Effect Of Hyperestrogen On The Functions Of Tight Junction In Human Placental Trophoblasts

Objective:Super-physiological levels of estrogen along with multiple follicular development in IVF cycles has been considered closely related to adverse pregnancy outcomes. Tight junction factor is the differentially expressed gene between normal and ART placentae, which was identified by human cDNA microarray in our laboratory. We propose that high estrogen in IVF cycle may in turn affect placental tight junctions and trophoblast cell invasion, causing related complications. Our previous experiments have established the methods of trophoblast cells culture in vitro, and confirmed the expression of tight junction factor in trophoblasts by immunofluorescence. Also we have already achieved to assess tight junction function by both Transepithelial Resistance and Paracellular Permeability measurement. This study is to test oestradiol(E2) levels in the maternal peripheral blood, villi and placenta in the first trimester and term parturition, as well as the estradiol level of umbilical artery blood in term delivery; To explore physiological and supra-physiological levels of estrogen on trophoblast cell tight junction and invasion ability. This study investigates the association of high level of estrogen in IVF cycle and adverse pregnancy outcomes, providing a new basis for optimizing ART procedure and improves ART security.Materials and Methods:(1) This study included 20 unwilling pregnant women who want to have early artificial abortion and 20 pregnant women of full term cesarean delivery. The levels of estradiol in the tissues of early abortion, peripheral blood of pregnant women and umbilical artery blood, placenta tissue of the women during labor were measured by chemiluminescence immunoassay(CLIA).(2) Culture the human trophoblast cell line Be Wo cells by Millicell system in vitro to establish cell monolayer model(3) Be Wo cells were exposed to the 17P-E2 with a series of concentration (OM、5×10-7M, 5×10-9M、5×10-10M) for different time (1h、3h、6h、12h、24h).At the certain time point, Transepithelial electrical resistance(TEER)and Paracellular permeability (CPP) were measured to evaluate the alteration of tight junction of trophoblast cells under estrogen conditions.(4) Make sure that different concentration estrogen deal with Be Wo cells for 12h has a most remarkable effect.Further,Western Blot were performed to analyze the expression of tight junction factors CLDN3,CLDN4,CLDN5,CLDN8,ZO-1,OCLN of BeWo cells dealed with different concentration estrogen 17β-E2 at protein level.respectively.(5) IF was utilized to detected the change of tight junction proteins after BeWo cells exposed to a certain concentration of 17β-E2 for 12h,and explore the possible causes which mediated tight junction disfunction by estrogen.(6) Culturing BeWo cells in vitro, BeWo cells were exposed to 17P-E2 with different concentrations for 12h.Then, CCK8 method was performed to test the cell vibility with BeWo celIs.Make sure that this criteria can be used in the subsequent invasion assay of BeWo cells.(7) Using the above-mentioned experimental conditions,the alternation of cell invasion was detected in the followed matrigel invasion assay.Western Blot were employed for the detection of protein expression of matrix metalloproteinases 2 and 9,and investigate the possible reasons of high level estrogen impaired invasion ability of trophoblast.Result:(1) Estradiol were lower in peripheral plasma from early pregnancy [(3.44±1.30) nmol/L] compared with that from third trimesters [(509.78± 97.82) nmol/L] (t=10.2,P<0.001);However, the concentration of estradiol was higher in villus obtained from first trimest [(30.82±13.91) pmol/mg] than that from third trimesters placenta[(17.21±5.37) pmol/mg] (t=4.1,P<0.001)(2) Culturing BeWo cells in Millicell chamber in vitro,after exposed to 17P-E2 of different concentrations(0M,5×10-7M,5×10-8M.5×10-9M,5×10-10M), TEER and CPP were detected at different time points(lh,3h,6h,12h,24h) to evaluate the alteration of tight junction.Result exihibited that there was no significant change of TEER and CPP at 1 and 3 hours time points, (P>0.05);After exposed to 5×10-7M,5×10-8M 17β-E2 for 6 hours,tight junction dysfunction of Be Wo cells was found with the phenomenon of decreased TEER and increased CPP(P<0.05);(3) Dealed with 5×10-7M,5×10-8M,5×10-9M 170-E2 for 12 and 24 hours respectively.tight junction function was impaired with the observation of decreased TEER and increased CPP(P <0.05). Different concentration estrogen deal with BeWo cells for 12h has a most remarkable influence on the tight junction function and exhibited a concentration-dependent manner to a certain extent.(4) Exposed to 17β-E2 of different concentrations for 12h,Western Blot was employed to test the expression of tight junction proteins.The result showed expression of OCLN was decreased by the interventation of 17P-E2 at concentrations of 5×10-7M,5×10-8M,5×10-9M and expression of CLDN3 was decreased at concentrations of 5×10-7 M, 5×10-8M(P<0.05).The protein expression of other tight junction factors showed no significant change (P>0.05).IF showed that the CLDNS had a shift from the cytoplasm to the nucleus.(5) Be Wo cells cultured in vitro and exposed to 17β-E2 of a series of concentration for 12h,CCK8 was used to test the cell viability. There was no obvious difference in viability after treatment of 17β-E2 for 12h(P>0.05).(6) Matrigel invasion assay was carried out to assess the invasion ability of treated Be Wo cells. Invasiveness of Be Wo cells was impaired afer exposed to 17β-E2 of concentrations of 5×10-7M and 5×10-8M severally for 12h(P<0.05),and the Western Blot showed a decreasing expression of MMP2 (P<0.05)and the expression of MMP9 showed no significant difference at the same condition(P>0.05).Conclusion:(1) The concentration of E2 in villus obtained from first trimester is higher; E2 in full-term birth placenta tissues is relatively lower.(2) By usage of BeWo cells,we found that high level estrogen can disturbe the tight junction function in placental trophoblast cells,suggesting that high level of estrogen in IVF cycle may affect the normal function of placenta through affecting the normal structure of tight junction in trophoblasts.(3) Invasiveness of BeWo cells was impaired by high level estrogen,indicating that high level of estrogen in IVF cycle may affect the invasion function of trophoblast cells and lead to placental disfunction,which may be involved in the occurrence of placenta-related pregnancy complications.(4) Our may provide a new basis for optimizing ART procedure and improves ART security.