Study Of Autophagy In Vascular Endothelial Cell In The Placenta Of Women With Intrahepatic Cholestasis Of Pregnancy

Introduction:Fetal sudden death is often found in pregnant women with Intrahepatic Cholestasis of Pregnancy (ICP) in late trimester, and the mechanism is still unknown. In this study, we observe increasing of placenta infarction, thrombosis and recanalization of thrombus in the fetal circulation in placentas from severe ICP with fetal sudden death. So we hypothesized that placental vascular endothelial cells were impaired by elevated bile acid, which may explain etiology of fetal sudden death in ICP.To verify the above hypothesis, we observe the pathological and ultrastructural changes of placentas from ICP women, detect related protein in placentas by immunehistochemical method, observe the effects of bile acid to HUVEC by in vitro Study.Materials and Methods:1. In Vivo Studies: ①Histological observation of placenta:13placentas paraffin specimens were collected from women with ICP and stillbirth.20placentas from pregnant women with severe ICP were recruited and20placentas from normal pregnant women served as control. The pathological changes of placentas were observed under light microscope after HE staining.②Placental vascular cell ultrastructural observation:Ultrastruture of placental vascular cells were observed by electron microscopy from ICP women, and compared to normal one.③Observation by immunehistochemical method:according to preliminary results, LC3A/B and vWF were detected by immunehistochemical. Correlational analyses were conducted.2. In Vitro Study:①HUEVC were treated or untreated with CG and harvested for ultrastructural observation.②HUEVC was stimulated by different concentration of CG, then analyze the expression of autophagy related protein LC3-II and beclin-1by western blotting.Results:1. Compare with normal placenta, we observe increasing of placenta infarction, thrombosis and recanalization of thrombus in the fetal circulation in placentas from severe ICP with fetal sudden death.2. By electron microscopy, the presence of autophagosomes in human placental vascular endothelical cells from ICP placenta was confirmed, autophagic vacuoles were significantly increasing in vascular endothelial cells from ICP women’s placenta.3. The location of LC3A/B were stained in the endothelium of the arterie and arteriole of stem villi, and in the syncytiotrophoblast that cover the stem villi and an emerging terminal villous in ICP placenta. The location of vWF were stained in the endothelium of the blood vessels and in the shedding of syncytiotrophoblast observed in the intervillous space. Significantly elevated H-score level of LC3A/B and vWF was associated with ICP, Kendall rank correlation coefficient is0.65(P<0.001).4. By electron microscopy, autophagic vacuoles were significantly increasing in HUVEC treated with CG.5. In HUVEC, CG display a concentration-dependent up-regulation on expression of LC3B-II and beclin-1. The expression of LC3B-II in100uM CG experimental group were significantly higher than the control group (P<0.01).Conclusion:1. Compare to normal, more infarction, thrombosis and recanalization of thrombus were found in ICP placentas showing thrombosis may be one of the important pathological changes of placenta when ICP fetal distress and even stillbirth occur.2. We observed increasing autophagic vacuoles in ICP placental vascular cells as well as CG treated HUVEC, identify elevated autophagy may be one of the pathophysiology mechanism of ICP. Elevated autophagy induced by high bile acid may be associated with microvascular thrombosis in human placenta which may explain etiology of fetal sudden death in ICP.