Effects Of Mifepristone On The Human Placental Villi During Early Pregnancy

Objective:1.To clarify the effects of mifepristone on the secretion of angiogenesis factor of human first trimester trophoblast,and to discuss the relationship of mifepristone and angiogenesis in villous stroma.2.To study the effects of mifepristone on proliferation and apoptosis of human first trimester trophoblastic cells.3.To study the effects of mifepristone on the induction of apoptosis of in vitro cultured BeWo and JEG-3 cell lines.Methods:1.The villous capillaries in the early pregnancy were stained with anti-CD34 and evaluated sterologically.2.Using immunohistochemistry and image analysis technique to study the expression of VEGF Ab-7,VEGF JH121,Ang-1 and TSP in the villous trophoblasts of mifepristone group and control group.3.The proliferative rate and apoptotic rate were evaluated by immunohistochemistry and TUNEL technique.The staining intensity and distribution of Caspase-3 was evaluated by image analysis technique.The expression of apoptotic related proteins, such as caspase-3,-7,-9 were detected by Western blotting technique.4.The BeWo and JEG-3 cell lines were cultured in vitro,the experimental group is divided into following 2 subgroups,(1) control group:BeWo and JEG-3 cell lines without mifepristone;(2) mifepristone group:cells were treated by 0,0.25,1,2.5,10 and 25μmol/L mifepristone respectively.Detecting the apoptotic and necrotic cell ratios by Annexin V-FITC/PI double staining flow cytometry.5.The ultrastructure of the villous tissue in mifepristone group(n=3) and control group(n=3) was investigated by transmitted electron microscope(TEM).Results:1.Effects of mifepristone on the stereological examination of the first trimester placental villiAlmost all villous capillaries of the first trimester villi were stained by CD34.In mifepristone group,the length density and volume density of villous capillaries were statistically significantly decreased than that in control group(P＜0.05).2.Effects of mifepristone on the secretion of angiogenesis factors in the first trimester trophoblastic cellsIn trophoblastic cells,the staining intensity of VEGF Ab-7 and Ang-1 in control group were statistically significantly higher than that in mifepristone group(P＜0.05). While the expressions of VEGF JH121 and TSP were predominantly decreased in control group than that in mifepristone group(P＜0.05).3.Influence of mifepristone on the proliferation and apoptosis in trophoblastic cellsAll villous specimen showed Ki67 and TUNEL positive expression.Ki67 mainly situated in cytotrophoblast,while TUNEL were mainly expressed in syncytotropho- blast.Using the number of positive nucleus in an unit area,the states of trophoblastic proliferation and apoptosis in the first trimester were examined.Compared with the control group,the trophoblastic proliferation in mifepristone group was decreased, while apoptosis was enhanced.4.Effects of mifepristone on the expression of apoptotic-related protein caspase-3,-7,and -9 in trophoblastic cellsIn trophoblastic cells,the staining intensity of caspase-3 in mifepristone group was significantly higher than that in control group(P＜0.05).Using western blotting technique,all the villous tissues expressed caspase-3.The detection rate of caspase-7 was higher in mifepristone than that in control group(75%vs 37.5%).And the detection rate of caspase-9 had the same outcome(50%vs 12.5%).5.Effects of mifepristone on the apoptotic state in vitro cultured BeWo and JEG-3 cell linesBeWo cells were treated with mifepristone for 24h,the number of apoptotic cells trended upwards obviously as mifepristone concentration increased.The ratios of cell apoptosis in different mifepristone group were higher than that in control group.The highest apoptotic rate of BeWo cells was found in 2.5μmol/L mifepristone group (8.03%vs 0.4%).JEG-3 cells were also treated with mifepristone for 24h.The number of live cells were trended downwards while the apoptotic cells upwards as mifepristone concentration increased.The ratios of cell apoptosis in different mifepristone group were higher than that in control group,and in 10μmol/L RU486 group the apoptotic rate was the highest(14.96%vs 4.69%).6.Ultrastructure of the villous tissues in mifepristone and control groupUltrastructure features of villous tissues in the normal early pregnancy:the free surface of syncytotrophoblast was rich in microvilli.The cytoplasm contained numerous mitochondria,rough endoplasmic reticulum(rER) and smooth endoplasmic reticulum(sER),scattered ribosomes and lipid droplets of various sizes.The cytoplasm of cytotrophoblast was rich in ribosomes,mitochondria which were often surrounded by numerous RER.The capillary endothelial cells were rich in cytoplasmic projections,and there were more cell-to-cell communication with the pericytes and stromal cells.Ultrastructural features of villous tissue in mifepristone group:the syncytophoblast shed off partly.The microvilli in the surface of the remained syncytotrophoblast became short and thin,even absent.The RER dilated, mitochondria shrinked and some even became vacuolization.The nucleus of syncytotrophoblast showed some apoptotic features including peripheral nuclear chromatin condensation.In the cytoplasm of cytotrophoblast,plenty ofα-glycogen accumulated,some mitochondria became vacuolizaed.The nucleus of endothelial cells revealed an apoptotic-like feature including peripheral nuclear chromatin condensation.The cytoplasm contained less cytoplasmic projections,and there were less cell-to-cell communications with the pericytes and stromal cells.Conclusions:1.Under the effects of mifepristone,villous angiogenesis and consequently might be disturbed,consequently it is harmful to the embryo' s survival.2.The secretory functions of trophoblastic cells are destroyed by mifepristone,the dynamic balance of angiogenic factors may be broken,and the angiogenesis will be destroyed.3.The balance of trophoblastic proliferation and apoptosis is important for the placental function.Mifepristone can blockade the trophoblastic proliferation and up-regulate the expression of caspases family proteins which can induce the cellular apoptosis.4.Mifepristone can induce the apoptosis of BeWo and JEG-3 cells in vitro, indicating that it can induce the apoptosis of human trophoblastic cells directly.5.In comparison to the control group,the trophoblastica cells of mifepristone group show morphological changes.Furthermore,these changes reflect the functional changes of the trophoblastic cells in secretion,proliferation and apoptosis.