| Background:As a vital component in maternal-fetal interface, placenta is a crucial guarantee in ensuring sufficient supply of nutrients and substance exchange between mother and developing embryo. During gestation period, endometrium decidualization and the normal exertion of decidua function play important roles in embryo implantation, the establishment and maintenance of pregnancy. Trophoblast cells are considered as an important component of placenta tissue, the substance exchange and oxygen supply are based on the interaction between maternal decidua and fetal trophoblast cells at the maternal-fetal interface.Both trophoblast cells and decidual stromal cells could expresss different kinds of chemokines to support trophoblast migration, meanwhile, trophoblast cells could also promote its migration and invasion ability through secretion of adhesion molecules and matrix metalloproteinase. Aberrant placenta development lead to many unexpected pregnancy-related disease including embryo growth restriction, gestational hypertension, preeclampsia, and spontaneous abortion. Possiable reasons include impaired invasion ability of extravillous trophoblasts and dysfunction of decidual stromal cells. There always affect the invasion of trophoblasts to maternal tissues, and eventually cause insufficient supply of blood, oxygen and nutrients to the developing fetus.Investigations showed that the incidence of infertility and spontaneous abortion were reached to 15%. As one of the kind of EEDs (Environmental Endocrine Disruptors), BPA (Bisphenol A) extensively exists in our daily life, like mineral water bottle, baby bottle, food packaging material, medical apparatus and instruments. Research reveal that BPA could be detected in maternal blood, umbilical cord blood and amniotic fluid. Once low-concentrations of BPA in environment enters into human body, it will enrich in follicular fluid. The concentration of BPA in adult female follicular fluid is three to four times higher than blood plasma. More and more evidences indicate that BPA is closely related to reproductive function damage.BPA possess the characteristics of disturb the effect of endogenous hormone and difficult to degradation, make it attracting extensive attention in influence on reproductive health. Studies show that maternal exposure to BPA causes change of sinus follicle number, the number of mature follicle and corpus luteum significantly decreased result in reproductive function damage. Experimentation on animals confirmed that BPA mainly disturb the effect of endogenous hormone, especially estrogen. As an important hormone on embryo implantation, the deviation of estrogen level may lead to implantation disorder, infertility and abortion. BPA could enhance the activity of natural killer cells, macrophages and cytotoxic T cells. It represent that BPA may destroy embryo implantation and influence normal embryo development through alter of endometrium immuno microenvironment. But the mechanism of BPA on the function of first-trimester decidual stromal cells and trophoblasts at the maternal-fetal interface is still unclear.Objective:To understand the effect of BPA on the function of first-trimester decidual stromal cells and trophoblasts in maternal-fetal interface and relative mechanism and provide theoretical basis and new clues for intervention and prevention of the female reproductive related diseases.Methods:1. The overall designWe had set up animal abortion model firstly and observed the effect of BPA on embryo implantation, calculate the number of embryo implantation. Secondly, in vitro cell invasion experiment was measured to observe the effect of BPA on decidual stromal cells in recruitment of trophoblasts. Furthermore, the expression of chemokines was measured in decidual stromal cells, to confirm whether BPA through affect the chemokines expression profile and in turn suppress the recruitment of trophoblasts. Different kinds of signaling molecules were detected to determine through which signaling pathway BPA could affect the function of decidual stromal cells. ERK was blocked through specific inhibitor to ensure its role in chemokines expression levels in decidual stromal cells after stimulation of BPA. Through transwell invasion assay to determine the role of ERK in recruitment of trophoblasts through decidual stromal cells. GPR30 and ERα/β were blocked by specific inhibitor, respectively, to ensure whether they were participated in the effect of BPA on trophoblasts