Expression Of CXCL12 In Peripheral Blood And Maternal-fetal Interface Of Women Who Have Miscarried And Preliminary Study Of CXCL12 Function

BackgroundAn early miscarriage is when the embryo or fetus has died and remained in the uterine cavity and failed to discharge in time during the first 12 weeks of gestation.This is considered to be a unique form of spontaneous abortion.The main reasons for this to occur are as follows:maternal immunological factors,prethrombotic state,chromosomal abnormalities(parental and embryos),reproductive tract anatomy endocrine factors,and male factors,etc.However,nearly 50%of pregnant women with spontaneous abortion cases are unable to identify the contributing cause.This is critical to identify,as spontaneous abortion may bring an economic burden to patients and their families and have a significant impact on patient’s physical and mental health.In recent years,studies have found regulatory networks of various chemokines and their receptors between the maternal and fetal interface-trophoblast,decidual stromal cells,and decidual immune cells.These cells play an essential role in regulating the proliferation and invasion of trophoblast cells,neovascularization,recruitment,migrationt,and homing of decidual immune cells.Successful pregnancy requires trophoblast cells,decidual stromal cells,and decidual immune cells between precise expression of chemokine and its receptors.Thus,the whole process of pregnancy immune regulation and its abnormal expression levels may break the balance of the immune tolerance state,leading to embryo implantation and placenta formation disorders,ultimately resulting in an abortion.CXCL12 and its receptors,CXCR4 and CXCR7,are both expressed in trophoblast cells of early pregnancy,and the CXCL12/CXCR4 and CXCR7 axis formed by them can regulate the proliferation and invasion of trophoblast cells,and CXCL12 can induce chemotaxis of CD56+CD16-NK cells in peripheral blood to the decidual tissue.Thus,immune tolerance at the maternal-fetal interface is preserved,enabling pregnancy to proceed.Therefore,it plays a crucial role in early pregnancy.This study explored the level of CXCL12 in the peripheral serum of women during early pregnancy,compared the expression levels of CXCL12,CXCR4 and CXCR7 in villus and decidual tissues of women who have miscarried,and the specific mechanism of CXCL12 regulation at the maternal-fetal interface was studied,the role of CXCL12 in the occurrence and development of miscarriage was discussed.Our study aimed to provide an experimental basis for the subsequent research,diagnosis,and treatment of miscarriage.Part 1 Expression of CXCL12 in peripheral blood of women who have miscarriedObjective:To analyze the expression level of CXCL12 in peripheral blood of healthy non-pregnant women,normal early pregnancy women and women who have miscarried,and investigate the clinical predictive value of CXCL12 in the occurrence of miscarriage.Methods:The levels of CXCL12 in peripheral blood of healthy non-pregnancy women(n=20),healthy early pregnancy women(n=20)and women who have miscarried(n=25)were determined by ELISA.Results:The expression level of CXCL12 in serum of women who have miscarried(n=25)was significantly lower than that of healthy early pregnancy women(n=20)by ELISA(P<0.001).Additionally,CXCL12 levels in healthy non-pregnant women(n=20)were significantly lower than those in early pregnancy women(P<0.01)and women who have miscarried(P<0.01).Conclusion:The significant increase of serum CXCL12 level after pregnancy may benefit normal pregnancy maintenance.The clinical application value of serum CXCL12 in predicting adverse pregnancy outcomes is worth further exploring.Part 2 Different expression of CXCL12,CXCR4 and CXCR7 at the maternal-fetal inter face in women who have miscarriedObjective:To investigate the expression levels of CXCL12 and its receptors CXCR4 and CXCR7 at the maternal-fetal interface(villus and decidua)of women who have miscarried and their possible significance.Methods:In this study,the expression levels of CXCL12,CXCR4 and CXCR7 in villus and decidual tissues of women who have miscarried(n=20)and healthy early pregnancy women(n=20)were detected by quantitative real-time polymerase chain reaction(qRT-PCR),Western blotting and immunohistochemistry.Results:1.The expression of CXCL12,CXCR4 and CXCR7 in villus tissues:The mRNA transcription levels,protein expression levels and average optical density(AOD)of CXCL12,CXCR4 and CXCR7 in villus tissues of healthy early pregnancy women were significantly higher than those in women who have miscarried(p<0.01);2.The expression of CXCL12,CXCR4 and CXCR7 in decidua tissues:There was no difference in the mRNA