Populational change of CD4^(+)CD25^(+)Treg cells is responsible for the synergistic effect of the combination of RAMP2 with baicalin in treating recurrent spontaneous abortion mouse models

Background: The absence of a safe and effective therapy for recurrent spontaneous abortion due to a maternofetal failure in immunological tolerance remains an intractable clinical obstacle for surgeons. Recently, traditional Chinese medicine has become a feasible alternative for certain diseases, including recurrent spontaneous abortion. However, because of the complex composition of the traditional Chinese medicine formula, its action mechanism remains unclear. Methods: We selected two isolated active ingredients (RAMP and baicalin) from the traditional Chinese medicine formula and used an abortion-prone CBA/J × DBA/2 model to simulate human RSA and compared the changes in fetal resorption rate, Treg cell percentage, and relevant cytokines before and after combination therapy. In addition, The mechanisms were preliminarily discussed using in vitro differentiation models. Results: In CBA/J × DBA/2 abortion-prone mice, the combination therapy resulted in a lower embryo resorption rate compared to that obtained with individual delivery of either RAMP or baicalin, thereby playing an embryo-protective role through the increase in Treg cells for the maintenance of maternal-fetal immune tolerance. In in vitro primary cell differentiation experiments, the concentration of Treg cells significantly increased from 11% to 17.9% after the combination therapy compared to that of the single administration group. Conclusion: the synergistic effects of RAMP and baicalin were responsible for Treg differentiation. The present study provides a solid basis for improving the applicability of traditional Chinese herbs in the treatment of recurrent spontaneous abortion.

Advances of Genetic Testing Technology in Etiology Diagnosis of Recurrent Spontaneous Abortion

Recurrent spontaneous abortion (RSA) is a complex and heterogeneous disorder with multiple etiologies. Genetic factors are thought to play an important role in the etiology of RSA. With recent advances in genetic testing technologies, there has been an increasing interest in using these tools to diagnose the etiology of RSA. This review discusses the different types of genetic testing methods, such as karyotyping, chromosomal microarray analysis, next-generation sequencing, and their applications in the diagnosis of the etiology RSA. The use of genetic testing in the diagnosis of RSA has the potential to improve the accuracy of diagnosis and the understanding of the underlying mechanisms of the disorder, which could lead to better management and treatment of affected individuals.

Up-regulated Expression of Tim-3/Gal-9 at Maternal-fetal Interface in Pregnant Woman with Recurrent Spontaneous Abortion

The relationship between T cell immunoglobulin domain and mucin domain protein 3（Tim-3）/Galectin（Gal）-9 pathway and recurrent spontaneous abortion（RSA） was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin（IL）-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas（P〈0.05） and healthy fertile non-pregnant controls（P〈0.05）, but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas（P〈0.05） and healthy fertile non-pregnant controls（P〈0.05）. It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.

Research advances of Chinese and Western medicine in the treatment of unexplained recurrent spontaneous abortion

Recurrent spontaneous abortion is a common disease in gynecology,and it seriously affects women's reproductive health and brings heavy burden and pain to society and families.The cause of recurrent spontaneous abortion is complicated,in addition to the well-defined genetic,anatomical,infection and endocrine factors,and there are still some unknown causes,which is called as unexplained recurrent spontaneous abortion,accounting for 40%of recurrent abortion.At present,there are a lot of researches on the treatment methods of the patients with the unexplained recurrent spontaneous abortion,which also shows that the treatment of traditional Chinese and Western medicine all have certain clinical application effect.Western medicine clinical methods mainly includes immunotherapy,immunosuppressive therapy,anticoagulation therapy,progesterone therapy,etc.Based on the experience of the professor and combined with many years of clinical practice,the author believes that the pathogenesis of this disease in traditional Chinese medicine is mainly due to impaired impulse and deficiency of Spleen,lack of qi and blood,can not nourishing the fetus;deficiency of Kidney Qi,blood flow was delayed,and blood stasis and could not raise the fetus.Clinical treatment is based on invigorating the kidney,tonifying spleen and nourishing blood,promoting blood circulation to remove blood stasis and dredging collaterals.Oral Chinese medicine combined with external acupuncture and moxibustion has achieved excellent effects in improving pregnancy rate.This article reviews the domestic and foreign methods of treating unexplained recurrent miscarriage in order to provide clinical reference.In the future,the combination of Chinese and Western medicine should become the main therapy to increase pregnancy rate.

