| backgroundRecurrent spontaneous abortion is defined as three or more consecutive fetal loss before 28 weeks of gestation.There is no consensus on the definition of recurrent spontaneous abortion in the world.The main differences lie in the number of abortions,the gestational age of abortion and whether biochemical pregnancy is included.The etiology of recurrent spontaneous abortion is complex and diverse,mainly including the following aspects:genetic factors,anatomical factors,endocrine factors,antiphospholipid antibody syndrome,environmental factors and immune factors.Immune factors include autoimmune factors and alloimmune factors.The Chinese Expert Consensus on diagnosis and Treatment of Spontaneous Abortion(2020 edition)recommends screening for autoimmune factors such as antiphospholipid syndrome,systemic lupus erythematosus,undifferentiated connective tissue disease,sjogren's syndrome,rheumatoid arthritis,and systemic sclerosis,but does not establish screening criteria for isoimmune factors.The correlation between immune risk factors and recurrent spontaneous abortion is becoming the focus of scientific attention.However,the immune factors are rarely mentioned in national guidelines,and there are still limitations in the study of immune factors in international guidelines.All guidelines on immune factors focus almost exclusively on screening for antiphospholipid syndrome,ignoring the fact that 50%of patients with recurrent spontaneous abortion cannot find a clear cause even after comprehensive and detailed screening for genetic,anatomic,endocrine,and environmental factors.Appropriate regulation of maternal-fetal immune tolerance is important for embryo implantation and successful pregnancy,however,the exact mechanisms that control physiological tolerance at the foetus-maternal interface remain unclear.It has been suggested that a series of mechanisms regulating maternal immune recognition and fetal antigen expression can prevent most pregnancy rejection,but when these mechanisms fail,pregnancy loss may occur.Over the past few decades,numerous studies have been conducted to elucidate the biomolecular mechanisms of endometrial embryo acceptance,as well as the specific cell types and cellular pathways involved in endometrial receptivity.However,our understanding of these mechanisms is still far from complete.In recent years,some studies on exosomes have found that exosomes play a biological role in regulating immune response.The functions,targeting and driving mechanisms of specific cell components in exosomes indicate that they play an important role in regulating intercellular communication,and also affect the immunity,proliferation and apoptosis of target cells.Exosomes are associated with immune response,viral pathogenicity,pregnancy,cardiovascular disease,central nervous system-related diseases,and cancer progression.Exosomes have a function in eliciting adaptive and innate immune responses,supporting their use in therapeutic development and playing a potential role in coordinating immune responses in response to infectious agents or cancer.Exosomes may also modulate immune responses by influencing gene expression and signaling pathways in recipient cells.Exosomes exhibit immunostimulating or immunosuppressive properties depending on their source and composition.There is growing evidence that microRNA(miRNA/miR)can be packaged into exosomes or microvesicles,and can also be loaded into high-density lipoproteins.These modes of action can protect miRNA from degradation and ensure its stability,so as to stably exist in body fluids,including saliva,urine,breast milk and blood,and participate in many biological activities,including cell proliferation,cell differentiation,cell migration,disease initiation and disease progression,etc.Due to the transportability of exosomes,among exosome transporter biomolecules,miRNAs have attracted increasing attention due to their highly conserved interspecies and extensive regulatory roles in gene expression.MiRNA plays an important role in embryo development and other important life processes.Defects in the biogenesis mechanism of miRNA or effector silencing factors will lead to serious embryo defects.Disruption of miRNA function or level affects the expression level of various target genes and further affects the function of many cells.Evidence has shown that a variety of mirnas can regulate cell proliferation and apoptosis.It is increasingly important to explore the important role of miRNA in recurrent spontaneous abortion and its expression in local or systemic pregnancy.At present,th e research on exosome in female reproductive system mainly focuses on the mechanism of exosome in gestational diabetes,preeclampsia,endometriosis and other related diseases,while the research on exosome in recurrent spontaneous abortion is limited.The mechanism of recurrent spontaneous abortion is very complex,and its immune etiology is an unsolved problem at present.The mechanism of recurrent spon taneous abortion is very complex,and its immune etiology is an unsolved problem at present.It has been found that abnormal immune factors can cause recurrent spontaneous abortion,and exosomes can regulate immune response through specific ways.Before our study,there were no reports about the correlation between exosomes and miRNA in exosomes and recurrent spontaneous abortion at home and abroad.Therefore,we will conduct experiments to investigate whether exosomes and their miRNA are correlated with mother-fetal immune tolerance.ObjectiveFirstly,animal experiments were carried out to study the effect of exosomes secreted by mouse mesenchymal stem cells on the absorption rate of aborted mouse embryos.To investigate whether exosome injection can improve the pregnancy outcome of aborted mice.Then,if this effect exists,further studies will be conducted on the pathways by which exosomes function:whether they function by influencing the secretion of systemic or local system-related cytokines,as well as the differences between systemic and local system-related cytokines.Finally,whether exosome injection can change the expression