| Objective:In recent years,the incidence of spontaneous abortion in clinic increased significantly,and there is an increasing trend year by year.The causes of spontaneous abortion are complex,including genetic,anatomical,endocrine,infectious,environmental,and immune factor.With the gradual increase of the incidence of adverse pregnancy,the need for research and development in the pathogenesis of spontaneous abortion and clinical diagnostic markers are becoming more and more urgent.With the rapid development in scientific researches,various omics research technologies,including genomics,transcriptomics,proteomics and lipidomics,are increasingly applied to the screening of pathogenic mechanisms and diagnostic markers of various diseases.Materials and Methods:To screen and validate the biomarkers for early diagnosis of spontaneous abortion,we established a research corhot of assisted reproductive women,collected the serum from pregnant women at 5th or 7th weeks of pregnancy,and then follow-uped to the birth.Obejectives were divided into abortion group and control group,according to whether abortion happened.The changes of proteins and lipids in the serum of patients were investigated.We adopted the untargeted relative quantitative lipidomics analysis platform based on UPLC-Orbitrap mass spectrometry system.Lipid Search software was used to identify and compare lipids in blood samples of patients with spontaneous abortion and controls.Support vector machine model,Gaussian Bayesian model(GB),decision tree model,neural network model and K-nearest neighbor model were used as classifiers,MGRFE,Lasso regression,decision tree and RFE feature selection methods were adopted to screen and analyze the differential levels of lipids.The isobaric tag for relative and absolute quantitation(i TRAQ)technique and proteomics was used to screen the proteins with different expression levels in the serum of patients with spontaneous abortion.ELISA and Western blot were used to verify the proteomics results,the receiver operating characteristic(ROC)curve was used to evaluate the sensitivity and specificity of the differential proteins,and the cutoff values were calculated.Further,we used biochemical and molecular biological methods to study the role of differentially expressed protein POF1 B in the occurrence and development of spontaneous abortion.Immunohistochemistry,q PCR and Western blot were used to detect the expression of POF1 B in the villus tissues of patients with spontaneous abortion.The human villus trophoblast cell line HTR-8/Svneo cells were cultured in vitro and used to construct POF1 B overexpressed cells.With apoptosis assay,CCK-8 assay,etc.,the effects of POF1 B overexpression on the proliferation and apoptosis of HTR-8/Svneo cells were determined.Meanwhile,HTR-8/Svneo cells overexpressing POF1 B were collected and analyzed by Illumina/Solexa sequencing for total transcriptomic and bioinformatics analysis,and downstream target genes of POF1 B were screened.q PCR and Western blot were used to verify the target genes.Chromatin immunoprecipitation(Ch IP)technique,RNA stability assay,apoptosis assay,and Western blot,etc.,were used to study the molecular regulation mechanism of downstream target genes.Results:A total of 1008 pregnant women were included in this study cohort,followed up to the outcome of abortion or fetal birth.There were 233 cases of spontaneous abortion.The multivariate logistic regression analysis showed that age,BMI,coffee,smoking,home decoration,uterine fibroids,uterine malformation,thyroid disease,and history of spontaneous abortion are risk factors for spontaneous abortion(P<0.05).A total of 1346 lipid compounds,belonging to 31 species,were identified in serum of patients with spontaneous abortion by lipidomics study.Among the support vector machine model,Gaussian Bayes model(GB),decision tree model,neural network model and K-nearest neighbor model classifiers with 5× cross validation,GB classifier has the best stability and accuracy,and was used as the classification model in the following feature selection process.The differences of MGRFE,Lasso regression,decision tree and RFE selection characteristics were compared.The calculation accuracy of ROC curve and AUC value were verified by stratified cross-validation.The results showed that MGRFE had the highest average AUC value among the four methods,and had higher accuracy and stability.With these machine learning strategies,we found there were 7 different lipid molecules in the serum of patients with spontaneous abortion,including SM(D39:1),SM(D41:2),SM(D18:1/24:2),DG(18:0/18:0),DG(22:4/18:2),PC(40:7)and LPE(18:1),belonging to sphingolipids,glycerols,glycerolipids and lecithin.The serum SMs level increased significantly,while the serum PC and LPE levels decreased significantly in pregnant women with abortion.The DG containing two saturated fatty acyl chains was decreased,but that containing two unsaturated fatty acyl chains was increased in the abortion group compared to the control group.By i TRAQ proteomic analysis,we identified 33 proteins with significantly different expression levels,including 12up-regulated and 21 down-regulated,between patients with spontaneous abortion and controls.By using the gene ontology enrichment analysis,we found that in the analysis of biological process(BP),molecular function(MF)or cellular component(CC),compared with the control group,there were significant differences in spontaneous abortion group.Through KEGG database,we also obtained the signal pathway that the differentially expressed proteins in serum of patients with spontaneous abortion.Serum of the spontaneous abortion group may change in chronic myeloid leukemia,prolactin signaling pathway,endocytosis and other signaling pathways.We also performed protein-protein interaction network analysis for proteins with significant expression differences in the serum of patients with spontaneous abortion.Twelve of the 33 differentially expressed proteins interacted with each other.Verification experiments,including Western blot and ELISA,found the serum levels of PSG9 and PSG11 were significantly reduced and that of POF1 B was significantly increased in the spontaneous abortion group compared with the control group.According to the ROC curve,it was suggested that POF1 B,PSG9 and PSG11 may be used for the diagnosis of spontaneous abortion.Mechanism of POF1 B in spontaneous abortion was further investigated.These results from cellular experiments suggest that POF1 b affects JDP2 level by regulating the stability of JDP2 m RNA,and the increased JDP2 regulates the downstream SPP1 expression through transcription,thereby increasing cell apoptosis and participating in abortion.Conclusion:Age,BMI,coffee,smoking,home decoration,uterine fibroids,uterine malformation,thyroid disease,and history of spontaneous abortion are shown to be risk factors for spontaneous abortion;SM(D39:1),SM(D41:2),SM(D18:1/24:2),DG(18:0/18:0),DG(22:4/18:2),PC(40:7)and LPE(18:1)might be used as lipid markers for early diagnosis of spontaneous abortion;POF1B,PSG9 and PSG11 can be used as protein markers for early diagnosis of spontaneous abortion;POF1B/JDP2/SPP1 pathway is involved in cell apoptosis,participating in the occurrence of spontaneous abortion. |
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