Relationship Between Follistatin Expression In Human First-trimester Trophoblast Cells And Spontaneous Abortion

Follistatin(FS),a monomeric glycoprotein,is named by inhibiting the secretion of follicle-stimulating hormone(FSH)from anterior pituitary,which is similar to the Inhibin.Macrophage,follicle cells and trophoblast cells,and so on,can secrete FS and activin A.FS neutralizes the biological functions of TGF-? superfamily and then is involved in various physical or pathological processes through combining with activin A,TGF-?1 and BMP,and so on.Successful pregnancy needs maternal-fetal interface to complete the following conditions in turn: endometrial decidualization,pre-implantation preparation,trophoblast cells invading into uterine decidua.Our previous results showed that the level of FS in serum from 20 spontaneous abortion patients is lower than that from normal early pregnancy,while the level of activin A from spontaneous abortion patients and whether FS can affect the viability of trophoblasts and be a warning indicator of spontaneous abortion are still lack of definite inclusion.First,in this study used ELISA to examine the level of FS and activin A in serum of normal early pregnancy and spontaneous abortion patients and then analyzed the relationship between the level of FS and activin A and spontaneous abortion.Next,our study used immunohistochemical staining to examine the levels of FS and activin A expression in villous tissue from normal early pregnancy and spontaneous abortion patients to confirm the relationship between those levels and spontaneous abortion.Furthermore,our study selected human villous trophoblast cell line HTR-8/SVneo cells to investigate the effect of FS on trophoblast cells in vitro through MTT,Brd U,RT-PCR,ELISA and so on.1.The levels of FS and activin A in serum of normal early pregnancy and spontaneous abortion patientsELISA method was used to detect the levels of FS and activin A in serum of normal early pregnant women and spontaneous abortion patients.The results showed that the level of FS in serum of spontaneous abortion patients was significantly lower than that of normal early pregnant women(n=40),which had statistical significance(P<0.05);while there is no statistical significance on the activin A level of serum between normal early pregnant women and spontaneous abortion patients.2.The expression of FS and activin A proteins in chorionic villiImmunohistochemical staining was used to analyze the expression of FS and activin A proteins in chorionic villi of normal early pregnant women and spontaneous abortion patients.The results showed that FS and activin A were localized in normal chorionic villi,mainly distributed in chorionic villi outer layer syncytiotrophoblast cells,rarely distributed in cytotrophoblast cells,and we found that the expression of FS in villi of normal early pregnant women was higher than that of spontaneous abortion patients,while the expression of activin A between the two groups had no significant change.3.The effect of FS and activin A on biological activities of human trophoblast cell line HTR-8/SVneo cellsHTR-8/SVneo cells were treated with different concentration gradient FS,activin A and the combination of the both,in vitro for 24 h.First,MTT and Brd U methods were used to investigate the proliferation capabilities of HTR-8/SVneo cells,and the results showed that FS can promote HTR-8/SVneo cells proliferation,while activin A had no significant influence.The effect of combining FS and activin A with the proportion of 4:1 could significantly promote the proliferation of HTR-8/SVneo cells.Next,ELISA was used to examine the levels of cytokine IL-6 and IL-10 in the supernatant of cultured cells.The results showed that FS promoted IL-6 release of HTR-8/SVneo cells,and did not alter IL-10 production.Furthermore,the m RNA of HTR-8/SVneo cells after being incubated in vitro for 24 h was extracted and RT-PCR was used to examine the expression level of IL-6,MMP2,TIMP2 m RNA.The results showed that FS upregulated IL-6 m RNA of HTR-8/SVneo cells,which was consistent with the results of ELISA.FS also upregulated MMP2 m RNA of HTR-8/SVneo cells,but did not influence TIMP2 m RNA.The previous studies have reported that when normal pregnancy and inflammation happen,the levels of FS and activin A in peripheral blood would be elevated.In this study,our results demonstrated that,FS and activin A during human early pregnancy are derived from chorionic villi and decidual tissue.The FS levels in serum and its expression in villi tissue from spontaneous abortion patients were lower than those in normal early pregnant women group,while the level of activin A had no significant change.The results further indicated that FS could promote the proliferation activity of HTR-8/SVneo cells in vitro,upregulate the expression of IL-6 and maintain the survival of chorionic trophoblast cells.Thus,low expression of FS may be associated with the pathological process of spontaneous abortion,the combination examination of FS and activin A in peripheral blood may be contributed to excluding inflammatory diseases,and have accessory diagnosis value for early spontaneous abortion.