Meta-Analysis of Bushen Huoxue Method in the Treatment of Recurrent Spontaneous Abortion Due to Prethrombotic State

Objective:To evaluate the effectiveness and safety of Bushen Huoxue prescription in the treatment of recurrent spontaneous abortion(RSA)due to prethrombotic state(PTS).Methods:Databases such as CNKI,WanFang,VIP,CBM,PubMed,Embase,and Cochrane Library were searched for randomized controlled trials on Bushen Huoxue prescription in treating RSA due to PTS from inception to March 2021;meta-analysis was performed by RevMan Version 5.3.0 following quality evaluation.Results:Seven trials were included,with 496 patients;the meta-analysis indicated that Bushen Huoxue prescription has advantages on the improvement of total clinical effective rate[RR=1.22,95%CI(1.10,1.35),Z=3.80(P=0.0001)],embryo survival rate at pregnancy of 12 weeks[RR=1.25,95%CI(1.10,1.41),Z=3.53(P=0.0004)],D-dimer levels[SMD=-1.59,95%CI(-2.20,-0.97),Z=5.07(P<0.00001)],and fibrinogen levels[MD=-1.00,95%CI(-1.29,-0.70),Z=6.61(P<0.00001)],but the statistical heterogeneity was significant;in terms of incidence of adverse reactions,there was no statistical difference between Bushen Huoxue prescription and western medicine.Conclusion:Compared with western medicine alone,Bushen Huoxue prescription alone or in combination with western medicine showed significant advantages in improving the overall clinical efficiency,embryonic survival rate at 12 weeks of pregnancy,and reducing D-dimer values as well as fibrinogen levels,without any significant difference in the incidence of adverse effects;however,the number of included studies is small and there are drawbacks,such as small sample size and low quality;therefore,high-quality clinical studies with large sample size and rigorous trial designs are needed in the future to provide a reliable basis for the effectiveness and safety of TCM in reducing the incidence of RSA due to prethrombotic state.

Small RNA sequencing revealed aberrant piRNA expression profiles in deciduas of recurrent spontaneous abortion patients

Piwi-interacting RNAs(piRNAs)is a novel class of non-coding RNAs.However,changes in piRNA expression profiles in recurrent spontaneous abortion(RSA)have not yet been investigated.The aim of this study was to identify differentially expressed piRNAs in deciduas of RSA patients.Decidua tissues were collected by curettage from recruited RSA patients and normal early pregnant(NEP)women with their informed consent.Small RNA sequencing was used to evaluate the differences in piRNA expression profiles between RSA and NEP.The present results demonstrated that the counts of total piRNA reads in RSA samples were increased compared with those in NEP samples(0.21%vs.0.11%).Differential expression analysis identified 29 upregulated piRNAs and 18 downregulated piRNAs in RSA samples.RT-qPCR further confirmed that the expression levels of uniq-109625,uniq-89328,uniq-50651 and uniq-4569 were decreased in 8 RSA tissues,compared with 13 NEP tissues.Otherwise,pi-22628 and uniq-173406 were increased in 8 RSA tissues.Based on GO term and KEGG pathway analysis,we speculate that these piRNAs regulate RSA by targeting extracellular matrix component pathway,cell adhesion pathway and focal adhesion pathway.PiRNAs may be involved in RSA pathogenesis by target genes function on adhesion and extracellular matrix component.

Investigation of FOXP3 (rs3761548) polymorphism with the risk of preeclampsia and recurrent spontaneous abortion: A systemic review and meta-analysis

Objective:To investigate the association between forkhead box P3(FOXP3)(rs3761548)polymorphism and the risk of preeclampsia and recurrent spontaneous abortion.Methods:Literature on the association of FOXP3 gene polymorphisms and susceptibility to preeclampsia and unexplained recurrent spontaneous abortion was retrieved by searching databases such as PubMed,Science Direct,Google Scholar and Embase from 2000 to 2021.The association measure was analyzed using an odds ratio(OR)and 95%confidence interval(CI).All the statistical analyses were executed using RevMan 5.4 software.Results:In the present meta-analysis,11 articles were analyzed.The pooled results showed no association between FOXP3 gene polymorphism(rs3761548)and preeclampsia risk in allelic,recessive,dominant and over dominant contrast models.FOXP3 gene polymorphism(rs3761548)showed an association with recurrent abortion in allelic,recessive and dominant models(OR 1.85,CI 1.59-2.14;OR 2.02,95%CI 1.56-2.62;OR 2.69,95%CI 1.50-4.83,respectively),while no association in the over dominant contrast model(OR 1.35,CI 0.87-2.10).Conclusions:In the present study,FOXP3 gene(rs3761548)polymorphism is associated with risk of recurrent spontaneous abortion but not preeclampsia.However,larger sample size and multiracial studies are needed in the future to confirm the findings.