of miRNA in recipient cells was investigated,and cell experiments were conducted to further verify whether there is immune correlation between the change of miRNA and recurrent spontaneous abortion.Methods1.Bone marrow mesenchymal stem cells were extracted and the fluorescence intensity of CD29,CD105,CD45,CD11b,SCA-1 and TER-119 on the surface of bone marrow mesenchymal stem cells was detected by flow cytometry.Exosomes secreted by bone marrow mesymal stem cells were isolated by supercentrifugation.The structure and morphology of exosomes were observed by transmission electron microscopy,and the expressions of TSG101 and CD63 were detected by Western-blot.2.The internationally accepted mouse models of normal pregnancy group and abortion group were selected,and the mice were mated in groups:?Normal pregnancy group(female CBA/J× male BALB/C mice)?Abortion model group(female CBA/J × male DBA/2 mice)?Exosome injection group(exosome+female CBA/J × male DBA/2 mice)The three groups of mice were cage mated in a ratio of 2:1,and the aborted mice were injected with 100ul exosome at the fourth day of gestation to study the effect of exosome injection on the embryo absorption rate of the three groups of pregnant mice.The immune cells of the decidua of pregnancy were isolated and the differences of CD4+,CD8+ and CD19+ cells in the decidua of pregnancy were detected by flow cytometry.3.CD4+T cells and macrophages were extracted from spleen and uterine decidua of pregnant mice in systemic system and local system.The changes of IL-4,IL-10,TNF-? and IFN-? in CD4+T cells and CD86,CD206,IL-10 and IL-12 in macrophages were detected by flow cytometry.To explore how exosomes affect the immune function of aborted mice,whether they play a role in the systemic system of mice or in the local system,and whether they play a role by affecting T cells or macrophages.4.Carry out the cell experiment.The CD4+in decidua of pregnant mice was detected by real-time fluorescence quantitative PCR The levels of miRNA in T cells of 6 groups(miR-101?miR-122?miR-146b?miR-125a?miR-125b?miR-34a)were closely related to the reproductive system.CD4+T cells were extracted from mice and transfected into CD4+T cells.The experiment was divided into three groups:?blank group,?miR-101-mimics group and ?miR-101-inhitibor group.48 h after transfection,the expression of miR-101 was detected by real-time fluorescence quantitative PCR,the proportion of Thl and Th2 cell subsets was detected by flow cytometry,and the changes of IL-4,IL-10,TNF-? and IFN-? cytokines were detected by supernatant.To investigate whether exosome injection can change the expression of miRNA in recipient cells and whether there is a correlation between the change of miRNA and recurrent spontaneous abortion.Result1.Mouse bone marrow mesenchymal stem cells and their secreted exosomes were extracted and identified.After exosome injection of pregnant mice,the embryo absorption rate of the exosome injection group was similar to that of the normal pregnancy group,and the embryo absorption rate of the exosome injection group was significantly lower than that of the abortion model group(p?0.01).2.The changes of CD4+,CD8+and CD 19+cells in the three groups of pregnant mice were compared.It was found that there were no obvious changes in CD4+and CD 19+cells in the three groups of pregnant mice.CD8+ cells in the abortion group were higher than those in the normal pregnancy group,while CD8+cells in the exosome injection group were significantly lower than those in the abortion group(p<0.05).3.CD4+T cells and macrophages were isolated from spleen.There were no significant differences in the levels of IL-4,IL-10,TNF-? and IFN-? in CD4+T cells between normal pregnancy group,abortion model group and exosome injection group(p>0.05).There were no significant differences in the levels of CD86,CD206,IL-10 and IL-12 in macrophages among the three groups(p>0.05).4.The expression of TNF-? and IFN-? in CD4+T cells in abortion,model group was significantly higher than that in normal pregnancy group,while the expression of IL-4 and IL-10 was significantly lower than that in normal pregnancy group(p?0.01).Compared with abortion model group,the expression of TNF-? and IFN-? in CD4+T cells decreased in exosome injection group,while the expression of IL-4 and IL-10 increased(p<0.05).Compared with the abortion group,the expression of IL-10 in macrophages was increased(p?0.01)and the expression of IL-12 was decreased(p<0.05)in the exosome injection group,but the expression of CD86 and CD206 was not affected.5.Compared with the abortion model group,the expression level of miR-101 in the exosome injection group was increased by 7.2 times(p?0.01),while the expression levels of miR-122,miR-146b,miR-125a,miR-125b and miR-34a were not significantly changed(p>0.05).6.CD4+T cell transfection experiment found that:Over expression of miRNA-101 increased the proportion of Th2 cell subsets,increased the secretion of Th2 cell related cytokines IL-4 and IL-10,decreased the secretion of Thl cytokines IFN-y and TNF-?,and decreased the Th1/Th2 ratio,while knockdown of miR-101 produced opposite results(p?0.001).In conclusion1.Exosome injection can reduce embryo absorption rate and improve pregnancy outcome of mice in abortion group.2.Exosome injection did not improve the pregnancy outcome of aborted mice by affecting the immune function of the whole system of the mice,but affected the production of local immune cells and cytokines in the decidua of the aborted mice.Exosomes induce CD4+T cells to migrate to Th2 type and macrophages to M2 type to induce maternal-fetal immune tolerance.3.Exosome injection improves pregnancy outcome in aborted mice by increasing miRNA-101 expression in CD4+T cells.Cell experiments confirmed that after transfection of CD4+T cells,the overexpression of Mir-101 can induce the differentiation of CD4+T cells into Th2 cells and the secretion of Th2 cytokines,which further proves that the increase of Mir-10 can induce the maternal-fetal immune tolerance and improve pregnancy outcome. |
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