal pregnant models were successfully constructed.2. The level of Tim-3protein and mRNA in placenta tissues of spontaneous abortion models was significantly higher than that in normal pregnant models (P＜0.05).3. The spontaneous abortion models had higher-level Tim-3+cells (%) on the trophoblast compared to the normal pregnant models (P<0.05)4. Tim-3espressed in the decidua of mice from both spontaneous abortion models and normal pregnant models, and mainly in the intracellular and cell membrane, the spontaneous abortion models had higher-level Tim-3protein compared to the normal pregnant models (P<0.05), there was no difference of the level of Tim-3mRNA between the two models (P>0.05)5.①There was no difference of the level of Tim-3+cells (%) on the decidual stromal cell between the two models (P>0.05).②The spontaneous abortion models had higher-level Tim-3+cells (%) on the decidual glandular epithelial cells compared to the normal pregnant models (P<0.05).③The spontaneous abortion models had lower-level Tim-3+cells (%) on the decidual lymphocytes cells compared to the normal pregnant models (P<0.05).6. There was no difference of the level of Gal-9mRNA in placenta tissues between the two moldes (P>0.05).7. The spontaneous abortion models had lower-level Gal-9+cells (%) on the trophoblast compared to the normal pregnant models (P<0.05). 8. Gal-9expressed in the decidua of mice from both spontaneous abortion models and normal pregnant, and mainly in the intracellular and cell membrane, there was no difference of the level of both Gal-9mRNA and protein between the two modles (P>0.05)9.①There was no difference of the level of Gal-9+cells (%) on the decidual stromal cell between the two modles (P>0.05).②The spontaneous abortion models had lower-level Gal-9+cells (%) on the decidual glandular epithelial cells compared to the normal pregnant models (P<0.05).③The spontaneous abortion models had lower-level Gal-9+cells (%) on the decidual lymphocytes cells compared to the normal pregnant models (P<0.05)[Conclusion]Tim-3and Gal-9were expressed at the maternal-fetal interface of both normal pregnant and RSA models. The abnormal expression of Tim-3and Gal-9might result in RSA. [Objective]Co-culture of trophoblast cells and lymphocytes (MLTR) from spleen and peripheral blood respectively in order to explore the relation between Tim-3-Gal-9pathway and.lymphocyte-mediated immunity.[Methods]1. FACS was used to investigate the expression of Tim-3and Gal-9on the lymphocytes from spleen and peripheral blood.2. ELISA was applied to the detection of IL-2/IFN-y produced by Thl and IL-4produced by Th2in the serum.3. Adding lymphocytes from spleen and peripheral blood to the trophoblast cells according to different modles, collected the supernatants at the same time of0,1,2,3,4.5,6,8and10day to examinate the proliferation index of lymphocytes and expressions of Tim-3and Gal-9on lymphocytes, meanwhile, the cytokines of Thl/Th2were tested by ELISA on1d,3d,6d and10d.[Results]1. The spontaneous abortion models had higher-level Tim-3+cells (%) and lower-expression Gal-9on peripheral blood lymphocyte compared to the normal pregnant models (P<0.05); there was no difference of the level of Tim-3+cells (%) and Gal-9+cells (%) on the splenic lymphocytes between the two modles (P>0.05)2. The spontaneous abortion models had lower-quantity IL-2and IFN-y produced by Thl compared to normal pregnancy modles (P<0.05), and no difference of IL-4 produced by Th2in the serum between the two modles (P<0.05).3. The proliferation index addressed the spontaneous abortion models had low proliferation of lymphocytes, but the normal pregnancy increased rapidly, not only splenic lymphocytes but also peripheral blood lymphocytes (P<0.05).4. The Tim-3expression on splenic lymphocytes of co-culture system from spontaneous abortion models was higher-expression at the beginning especially followed by lower-expression later, with the Gal-9expression on the contrary (P<0.05). The Tim-3/Gal-9expression on the peripheral blood lymphocyte of co-culture system were consistent with the splenic lymphocytes (P<0.05).5. The spontaneous abortion models had higher-level IL-2/IFN-y but lower-expression IL-4in the co-culture system (P<0.05), not only the co-culture system formed by peripheral blood lymphocytes and trophoblast cells but also the splenic MLTR (P＜0.05)[Conclusion]The Thl/Th2cytokines might have a close link with the outcome of pregnancy. The dysregulation of Tim-3and Gal-9could result in imbalance of Th1/Th2, then might lead to RSA.