Correlation of BDNF Gene Polymorphism and Psychological Nursing Intervention in Patients with Recurrent Spontaneous Abortion

Objective: To investigate the correlation between recurrent spontaneous abortion patients and brain derived neurotrophic factor BDNF gene polymorphism and the mechanism of BDNF and recurrent spontaneous abortion under stress state in order to provide theoretical basis for nursing psychological intervention of patients with recurrent abortion. Methods: Medical coping Questionnaire (MCMO) and post-traumatic stress Diagnostic Scale (POST-traumatic stress Scale) were adopted Dissorder (PTSD) (PCL-c) scale was used to diagnose the psychological stress of recurrent spontaneous abortion in our hospital, and the BDNF gene polymorphism and the correlation factors of METHYLation in BDNF promoter region were studied in 30 cases (control group) and normal control group. Results: The MCMQ score of the case group and the control group was significantly lower than that of the conventional group (P Conclusion: There are significant differences in PTSD between the case group and the control group, which are related to methylation in the PROMOTER region of BDNF and SNP g-712A of BDNF gene. AG patients in the case group are more susceptible to anxiety and depression, and GG PTSD is more severe in the case group. BDNF promoter methylation and G-712A were independent risk factors for PTSD in the case group.

Association between p53 Polymorphism at Codon 72 and Recurrent Spontaneous Abortion

p53 gene plays an important role in apoptosis, which is necessary for successful invasion of trophoblast cells. The change from an arginine（Arg） to a proline（Pro） at codon 72 can influence the biological activity of p53, which predisposes to an increased risk of recurrent spontaneous abortion（RSA）. In order to investigate the association between p53 polymorphism at codon 72 and RSA, we conducted this meta-analysis. Pubmed, Embase and Web of science were used to identify the eligible studies. Odds ratio（OR） with 95% confidence interval（CI） was used to evaluate the strength of the association. Six studies containing 937 cases of RSA and 830 controls were included, and there was one study deviated from Hardy-Weinberg equilibrium（HWE）. There was a significant association between p53 polymorphism at codon 72 and RSA in recessive model（Pro/Pro vs. Pro/Arg＋Arg/Arg; OR=1.60, 95% CI： 1.14–2.24） and co-dominant model（Pro/Pro vs. Arg/Arg; OR=1.47, 95% CI： 1.02–2.12） whether the study that was deviated from HWE was eliminated or not. A significant association was observed in allelic model（Pro vs. Arg; OR=1.28, 95% CI： 1.04–1.57） after exclusion of the study that was deviated from HWE. No association was noted in recessive model（Pro/Pro＋Pro/Arg vs. Arg/Arg; OR=1.05, 95% CI： 0.86–1.30） and co-dominant model（Pro/Arg vs. Arg/Arg; OR=0.96, 95% CI： 0.77–1.19）. Subgroup analysis by ethnicity also indicated a significant association between p53 polymorphism at codon 72 and RSA in Caucasian group. No heterogeneity and publication bias were found. Our meta-analysis implied that p53 polymorphism at codon 72 carries high maternal risk of RSA.