recruitment through decidual stromal cells. Through transwell invasion assay and wound healing test to detect whether BPA could directly affect the invasion and migration ability of HTR-8/SVneo. Finally, the effect of BPA on the expression of adhesion molecule, matrix metalloproteinase and the tissue inhibitor of matrix metalloproteinase in HTR-8/SVneo.2. The effect of BPA on embryo implantation,The 8 weeks old ICR virgin females were mated naturally with untreated young males, they were checked each morning at 8:00AM-9:00AM, when a vaginal plug was appear, that day was considered as gestation day 0.5. The plugged females were randomly distributed into 2 groups, the mice in BPA group were given by gavage once a day at 8:00AM-9:00AM from gestation day 0.5 to gestation day 4.5. The other group of mice were totally exposured to DMSO at the same time. At gestation day 5.5, the mice were given intravenously trypan blue through tail vein. After 10 minutes, obtain the uterin and calculate the implantation sites.3. The effect of BPA on decidual stromal cells in recruitment of trophoblastsAfter DSC incubated with BPA for 72h, replace the medium with fresh complete medium, put the isolated primary trophoblasts or HTR8/SVneo in the upper chamber of trans wells which precoated with Matrigel. After incubated for 24h, observe the cells with an inverted microscope, calculate cells migrated to the other side of the inserts.4. The effect of BPA on chemokines expression in decidual stromal cellsThe expression of chemokines IL-6, CXCL6, CXCL8, CCL11 were detected by Real Time-PCR, and CXCL8 was also measured by ELISA.5. The signaling pathway affected by BPA in decidual stromal cells in recruitment of trophoblastsThe decidual stromal cells were stimulated with BPA, cell protein was isolated and the effect of BPA on the phosphorylation of JNK, ERK, AKT and p38 in decidual stromal cells was measured by western blotting. ERK was blocked through specific inhibitor U0126 to ensure its role in chemokines expression levels in decidual stromal cells after stimulation of BPA. Through transwell invasion assay to determine the role of ERK in recruitment of trophoblasts through decidual stromal cells. GPR30 and ERα/β were blocked by specific inhibitor G15 and ICI 182,780, respectively, to determine whether they were involved in the effect of BPA on trophoblasts recruitment through decidual stromal cells.6. The effect of BPA on invasion and migration ability in HTR-8/SVneoThrough transwell invasion assay and wound healing test to detect whether BPA could directly affect the invasion and migration ability of HTR-8/SVneo.7. The effect of BPA on protein expression profile in HTR-8/SVneoThe human extravillous trophoblast cell line HTR-8/SVneo were stimulated with BPA, The protein were isolated and the expression of adhesion molecule, matrix metalloproteinase and the tissue inhibitor of matrix metalloproteinase were detected by western blotting.Results:BPA significantly inhibit the numbers of embryo implantation. In vitro cell invasion experiment indicate that BPA could affected the recruitment of trophoblasts through inhibit the expression of chemokine CXCL8 in decidual stromal cells. BPA also affected the expression of IL-6, CXCL6 and CCL11. BPA significantly activated the phosphorylation of many signaling molecules including Akt, ERK and p38. The inhibition of BPA on CXCL8 expression and trophoblasts recruitment was recovered when blocked ERK with specific inhibitor U0126 in dicidual stromal cells. The inhibition of GPR30 and ERα/β were performed through specific inhibitor G15 and ICI 182,780, respectively, and the inhibition of BPA on CXCL8 expression and trophoblasts recruitment were also recovered. Moreover, BPA could directly inhibited the invasion and migration ability of HTR-8/SVneo through affected the expression level of adhesion molecule, matrix metalloproteinase and the tissue inhibitor of matrix metalloproteinase.Conclusion:In maternal-fetal interface, after binds to the membrane receptor GPR30 on the surface of decidual stromal cell or nuclear receptor ERα/β, BPA up-regulated the phosphorylation level of signaling molecue ERK and inhibited CXCL8 expression in decidual stromal cells, therefore suppressed the recruitment of trophoblasts. Besides, BPA could also directly inhibited the invasion and migration ability of HTR-8/S Vneo through affected the expression level of a variety of protein in HTR-8/SVneo. |
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