Effect Of Mifepristone On Human 7-9 Gestational Weeks Placental Villi

Objectives:1.To investigate the synthesis and regulation of prostaglandins, oxidative stress and microvascular changes in the mesenchyma of human placental villi in order to explain the possible mechanism of mifepristone used for medical abortion.To observe the effect of mifepristone on the synthesis and expression of PGF_2α,the balance of oxidation/antioxidation,the configuration and number of the mesenchyma micrangium in human placental villi during 7-9 week's pregnancy.2.To improve the knowledge of action mechanism of mifepristone and rationally expand the use of mifepristone in abortion,offer theoretical basis and lab reference for its application even in other fields.Methods: 1.To study the effect of mifepristone on synthesis and expression of PGF_2α in human placental villi during early pregnancy(7-9 weeks). The patients were divided into mifepristone group(n=12) and induced abortion group(n=12) randomly.Immunohistochemistry (streptavidin-peroxidase,S-P) was used to detect the expression of COX-2,COX-1 and NF-κB in human placental villous trophoblast between mifepristone group and induced abortion group.The expression level of PGF_2α was determined by enzyme linked immunosorbent assay(ELISA).2.To investigate the effect of mifepristone on oxidation/antioxidation balance of human placental villi during early pregnancy(7-9 weeks). Colorimetry was used to detect the level of MDA,T-AOC,CuZn-SOD and MDA/T-AOC in above mentioned groups.3.Immunohistochemistry(streptavidin-peroxidase,S-P) was used to locate and detect the expression level of HO-1 in trophoblast of two groups.The protein expression level of HO-1 was determined by Western blot;Reverse transcription-polymerase chain reaction (RT-PCR) was applied to measure the mRNA level of HO-1 in two groups;Thus we can investigate the possible mechanism of mifepristone by analyzing interactions among PGF_2α synthesis and regulation,oxidation stress and mesenchyma microvacular changes of human placental villi through HO-1. 4.To study how mifepristone affect mesenchyma micrangium of human placental villi during early pregnancy(7-9weeks). Immunofluorescence histochemistry and Laser Scanning Confocal Fluorescence Microimaging System were used to detect configuration and number of vascular fluorescence labeled by CD34 in two groups.Results:1.COX-2 was strongly expressed mainly in cytoplasm of syncytrotrophoblast on the surface of villi,weakly expressed in cytoplasm of cytotrophoblast,and the expression in mifepristone group was significantly higher than that in artificial induced group (P＜0.01).NF-κB was strongly expressed mainly in cytoplasm of syncytrotrophoblast,weakly expressed in cytoplasm of cytotrophoblast, and the expression in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).COX-1 was strongly expressed in the cytoplasm of syncytrotrophoblast and cytrotrophoblast in both mifepristone group and induced abortion group.No statistically significant difference was found between two groups(P =0.064,P＞0.05).The expression of PGF_2α in villi in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).2.The villus level of MDA in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).The villus level of T-AOC in mifepristone group was significantly lower than that in induced abortion group(P＜0.01).The villus level of CuZn-SOD in mifepristone group was significantly lower than that in induced abortion group(P＜0.01).The villus level of MDA/T-AOC in mifepristone group was significantly higher than that in induced abortion group(P＜0.01)3.HO-1 was expressed mainly in cytoplasm and neucleus of trophoblast, partly in cytoplasm and neucleus of vascular endothelial cells and mesenchyma cells.The expression of HO-1 in trophoblast in mifepristone group was significantly higher than that in induced abortion group(P＜0.01).Using Western blot,the protein level of HO-1 was significantly higher in mifepristone group than that in induced abortion group(P＜0.01).When detected by RT-PCR,the mRNA level of HO-1 was significantly higher in mifepristone group than that in induced abortion group(P＜0.01)4.Clear and bright green fluorescence was found in the vascular endothelial cells labeled by FITC-CD34 in both groups.In induced abortion group,the capillary configuration was regular.The lumen was distinct and normal in size.While in mifepristone group,the number of mesenchyma micrangium increased.The configuration was irregular.The lumen was twisted and dilated in various degrees. The microvascular length density(Lv) was significantly higher in mifepristone group than that in induced abortion group(P=0.043,P＜0.05.The microvascular volume density(Vv) was significantly higher in mifepristone group than that in induced abortion group(P = 0.031,P＜0.05).VEGF was expressed in the cytoplasm of trophoblast and vascular endothelial cell in above mentioned groups, and the expression in mifepristone group was significantly lower than that in induced abortion group(P＜0.01).Conclution:1.NF-κB is activated by mifepristone,the expression of COX-2,and more PGs(esp.PGF_2α) are synthesized,which consequently induces vessel contraction,degeneration and necrosis of decidua tissue and the villus blood supply is affected directly or indirectly.The inhibition of HO-1 produces more ROS by antioxidation stress which further aggrevates the oxidative damage and leads to uterine smooth muscle contraction,thus interrupts and hampers pregnancy.2.There is oxidative stress in human villi during early pregnancy (7-9weeks).Mifepristone can further destroy the balance of oxidation/antioxidation,produce ROS and activate NF-κB, synthesize and express more PGs(esp.PGF_2α),then vessel contraction leads to the potential loss of vascular endothelial cells and mitochondrial,which produces more ROS,and oxidation/antioxidation balance is further destroyed.3.Mifepristone promotes to produce ROS,which can increase HO-1mRNA transcription and protein synthesis through activational NF-κB in promoter region of HO-1 gene.To resist the effect of mifepristone,HO-1 decreases oxidative stress,dilates vessel to improve blood supply in placenta and infant growth and development,protects cell,inhibits inflammation,aggregation of platelet and apoptosis.The result of competition between PGF_2α and HO-1 decides whether prgnancy continues.If the balance of oxidation/antioxidation is maintained in a new degree,the pregnancy will continue.On the contrary,the vessel contracts,the endothelial cells are damaged,ROS is produced,the oxidation/antioxidation balance is completely destroyed.4.24-48h after taking mifepristone 150mg(without adding misoprostol), the synthesis and expression level of PGs are increased and the balance of oxidation/antioxidaiton is destroyed in human placental villi during early pregnancy(7-9weeks).HO-1 mRNA and protein synthesis are also increased to resist the effects of mifepristone mentioned above.The number of capillary in villi is increased.The lumen is twisted and dilated in various degrees.Probably it is a compensatory rather than an exhausting phenomenon.Then PG is needed to inhibit the compensatory,effect of HO-1.Therefore,the usage time and dosage of mifepristone should be extended or increased in order to complete the medical abortion.