transcription levels of CXCL12,CXCR4 and CXCR7 between the two groups(p=0.49,p=0.72,p=0.28).The protein expression levels and average optical density(AOD)of CXCL 12,CXCR4 and CXCR7 in decidual tissues of healthy early pregnancy women were up-regulated compared with those women who have miscarried,and the differences were statistically significant(p<0.01);3.CXCL 12 mRNA transcript levels,protein expression levels and average optical density(AOD)in villus of healthy early pregnancy women and women who have miscarried were higher than those in decidua tissue(p<0.01);4.CXCR4 and CXCR7 mRNA transcript levels,protein expression levels and average optical density(AOD)in decidua tissues of healthy early pregnancy women and women who have miscarried were higher than those in villus tissues(p<0.01).Conclusion:The overall level of CXCL12/CXCR4,CXCR7 axis was significantly down-regulated in villus and decidua tissues of women who have miscarried,suggesting that its level change maybe closely related to the occurrence and development of miscarriage.Part 3 Effect of CXCL 12 on trophoblast cells andendometrial stromal cellsObjective:To investigate the specific mechanism of CXCL 12 regulation at the maternal-fetal interface and explore the role of CXCL 12 in the occurrence and development of miscarriage.Our study will provide new ideas for the prevention,diagnosis and treatment of miscarriage.Methods:Decidual immune cells were isolated from healthy early pregnancy women and women who have miscarried.Flow cytometry was used to analyze the subtypes of decidual immune cells and the proportion of CXCR4 and CXCR7 positive cells in the two groups.The silencing and overexpression system of CXCL 12 were established using HTR-8/SVneo cell line.The cell viability was determined by MTT assay.The horizontal migration ability of HTR-8 cells was determined by cell scratch assay.The invasion ability of HTR-8 cells was tested by Transwell matrigel invasion assay.Finally,recombinant human CXCL12 was used to stimulate endometrial stromal cells to explore the role of CXCL12 in endometrial decidualization.Results:1.The subtype distribution of decidual immune cells:the distribution ratios of CD56+NK cells and CD49a+NK cells in healthy early pregnancy women were significantly higher than those in women who have miscarried,and the differences were statistically significant(p<0.01,p<0.01);2.The positive expression of CXCR4 and CXCR7 in decidual immune cells:the positive expression rates of CXCR4 and CXCR7 in decidual immune cells of healthy early pregnancy women were significantly higher than those of women who have miscarried,and the differences were statistically significant(p<0.05,p<0.01);3.CXCL12 overexpression promoted the proliferation of HTR-8 cells:Compared with the Control cDNA group,CXCL12 overexpression significantly promoted the proliferation of HTR-8 cells(p<0.01),and compared with the Control siRNA group,CXCL12 silencing significantly inhibited the proliferation of HTR-8 cells(p<0.01).4.CXCL12 overexpression significantly promoted the migration ability of HTR-8 cells:At 24 hours after cell scratch,the cell migration rate of the CXCL12 overexpression group was significantly higher than that of the Control cDNA group(p<0.01),and the cell migration rate of CXCL12 silencing group was significantly lower than that of the Control siRNA group(p<0.05).5.CXCL12 overexpression significantly promoted the invasiveness of HTR-8 cells:compared with the Control cDNA group,the invasiveness of HTR-8 cells overexpressing CXCL12 was significantly increased(p<0.01),and the invasiveness of HTR-8 cells silenced CXCL12 was significantly down-regulated(p<0.01).6.Decidualization of endometrial stromal cells:Recombinant human CXCL12 can significantly promote decidualization of endometrial stromal cells,and this process can be inhibited by CXCR4 neutralizing antibodies to a certain extent.Conclusion:CXCL12 plays a more positive role in pregnancy,including promoting the proliferation,migration and invasion of trophoblast cells,and promoting the decidualization process of endometrial stromal cells.Down-regulation of CXCL12/CXCR4,CXCR7 axis may directly or indirectly affect the proliferation,migration and invasion ability of trophoblast cells.This will lead to increased apoptosis of trophoblast cells,insufficient depth invasion of maternal decidua,shallow placental implantation,incomplete decidualization of endometrium,difficult implantation of embryos,dysplasia of embryos and so on.This may be an important reason for the occurrence and development of miscarriage.Our study suggests that CXCL12 supplementation during pregnancy may be an effective prevention and treatment strategy for miscarriage.