The Role And Mechanism Study Of Growth Differentiation Factor15(GDF15)-regulated Trophoblast Invasion In Recurrent Pregnancy Loss

Background:Recurrent pregnancy loss(RPL)is defined as the failure of two or more clinically recognized pregnancies before 20 weeks of gestation.The incidence of RPL is 1%to 5%.It brings tremendous pain and suffering to prospective mothers and their families.Although aneuploid embryos are the most common cause of RPL the etiology of approximately 60%of RPL cases remains unexplained,and these cases are referred to as unexplained RPL(URPL).URPL is a distressing pregnancy disorder that requires further discovery of its etiology.The establishment and maintenance of maternal-fetal interface is crucial for a normal pregnancy.The maternal-fetal interface refers to the endometrium and peripheral tissues during pregnancy,including decidua parietalis,decidua basalis and trophoblast.It is the interface of direct"dialogue" between mother and fetus,involving a series of complex physiological processes,such as early placentation and endometrium decidualization.Extravillous trophoblast(EVT)is an invasive cell located outside the villi during the early stage of placental development.Insufficient EVT invasion during early placentation has been shown to contribute to RPL.However,the regulatory factors and underlying molecular mechanisms of this process and their involvement in RPL pathogenesis have yet to be fully revealed.Growth differentiation factor 15(GDF15).member of the transforming growh factor-β(TGF-β)superfamily,is highly expressed in human placenta tissue.GDF 15 levels have been associated with pregnancy complications,including early pregnancy loss,preeclampsia and hyperemesis gravidarum.GDF 15 has been reported to regulate trophoblast cell invasion,but its role and molecular mechanism in regulating EVT invasion and pregnancy maintenance are still unclear.Objective:To clarify the transcriptomic expression profile of villus tissue in patients with URPL;to further explore the function and potential molecular mechanism of differentially expressed gene GDF 15 in the process of establishment and maintenance of maternal-fetal interface;to elaborate the occurrence and development mechanism of URPL and facilitate the development of preventive and/or therapeutic strategies for RPL.Methods:Clinical first-trimester villous and serum specimens from normal pregnancies and URPL patients were subjected to RNA sequencing and ELISA analyses to find differentially expressed genes.Villus explants,HTR-8/SVneo cell lines,and human primary EVT cells were treated with small interfering RNA mediated GDF15.TranswellTM invasion assays,woundhealing assays,and TranswellTM migration assays were used to investigate the function of EVT.Transcriptomic sequencing was performed on HTR-8/SVneo cells mediated by small interfering GDF 15 to screen downstream potential target genes,and the relevant molecular mechanisms were studied by Western blot,RT-qPCR and other experiments.The classic Lipopolysaccharide(LPS)-induced abortion mouse model was tested in vivo,Results:Differential gene GDF15 was screened based on the transcriptome sequencing results of villus tissues of normal pregnant women and patients with URPL.We found aberrantly decreased GDF15 levels in both first-trimester villous and serum samples from URPL patients compared with normal pregnant women.Small interfering RNA mediated GDF15 knockdown villi explants inhibited the invasion of villi explants,and s siRNA-mediated GDF15 knockdown HTR-8/SVneo cell line and human primary EVT cells inhibited the invasion and migration of trophoblast cells,and were not affected by the proliferation and apoptosis of trophoblast.Transcriptomic sequencing was performed on HTR-8/SVneo cells with small interfering RNA mediated GDF15 knockdown treatment to screen out the downstream potential target gene Jagged-1(JAG1).Subsequent studies found that GDF15 knockdown by small interfering RNA in human trophoblast cells reduced the expression of βcatenin,blocking the interaction between β-catenin and TCF4,down-regulated the expression of jagge-1,and then inhibited the activation of NOTCH3/HES1 pathway.Notably,JAG1 overexpression rescued GDF15 knockdown-inhibited EVT invasion.Moreover,in the mouse model of LPS-induced abortion,reduced serum and placental GDF15 levels were observed.Importantly,embryonic resorption in mice was significantly rescued by in vivo recombinant GDF15 protein administration.Conclusions:Insufficient GDF15 expression at the first-trimester maternal-fetal interface impairs EVT invasion by suppressing the JAG1/NOTCH3/HES1 pathway,thus participating in the pathogenesis of URPL.Moreover,GDF15 is a potential diagnostic and therapeutic target for clinical management of URPL.