Animal study on CD4^(+) CD25^(+) regulatory T cells for treating female mouse with recurrent spontaneous abortion

Objective:To explore immunotherapy effectiveness of the CD4^(+)CD25^(+) regulatory T cells for treating female mouse with recurrent spontaneous abortion(RSA)by animal experiments.Methods:Mononuclear lymphocytes were isolated from the blood(instead of cord blood)of new-born baby of KunMing Bai mouse or BALB/c male mouse with normal birth ability(as unrelated third party blood source)by density gradient centrifuga-tion method.The CD4^(+)CD25^(+) regulatory T cells were selected by magnetic-activated cell sorting from mononuclear cells of cord blood cells.CBA/J female mouse copulated with DBA/2J male mouse was utilized as RSA animal model.Pregnant RSA mice were injected different types of lymphocytes through tail vein.Independent sample t-test was used to analyze the data from each group.Results:The proportion of CD4^(+)CD25^(+)T cells in CD4^(+)T cells was(17.49±0.60)%in CD4^(+)CD25^(+) regulatory T cells injection group,which was statistical significant higher than that of mononuclear lymphocyte injection group(14.68±0.83)%,sterile PBS group(9.54±0.85)%or no injection group(9.28±0.68)%(p<.05,t-value was 4.754,13.242 and 15.621,respec-tively).The Foxp3 relative protein expression level of CD4^(+)CD25^(+) regulatory T cells injected group was 5.85±0.45,which was also significant higher than that of mononuclear lymphocyte injection(2.86±0.54),sterile PBS group(1.08±0.16)or no injection group(1.00±0.00)(p<.05,t-value was 7.276,17.227 and 18.635,respectively).Finally,two times of CD4^(+)CD25^(+)T cell injected group at the 4 th and 8 th day had well effect for RSA mouse,and embryo sorption rate was(4.92±0.08)%,which significant lower than that of two times of mononuclear lymphocyte injected group(13.07±0.06)%,sterile PBS group(23.11±0.12)%,or no injection group(25.47±0.11)%(p<.05,t-value was-2.603,-4.012 and-4.700,respectively).Conclusions:Pregnant mouse with RSA injected CD4^(+)CD25^(+)T cells several times for immunotherapy can get better effec-tiveness than that of pregnant mouse injected traditional mononuclear cells.

Efficacy of Different Treatment Regimens for Antiphospholipid Syndrome-related Recurrent Spontaneous Abortion

Background： Antiphospholipid syndrome （APS）-related immune factors are considered as an important cause of recurrent spontaneous abortion （RSA）. Anticoagulant and anti-inflammatory treatments are believed to effectively improve adverse pregnancy outcomes by affecting the abnormal autoimmune response of the maternal-fetal interface. The aim of this study was to observe the clinical characteristics and treatment outcomes of anticoagulant regimens and anti-inflammatory plus anticoagulation regimens for APS-related RSA. Methods： APS-related RSA cases from September 2011 to September 2016 at Peking University Third Hospital were retrospectively analyzed. The patients were assigned to study group （anti-inflammation plus anticoagulation） and control group （simple anticoagulation）, The incidence of repeat abortion, the incidence of placental dysfunction, the gestational weeks of pregnancy, and the mean weight of the fetus were observed. Results： The pregnancy and neonatal outcome indicators of the repeat pregnancy loss rate （ 11. 11% vs. 22.70%）, placental dysfunction-related diseases （6.35% vs. 15.60%）, the mean birth weight of infants born after 24 weeks gestation （3152.4 ± 844.67 g vs. 2765.76 ± 816.40 g）, full-ternl delivery weight （3456.28 ±419.79 g vs. 3076.18±518.79 g）, the proportions of low birth weight infants （ 12.70% vs. 21.98%）, and small for gestational age （6.35% vs. 14.18%） differed significantly between the study and control groups （all P 〈 0.05）. The incidence of preterm delivery, term delivery, and stillbirth was not significantly different between the two groups, and there was no significant difference between the study and control groups in gestational age at birth （37.6 ± 3.3 weeks vs. 36.9 ± 3.2 weeks： P 〉 0.05）. Conclusion： The anti-inflammatory and anticoagulation regimen is more effective than the simple anticoagulation regimen in the treatlnent of APS recurrent abortion.

