| Background:Trophoblast is an embryonic tissue which exerts a crucial role during implantation and placentation. At the time of implantation, the trophoblast cells of the embryo adhere and then invade into the maternal endometrium and eventually establish placentation. Defects in trophoblast invasion are associated with severe pregnancy-related complications affecting both mother and foetus. Reduced trophoblast invasion, and failure of the remodelling of maternal spiral arteries, is observed in pregnancies complicated with pre-eclampsia, spontaneous abortion and/or intrauterine growth restriction. The invasion of trophoblast is mainly regulated by cytokines, integrin, adhesion molecules, matrix metalloproteinases, et al. Biochemically, the trophoblast achieve this active invasive potential by producing high amounts of MMPs to degrade the extracellular matrix.In addition, in the immune microenvironment of the maternal-fetal interface, there are a variety of cells and molecules involved in immunotolerance so that the fetus is prevented from the maternal rejection. Maternal decidual immune cells play a critical role in maintaining immunotolerance. In the second trimester, decidual macrophages displays the M2 macrophages that have the high levels of IL-10, and low levels of IL-1β so that to maintaining immunotolerance.As a kind of environmental endocrine disruptors(EEDs), triclosan(TCS) is often found in toothpaste, soap and shampoo because of its broad antibacterial activity. A large number of experimental data have shown that TCS may lead to embryo implantation failure, abnormal embryo development and spontaneous abortion.TCS could permeate into cells by binding to the estrogen receptor since its structure is similar to estrogen. And TCS also inhibit embryo development by affecting the expression and secretion of estrogen and thyroxin. However, the mechanism how TCS induces embryo implantation failure and spontaneous abortion is remain unclear.Objective:To explore the effect of TCS on inhibition of trophoblast invasion and relative mechanisms and spontaneous abortion, providing the theoretical basis and new clues for intervention and prevention of the related female reproductive diseases.Methods:1. The overall designFirst, we had set up an animal abortion model in first-trimester and observed the effect of TCS on embryo implantation by calculating the numbers of implanted embryos. Meanwhile, the expression of MMP-2 and MMP-9 in the uterus was measured. Second, in vitro, cell invasion experiment was performed to observe the effect of TCS on HTR-8/SVneo invasion. And we also measured whether the expression of MMP-2 and MMP-9 was inhibited in HTR-8/SVneo. Furthermore, different kinds of signaling molecules were detected to determine through which signaling pathway TCS could affect the trophoblast invasion. After silencing SOCS3 by SOCS3 siRNA and activating the JAK2/STAT3 pathway, we observed the effect of TCS on HTR-8/SVneo invasion through transwell invasion assay, and measured the expression of MMP-2 and MMP-9.In addition, we also had set up an animal abortion model in second-trimester and observed the effect of TCS on abortion. And then, we measured the expression of TNF-α, IL-6, IL-1β, CD206, IL-10 and Yml.2. The effect of TCS on embryo implantationThe 6-8 weeks old ICR virgin females were mated naturally with males before they were checked at 8:00AM and 8:00PM everyday. Gestation day 0.5 was identified when a vaginal plug appeared. The plugged females were randomly distributed into the TCS group and the DMSO group. The mice were given TCS or DMSO by gavage once a day from gestation day 0.5 to gestation day 4.5. At gestation day 5.5, the mice were given trypan blue through tail vein. After 10 minutes, the uterus was isolated and calculated the implantation sites.3. The effect of TCS on trophoblast invasionHTR8/SVneo was put in the upper chamber of transwells which precoated with matrigel and incubated with TCS for 24h. Cells invading to the other side of the inserts were observed and calculated by an inverted microscope.4. The effect of TCS on trophoblast migrationThe effect of TCS on trophoblast migration was detected by wound healing test.5. The effect of TCS on uterine matrix metalloproteinasesThe expression of uterine matrix metalloproteinases MMP-2 and MMP-9 was measured by Western blotting.6. The effect of TCS on the expression of MMPs in HTR-8/SVneoThe expression of matrix metalloproteinases MMP-2, MMP-9, CD 146 and integrin Pi in HTR8/SVneo was measured by Western blotting.7. The signaling pathway affected by TCS in HTR-8/SVneoThe effect of TCS on the phosphorylation of ERK, AKT and STAT3, as well as the expression of SOCS3 in HTR8/SVneo cells were measured by Western blotting. After silencing SOCS3 by SOCS3 siRNA and activating the JAK2/STAT3 pathway, we observed the effect of TCS on HTR-8/SVneo invasion through transwell invasion assay and measured the expression of MMP-2 and MMP-9.8. The effect of TCS on abortion in second-trimesterThe 6-8 weeks old ICR virgin females were mated naturally with males, and they were checked at 8:00AM and 8:00PM everyday, 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 TCS group were given by gavage once a day from gestation day 0.5 to gestation day 14.5. The other group of mice were totally exposured to DMSO. Isolated the uterin and calculated the embryo loss rate.9. The effect of TCS on macrophage polarizationThe expression of TNF-a, IL-6, IL-1β, CD206, IL-10 and Yml were measured by Q-PCR after isolating the decidua.Results:TCS significantly reduced the numbers of implanted embryos and down-regulated the expression of MMP-2 in uterine. In vitro, TCS affected trophoblast invasion, migration and the expression of MMP-2. TCS activated the phosphorylation of ERK and AKT, but inhibited the phosphorylation of STAT3 by activating the expression of SOCS3. Trophoblast invasion and the expression of MMP-2 were both recovered by silencing SOCS3. Moreover, TCS significantly increased the embryo loss rate in second-trimester and enhanced the expression of pro-inflammatory cytokines.Conclusion:In first-trimester, TCS down-regulated the phosphorylation of STAT3 by activating the expression of SOCS3, and inhibited the expression of MMP-2, therefore affected the trophoblast invasion, and eventually led to a decline on the rate of embryo implantation. In second-trimester, TCS increased the embryo loss rate by affecting macrophage polarization. |
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