| Background:Recurrent spontaneous abortion(RSA)refers to the loss of embryos(weight≤500 g)before 20 weeks of gestation for at least two consecutive times with the same partner.With the postponement of the average childbearing age of Chinese women in childbearing age and implementation of the two child policy,the proportion of the elderly,the endocrine abnormalities and women with assisted reproductive technology has increased significantly,which lead to the incidence of RSA has increased gradually.The etiology of RSA is very complex.At present,Femal etiology include genetic,anatomical,endocrine,infection,coagulation and immune factors,among which immune factors are the focus of reproductive immunology.After systematic etiology screening,nearly 30%of patients still have unclear etiology,which is called unexplained recurrent spontaneous abortion(URSA).Pregnancy is similar to semi-allogeneic transplantation.Successful pregnancy requires maternal immune system to achieve immune tolerance to paternal antigen carried by embryo.Most experts believe that URSA is related to alloimmune imbalance,in which T-cell immune disorder and dysfunction play an important role,but the mechanism of T-cell immune tolerance is unclear.The treatment of URSA is mainly immunomodulatory.Some studies believe that LMWH can improve the pregnancy outcome in URSA patients,but the treatment mechanism is unclear.Follicular helper T cells(Tfh)are newly found independent T cell subsets,which play an important role in transplantation rejection,autoimmune diseases and tumor immunity.Pregnancy favors the expansion of Tfh in peripheral blood,while Tfh in URSA was significantly higher than that in healthy pregnancy.We speculate that the increase of Tfh cells in pregnancy may contribute to the success of pregnancy,but excessive Tfh cells may lead to abortion.At present,the study of Tfh cells in URSA focuses on the percentage of Tfh cells,and the specific mechanism of the interaction between Tfh cells and trophoblasts in the maternal fetal interface has not been fully investigated.Exosomes are vesicles of membrane structure that are released,which contains lots of information molecules,such as proteins,lipids,and nucleic acids.The phospholipid bilayer membrane structure of exosomes can protect the carrying information from being degraded by enzymes,and make it stably exist in the peripheral blood.The exosomes of maternal peripheral blood can carry a series of information related to trophoblast proliferation,differentiation,apoptosis,angiogenesis and embryo development.Our previous study found that after LMWH treatment,the expression of miR-18a-3p and miR-17-3p in serum exosomes of URSA patients changed significantly,making them return to the level of healthy women of first trimester.LMWH can improve the biological function of trophoblast in vitro.Mir-18a-3p and mir-17-3p belong to mi-17-92 gene cluster.Mi-17-92 gene cluster is very important for the differentiation and long-term survival of Tfh cells.We speculated that LMWH might improve the function of trophoblast and change the content of related miRNA in exosomes,so as to improve the abnormal Tfh of peripheral blood in URSA patients.Objective:This study is divided into three parts:the first part is to study the changes of CD4~+CXCR5~+Tfh cells in decidua and peripheral blood of healthy pregnant women during pregnancy.The second part is to compare the percentage of Tfh cell in peripheral blood of first trimester in healthy women,URSA without LMWH treatment and URSA after LMWH treatment;To compare the percentage of Tfh cell in decidua between URSA and healthy women in first trimester.The third part is to explore the mechanism of LMWH in improving the abnormal Tfh in URSA patients.Methods:Part 1:In the first part,the percentage of Tfh subsets in peripheral blood and decidua of healthy women in first trimester,second trimester,third trimester and before delivery were detected by primary cell extraction and flow cytometry.A total of 80healthy pregnant women were divided into 4 groups according to the gestational weeks,20 cases in each group:first trimester;second trimester;third trimester;before delivery,flow cytometry was used to detect the percentage of CD4+CXCR5+Tfh in CD4+T cell subsets in peripheral blood;PD1~-ICOS~+Tfh,PD1~+ICOS~+Tfh,PD1~+ICOS~-Tfh and PD1-ICOS-Tfh in CD4+CXCR5+Tfh subsets;CXCR3-CCR6+Tfh,CXCR3+CCR6-Tfh and CXCR3~-CCR6~-Tfh as a percentage of CD4~+CXCR5~+CD45RA~-cells.The decidua tissue were collected from 25 cases,including 13 cases of first trimester and 12 cases of before delivery.The DICs were extracted and the percentage of the above Tfh subsets in DICs was detected by flow cytometry;Part 2:The percentage of Tfh subsets in decidua and peripheral blood was detected by primary cell extraction and flow cytometry.A total of 60 first trimester women,among them 30 healthy women(healthy control group),30 URSA patients without LMWH treatment(URSA before treatment group),after LMWH treatment,Tfh cell in URSA were tested again(URSA after treatment group).The percentage of Tfh in peripheral blood were also measured.The decidua tissues of 10 URSA women(D-URSA group)and 10 healthy women(D-control group)were collected,the primary immune cells of decidua were extracted,and the above Tfh subsets of DICs were detected by flow cytometry.RT-PCR was used to detect the mRNA expression