Association of Estrogen Receptor I Genetic Polymorphisms with Recurrent Spontaneous Abortion Risk

Background： Estrogen is one of the most important reproductive steroidal hormones and plays a critical role in the maintenance of pregnancy, and its function is mediated by estrogen receptor 1 （ESR 1）. The polymorphisms ofESR 1 were involved in recurrent spontaneous abortion （RSA）; however, the association between ESRI polymorphisms and RSA remains controversial. The present meta-analysis was aimed to clarify the association between ESRI Pvull （-397C/T, rs2234693） and Xbal （-351 A/G, rs9340799） polymorphisms and the risk of RSA. Methods： All the included articles were retrieved from PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, and Wanfang Med Online Database up to January 3,2018. Data were processed in the Stata 12.0 software. The odds ratios （ORs） and 95% confidence intervals （95% Cls） were calculated using fixed-effects models （FEM）/random-effects models （REM）. Results： Seven case-control studies with 836 cases and 1164 controls were included in the study. Generally, the ESR 1 polymorphisms were not associated with RSA in any of the genetic analysis models. However, it was found that as rs9340799 polymorphism was related to increased risk of RSA in non-Asian group in the homozygous genetic model （OR = 2.40, 95% CI = 1.05-5.50, P = 0.039）. Moreover, in Asian group, rs9340799 polymorphism was found to be related to decreased RSA risk in both the heterozygous model （OR = 0.53, 95% CI = 0.33-0.85, P = 0.009） and the dominant genetic model （OR=0.55, 95% CI = 0.30 0.98, P = 0.042）. Conclusions： Generally. there was no significant association between the polymorphisms of ESRI and the risk of RSA. However, subgroup analysis indicated that ESR1 rs9340799 polymorphism was related to increased RSA risk in the non-Asian group while associated with decreased RSA risk in Asian group.

Role of cyclooxygenase-2 signaling pathway dysfunction in unexplained recurrent spontaneous abortion

Background Experimental evidence indicates that cyclooxygenase-2 （COX-2） plays a critical role in blastocyst implantation; however, little is known of the role of COX-2 in unexplained recurrent spontaneous abortion （URSA）. Methods We evaluated the expression level and potential signaling pathway of COX-2 in 30 cases of URSA who were excluded the abnormality of chromosomes, anatomy, endocrine, infectious, autoimmune diseases and in 30 normal pregnancies. Results The mRNA and the protein expression level of COX-2 in the URSA group （-0.238±0.848, 0.368±0.089, respectively） were significantly lower than that in the control group （1.943±3.845, 1.046±0.108, respectively） （both, P 〈0.01）. The expression of prostaglandins PGF2a, PGD2, PGE2, and PGI2, in the URSA group （（2326.0±295.6） pg/ml, （2164.0±240.5） pg/ml, （238.7±26.4） pg/ml, （2337.0±263.0） pg/ml, respectively） were significantly lower than that in the control group （（3450.0±421.7） pg/ml, （3174.0±415.6） pg/ml, （323.5±43.8） pg/ml, （3623.0±460.4） pg/ml, respectively） （P 〈0.05）. The mRNA expression level of PPARI3 and RXRa （0.859±0.653, -0.172±0.752, respectively） in URSA group was significantly lower than that in the control group （1.554±1.735, 0.777±2.482, respectively） （both P 〈0.05）. The mRNA and protein expression levels of vascular endothelial growth factor-A （VEGF-A） in the URSA group （2.010±1.522, 0.35±0.46） was significantly lower than that in the control group （4.569±2.430, 0.750±0.350） （both P 〈0.05）. Conclusions COX-2 and the COX-2-derived PGI2 signaling pathway possibly play an important role in successful embryo implantation, and their decreased expression may result in URSA. The decreased expression may influence the expression of VEGF-A which interferes with placental angiogenesis causing failure of embryo implantation, leading to spontaneous abortion.

The imbalance of T-cell immunoglobulin and ITIM domain and CD226 on regulatory T cell in recurrent spontaneous abortion patients

Objective:To analyze the proportion of peripheral regulatory T cells(Tregs)and the expression of the immune checkpoint molecules T-cell immunoglobulin and ITIM domain(TIGIT)and CD226 on Tregs in patients with recurrent spontaneous abortion(RSA).Methods:The proportion of CD3+CD4+CD25+Foxp3+Tregs and the expression levels of CD226 and TIGIT on Tregs in 30 normal pregnant women and 28 patients with RSA were determined via flow cytometry.Results:The proportion of Tregs in the RSA group(4.41%±1.54%)was significantly lower than that in the control group(5.27%±1.52%,P=0.0374).Compared with the normal pregnant women,patients with RSA showed decreased TIGIT expression(54.75±9.70%vs.63.07±12.48%,P=0.0066)and increased CD226 expression on Tregs(25.59%±8.22%vs.20.46%±6.97%,P=0.0168).The ratio of CD226 to TIGIT in the RSA group(0.48±0.19)was higher than that in the control group(0.34±0.15,P=0.0027).The proportion of TIGIT+CD226+Tregs was significantly lower in patients with RSA(9.30%±4.95%vs.13.43%±4.72%,P=0.0020)than in the controls.Conclusions:Patients with RSA show a reduced proportion of Tregs and an imbalance in the expression of TIGIT and CD226 on Tregs.

