The Association Of Complement And Coagulation Cascade Pathway With Unexplained Recurrent Spontaneous Abortions

Unexplained recurrent spontaneous abortion(URSA)refers to recurrent spontaneous abortion that excludes chromosomal abnormalities,anatomical abnormalities,endocrine disorders,reproductive tract infections,autoimmune diseases and other factors,with a incidence of about 1%?5%,which seriously affects the physical and mental health of women of child-bearing age and their family happiness.So far,the pathogenesis of URSA remains unclear.Over the past 40 years,more and more studies have shown that the occurrence of URSA is closely related to gene mutations,changes in protein expression levels and metabolic abnormalities.Although these studies have advanced the understanding of the pathogenesis of URSA,some of the conclusions are contradictory,and abnormal expression levels of individual molecules do not explain the complex biochemical processes of the disease.Therefore,it is necessary to make use of the latest achievements in the field of biotechnology--omics technology platform--to further explore the pathogenesis and specific therapeutic targets of URSA comprehensively and systematically from the level of transcriptome and proteomics,and verify them in the animal model of URSA.Isobaric tags for relative and absolute quantitation(i TRAQ)is an analytical technique for identifying and quantifying a variety of protein samples with high throughput and high accuracy,which can observe the dynamic changes of key molecules.It has been widely used in quantitative proteomics research.Bioinformatics analysis can provide the biochemical processes and signal transduction pathways involved in different protein molecules and provide new ideas for the study of the pathogenesis of diseases.Clinical studies have found that URSA patients with high intercouple human leukocyte antigen(HLA)compatibility can significantly reduce the risk of miscarriage by using allogeneic lymphocyte immunotherapy(LIT),which USES peripheral blood lymphocytes from the husband or a third party to treat URSA patients before and during pregnancy.LIT treats URSA patients with a pregnancy success rate of up to 90%.CBA/J and DBA/2J in the URSA mouse model were both inbred strain mice,and the mating characteristics of these two strains were repeated abortion,and the abortion rate was relatively constant.At the same time,this model abortion belongs to peri-implantation abortion,that is,it occurs before and after embryo implantation in early pregnancy,which is similar to the time of URSA abortion.At present,this model has already been introduced into URSA research at home and abroad,and animal sources are relatively sufficient,reliable and economical,which can ensure large-scale and in-depth experimental research.Therefore,studies using this model will help reveal the pathogenesis of URSA and the mechanism of action of LIT in treating URSA.This study first quantitatively analyzed the changes in protein molecular expression levels in URSA patients before and after LIT treatment by proteomics technology,and screened out significantly different protein molecules.Then,bioinformatics analysis was used to identify the key metabolic pathway with the highest concentration of differentially expressed proteins,and to explore the relationship between this metabolic pathway and repeated pregnancy failure of URSA.Finally,m RNA transcription and protein expression levels of differentially expressed protein molecules in key metabolic pathways were verified in the URSA mouse model.This study will have important implications for elucidating the pathogenesis of repeated pregnancy failure in URSA,and will also provide new targets for evaluating the mechanism of action and efficacy of LIT in treating URSA.Therefore,this study is divided into the following two parts for in-depth study: Part I plasma proteomics study of complement and coagulation cascade pathway affecting URSA Objective: Significant differences in protein expression before and after LIT in URSA patients were found to investigate the biochemical processes and metabolic pathways involved in significantly different proteins.Methods: The protein expression levels in the plasma of URSA patients before and after LIT were quantitatively analyzed by i TRAQ combined with liquid chromatography-mass spectrometry(LC-MS).The screening conditions for significantly differentially expressed proteins were > 1.2 and p < 0.05 in at least 3 replicates.Gene ontology(GO)enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis were conducted to analyze the biochemical processes and key metabolic pathways involving significantly different proteins.Results: A total of 97 differential proteins were identified by i TRAQ quantitative analysis,including 53 up-regulated proteins and 44 down-regulated proteins.GO enrichment function analysis showed that significantly different protein molecules were mainly involved in extracellular,lipid binding and transport biochemical processes.In KEGG enrichment analysis,complement and coagulation cascade pathway were the key metabolic pathways with the most significant enrichment of differential proteins(p = 0.0001),among which coagulation factor V(F5),fibrinogen(FIB),coagulation factor XIII(F13),complement C5(C5),heparin cofactor II(HC II),protein S(PS)and protein C(PC)were significantly up-regulated after URSA treatment.Coagulation factor X(F10)and C1 inhibitors(C1INH)were significantly down-regulated.Conclusion: Patients with URSA showed abnormal expression of multiple proteins involved in multiple biochemical processes before and after LIT.The complement and coagulation cascade pathway was the most significantly enriched metabolic pathway,suggesting that LIT may prevent embryo loss by modulating the complement and coagulation cascade pathway in URSA patients.Key molecules in the complement and coagulation cascade pathway are expected to be biological targets for early diagnosis of URSA and LIT efficacy evaluation.Part II: Validation of the animal model of complement and key molecules in the coagulation cascade pathway affecting URSA Objective: A mouse model of unexplained recurrent spontaneous abortion with was established and lymphocyte immunotherapy was performed.To investigate the differences in expression levels of transcription and protein in decidua cells and plasma of pregnant mice in LIT group and control group,respectively,and to explore the relationship between complement and coagulation cascade pathway and its key molecules and URSA,as well as the biological markers for LIT efficacy evaluation.Methods: The URSA mouse model and experimental group were established: female CBA/J and male DBA/2J mice were cage mated at a ratio of 2:1.The 24 CBA/J mice were established into the URSA mouse model control group(CBA/J,12)and LIT group(CBA/J,12).The mice were closed at 8pm,and the female's vaginal plug was observed at 9am the next day.In the LIT group,female mice four days pregnant were treated with lymphocyte immunotherapy.The two groups dissected the uterus and observed embryo loss rates in pregnant mice on 14 days to determine whether LIT had successfully reduced the rate of embryo loss in URSA mice.Then,m RNA levels of F5,F10,FGA,FGB,FGG,F13A1,HC II,C5,and C1 INH in decidua cells of CBA/J pregnant mice from LIT group and control group were detected by real-time fluorescence quantitative PCR(rt-qpcr).Protein expression levels of coagulation factors F5,F10,FGA,FGG,F13A1,C5 and C1 INH in plasma of pregnant mice from LIT group and control group were detected by enzyme-linked immunosorbent assay.The diagnostic value of each index was analyzed by ROC curve,and the correlation between each index was analyzed by Pearson correlation coefficient.Results: The embryonic absorption rate of URSA mice in the LIT treatment group was significantly reduced(p<0.05).Compared with the control group,the m RNA contents of coagulation factors F5,F10,FGA,FGB,FGG,HCII,F13A1 and complement molecules C5 and C1 INH in the slough cells of URSA pregnant mice in the treatment group were significantly increased(p<0.0001).Meanwhile,the plasma levels of F10,FGG,FGA,F13A1,C5 and C1 INH in pregnant mice in the treatment group were significantly increased(p values were 0.02,<0.000,0.037,0.03,0.033 and <0.000,respectively),and the F5 expression level was significantly decreased(p<0.05).The AUC area of F5,F10,FGG and C1 INH combined diagnosis was 0.972(p = 0.00086).95%CI,0.912 to 1.0)had a specificity of 100% and a sensitivity of 91.7%.Pearson correlation analysis results showed that C1 INH was not correlated with FGG,F5 and FGG,F10 and F5,FGG and F13 in the control group.C1 INH in the LIT treatment group was associated with FGG(r = 0.601,p = 0.039),F5 with FGG(r = 0.650,p = 0.022),F10 with F5(r = 0.632,p = 0.028),and FGG with F13(r = 0.637,p = 0.026).Conclusion:(1)LIT treatment of the URSA mouse model significantly reduced the embryo loss rate.(2)Analysis of differences in protein and transcriptional levels confirmed that complement and coagulation cascade pathways are involved in the molecular mechanism of lymphocyte immunotherapy for URSA,and key molecules can be used as potential biological targets to evaluate the efficacy of LIT.(3)This study initially found an interaction between the coagulation and complement cascade pathways,which may be related to the pathogenesis of URSA and the mechanism of LIT therapy.To sum up,the following conclusions can be drawn from the above two parts:1.Complement and coagulation cascade pathways were the most significantly enriched metabolic pathways of URSA before and after LIT treatment,suggesting that this combined metabolic pathway is closely related to the pathogenesis of URSA.2.Changes in transcriptional and protein levels in animal models before and after LIT therapy confirmed that complement and coagulation cascade pathways were involved in the pathogenesis of URSA.LIT may prevent embryo loss by modulating complement and coagulation cascade pathways in patients with URSA.3.Key molecules in complement and coagulation cascade pathways,such as F5,F10,FGG,and C1 INH,are expected to be biological targets for early diagnosis of URSA,LIT efficacy evaluation,and URSA prognosis.4.Coagulation and complement cascade pathways interact and may be involved in the pathogenesis of URSA and the treatment mechanism of LIT.