of Tfh cells(CXCR5,bcl-6,PD-1,ICOS,CXCR3 and CCR6)in decidua of D-control group and D-URSA group.Part 3:1.The peripheral blood of URSA without LMWH treatment were collected and PBMC was extracted.Flow cytometry was used to detect the percentage of Tfh before and after treatment of PBMC with LMWH at concentrations of 0IU/ml,0.25IU/ml,0.5IU/ml,1.0 IU/ml,2.0 IU/ml,2.5 IU/ml and 5.0 IU/ml.2.PBMC was cultured in the supernatant of HTR-8/SVneo cells(control group)and LMWH treated HTR-8/SVneo cells(treatment group).The percentage of Tfh before and after treatment was detected by flow cytometry.3.Transmission electron microscope,Nanosight Tracking Analysis and western blot(CD63,CD81,ALIX,PLAP)were used to identify characteristics of exosome.of LMWH treated HTR-8/SVneo cells(Exo+group),Supernatant of LMWH treated HTR-8/SVneo cells without exosome(Exo-group)and exosome of supernatant of LMWH treated HTR-8/SVneo cells(Exo group)treated PBMC respectively,flow cytometry detected the percentage of Tfh before and after treatment.Exosome of the supernatant of HTR-8/SVneo cells and LMWH treated HTR-8/SVneo cells were used to treat PBMC respectively,flow cytometry detected the percentage of Tfh before and after treatment.Total RNA in exosome of supernatant of HTR-8/SVneo cells and LMWH treated HTR-8/SVneo cells was extracted.RT-PCR was used to detected the expression of miR-17-3p and miR-18a-3p in exosome.Results:Part 1:1.The analysis of clinical data showed that the age,BMI and gestational age were comparable between groups.2.With the development of gestation,the proportion of CD4~+CXCR5~+Tfh cells in CD4~+T cells increased gradually(P=0.001),and the percentage of PD1~-ICOS~+Tfh and PD1~+ICOS~+Tfh in CD4~+CXCR5~+Tfh decreased significantly(P<0.01).The percentage of PD1~-ICOS~-Tfh in CD4~+CXCR5~+Tfh increased significantly with the development of gestation(P=0.005).The percentage of CXCR3~-CCR6~-in CD4~+CXCR5~+CD45RA~-Tfh cells reached the peak in third trimester(P<0.0001).3.The proportion of PD1~+ICOS~-Tfh in CD4~+CXCR5~+Tfh cells in first trimester was significantly higher than that before delivery(P<0.0001).The percentage of PD1-ICOS-Tfh in CD4+CXCR5+Tfh cells in first trimester was lower than that before delivery(P=0.0009).The percentage of CCR6~-CXCR3~-Tfh cells to CD4+CXCR5+CD45RA-Tfh cells in decidua before delivery was significantly higher than that in first trimester(P=0.0336).Part 2:1.The general data of the subjects are comparable between groups.2.The percentage of CD4~+CXCR5~+Tfh cells in peripheral blood of URSA was significantly higher than that of healthy control group(P=0.04);the percentage of CD4~+CXCR5~+Tfh cells in peripheral blood of URSA with LMWH treatment group was not significantly different from that of healthy women(P=0.26).The percentage of PD1~+Tfh cells in peripheral blood of URSA group was significantly higher than that of healthy women(P=0.02).The percentage of CCR6-CXCR3+Tfh cells in peripheral blood of healthy women was significantly lower than that of URSA patients(P=0.0138).3.The percentage of CD4+CXCR5+Tfh cells in decidua of D-control group was significantly lower than that of D-URSA group(P=0.006).Compared with D-control group,the percentage of PD1~-ICOS~+Tfh and PD1~+ICOS~+Tfh in CD4~+CXCR5~+T cells in D-URSA group increased significantly(P<0.05).Compared with D-control group,the ratio of CXCR3~+CCR6~-Tfh to CD4~+CXCR5~+CD45RA~-Tfh cells in D-URSA group increased significantly(P=0.010),and the ratio of CXCR3-CCR6-Tfh to CD4+CXCR5+CD45RA-Tfh cells decreased significantly(P=0.002).Part 3:1.The percentage of CD4+CXCR5+Tfh in CD4+T cells before and after PBMC treatment with LMWH of 0IU/ml,0.25 IU/ml,0.5 IU/ml,1.0 IU/ml,2.0 IU/ml,2.5IU/ml and 5.0 IU/ml had no significant change(P>0.05).2.Compared with the control group,the percentage of CD4~+CXCR5~+Tfh in CD4~+T cells and the percentage of PD1+Tfh in CD4+CXCR5+Tfh cells decreased significantly(P<0.05).3.The results of nanoparticle tracing analysis showed that the peak value of the exosomes was 130 nm,and the exosomes showed a round cup vesicular structure under TEM.Western blot showed that the exosomes expressed the key surface markers of CD81,CD63,Alix and PLAP,which were consistent with the theoretical characteristics.The percentage of CD4+CXCR5+Tfh cells in Exo+group and Exo group decreased significantly after PBMC treatment(P<0.05),but there was no significant change in Exo-group before and after PBMC treatment(P>0.05).4.The expression of miR-18a-3p in Exo+group was significantly higher than that in Exo-group(P<0.05).There was no significant difference in the expression of mir-17-3p between the three groups(P>0.05).Conclusion:Part 1:The PD1-ICOS-Tfh and CXCR3-CCR6-Tfh cells are related to the maintenance of immune tolerance in the second and third trimester.The PD1+ICOS-Tfh cells are more inclined to regulate the immune balance in the first trimester.Part 2:The percentage of Tfh cells in the peripheral blood of URSA patients increased significantly,especially the PD1+Tfh and CCR6-CXCR3+Tfh.LMWH can significantly reduce the abnormal Tfh level of URSA patients,especially PD1~+Tfh.Part 3:LMWH may affect the percentage of Tfh in URSA patients by changing the content of miR-18a-3p in exosome secreted by trophoblasts.The specific mechanism needs to be further studied. |
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