Treatment of Integrated Traditional and Western Medicine in Recurrent Spontaneous Abortion of Immune Abnormality Type

Objective: Immune abnormality type of recurrent spontaneous abortion (RSA) is an important etiological type, major findings of that are the abnormal increases in auto and/or allo-antibodies of anti-zona pellucida, anti-phospholipid and anti-ABO blood group. The purpose of the present study is to assess the clinical values of integrated traditional and western medicine in treating the immune abnormality type of RSA.Methods: The aborters were treated by Zhibai Dihuang Pills for anti-zona pellucida antibodies, and clearing evil Heat, removing Dampness, replenishing blood and activating circulation by Chinese medicinal herbs recipe for anti-phospholipid and anti-ABO group antibodies.Results: After they had been treated by these recipes, 92.3% became pregnant with normal delivery. It was found in dynamic investigation that level of the antibodies turned to decrease gradually during the treatment, and that of anti-phospholipid and anti-ABO group antibodies increased again at beginning of pregnancy and then decreased gradually with continuing the treatment.Conclusion: Chinese herbs recipe could be used to treat RSA of immune abnormality-type by way of decreasing the elevated levels of auto and/or allo-antibodies.

Pathogenesis,diagnosis,and treatment of recurrent spontaneous abortion with immune type

Recurrent spontaneous abortion(RSA),defined as three or more consecutive pregnancy losses before 20 weeks of gestation,is difficult to treat in the clinical setting.It affects 1%–5%of women of reproductive age.In the investigations of immunopathogenesis,diagnosis,and treatment of RSA since the late 1980s,it was found that RSA was associated with abnormal maternal local or systemic immune response.The pathogenesis of autoimmune RSA was mainly associated with antiphospholipid antibody(APA),while that of alloimmune RSA was due to the disturbance of maternofetal immunological tolerance.Systemic etiological screening process and diagnosis systems of RSA with immune type were developed,and anticardiolipin(ACL or ACA)+anti-β2-GP1 antibody combining multiple assays for effective diagnosis of RSA with autoimmune type was first established.According to the dynamic monitoring of clinical parameters before and during gestation,low-dose,short-course,and individual immunosuppressive therapy and lymphocyte immunotherapy for RSA with immune type were carried out.The outcomes of the offsprings of patients with RSA were followed up,and the safety and validity of the therapies were confirmed.The research achievement leads to great progress in the diagnosis and treatment of RSA in China.

The Effect of Lymphocyte Immunotherapy on CD80^+ Cells at the Fetomaternal Interface and Cyesis Result of Mice Model of Spontaneous Abortion

Objectives To explore the relationship between CD80 expression on lymphocytes at the fetomaternal interface and the fertility characteristics in CBA/J×DBA/2 mice as a model of recurrent spontaneous abortion (RSA) and to investigate the effects of lymphocyte immunotherapy (LIT) on the level of CD80 expression. Materials & Methods The characteristics of fertility in CBA/J×DBA/2 mice were observed in a 120 day period and compared with four normal fertile groups. In another 15 pairs of CBA/J×DBA/2 breedings, resorption rate on day 13 of pregnancy were calculated and the proportion of CD80 + cells at the fetomaternal interface were determined by using two color flow cytometric analysis, mainly stained with CD80 FITC and CD45 PE. In order to determine the identity of CD80 + cells, the expression levels of CD3(T cell marker), DX5(NK cell marker), and MHC II(antigen presenting cell marker) were detected in this cell population. Furthermore, the resorption rate and the proportion of CD80 + cells among CBA/J×DBA/2 breedings with and without immunotherapy were also determined and compared with normal fertile controls. Results The characteristics of abortion in CBA/J×DBA/2 mice were recurrent abortion on about day 10 of gestation. The resorption rate in CBA/J×DBA/2 mice was significantly higher than that in BALB/c×DBA/2 mice (30.8%±16.6% vs. 7.7%±6.7%, P<0.01). Accordingly, the proportion of CD80 + cells evaluated at the fetomaternal interface in CBA/J×DBA/2 mice was also significantly higher (11.7% ±5.8% vs. 3.9%±1.8%, P<0.01). Resorption rate of CBA/J×DBA/2 mice underwent of LIT was significantly lower than that without LIT, and this decreased rate was correlated with decreased proportion of CD80 + cells. Conclusion In CBA/J×DBA/2 mice model, the characteristics of abortion seem to be peri implantation embryo resorption. A correlation between early embryonic waste and higher CD80 proportion at the fetomaternal interface suggests that CD80 + cells may be an important determinant in recurrent peri implantation abortion.

VEGFR2 mediated RAS/MAPK signaling pathway to explore the mechanism of Bushen Antai Granule in the treatment of recurrent abortion

Objective:To investigate the effect of Bushen Antai Granule on the mRNA and protein expression of Ras protein/mitogen activated protein kinase mediated by vascular endothelial growth factor receptor 2 with small interference RNA interference.Methods:Method to construct the placenta microvascular endothelial cells,and the preparation of kidney fetus granule drug-containing serum,select the best drug-containing serum concentration,it can be divided into normal group,the serum siRNA-NC normal serum group,drug serum,siRNA normal serum group,siRNA drug serum group,using real-time fluorescent quantitative PCR,Western blotting,immunofluorescence test respectively the RAS/MAPK mRNA and protein expression.Results:Results there was no significant difference in Ras and MAPK mRNA and protein expression between the normal group and the negative control group(P>0.05).The mRNA and protein expressions of Ras and MAPK in the drug serum group were significantly higher than those in the normal serum group(P<0.01).Ras and MAPK mRNA and protein expression were significantly decreased in siRNA1 normal serum group compared with normal serum group(P<0.01).Ras,MAPK mRNA and protein expression in siRNA1 drug serum group were significantly different from that in siRNA1 normal serum group(P<0.01).Conclusion:Conclusion The therapeutic effect of Bushen Antai Granule on recurrent abortion may be realized by upregulation of RAS/MAPK mRNA and protein expression.

Mechanism of treating recurrent abortion with “Peiyuan Bushen Antai formula” based on network pharmacology and molecular docking

Objective:To study the effective compound action target signal pathway of Peiyuan bushenan taifang(PYBSATF)has achieved good clinical efficacy in the treatment of recurrent spontaneous abortion(RSA),but its mechanism has not been clarified because of its complex components.In this study,network pharmacology is applied to study the effective compounds,targets and signal pathways of PYBSATF in the treatment of RSA,so as to reveal its pharmacological mechanism of action in the treatment of recurrent spontaneous abortion.Methods:The Traditional Chinese Medicine Systems Pharmacology database(TCMSP)and CNKI are used to obtain the main compounds and drug action targets of PYBSATF.Genecards and the Online Mendelian Inheritance of Man databases(OMIM)are searched to collect the known genes related to RSA,so as to construct compound-target network and screen out the common target proteins and main active compounds.We also use string database to construct a visual protein-protein interaction network(PPI).Cluster Profiler and R software were used to analyze the common targets of drugs and diseases for GO function analysis and KEGG pathway enrichment analysis.Finally,the compound and the protein sequences were conducted according to the node parameters,so that the core protein and core compounds are used to perform molecular docking.Results:186 potential active components and 65 predicted action targets of PYBSATF were screened out.At the same time,1658 genes were also screened out to be closely related to RSA,among which 65genes overlaped with PYBSATF targets and were considered to be related to way of treatment.PPI network showed that VEGFA,IL6,EGFR,MAPK8 and ESR1were the core targets of PYBSATF for the treatment of RSA.GO and KEGG enrichment analysis obtained 93 biological processes of cells(P<0.01)and 87 signaling pathways(P<0.01).PYBSATF played a pharmacological role through a variety of pathways including anti-inflammatory,anti-apoptosis,promoting proliferation and angiogenesis.Molecular docking showed that most active components and key targets of PYBSATF had strong efficiency.Conclusion:Through the study of network pharmacology,it predicted that PYBSATF might treat RSA through multiple targets and multiple signal pathways.Significantly,the predictive targets may be potential targets for treatment of RSA.