Community Characteristics Of Microbes And Bioinformatics Analysis Of Identifying Functional Gene In Pre-eclampsia

Background and objectivePre-eclampsia(PE)is the second leading cause of maternal death worldwide and its pathogenesis is still unclearly.At present,it is believed to be related to oxidative stress,immune system,environmental genetic factors and imbalance of microbial flora,etc.Many recent reports suggest that PE is correlated with microflora,which comes from various parts of the human body such as oral cavity,intestinal tract,placenta,amniotic fluid and genitourinary system.Recently,gene expression studies related to human diseases have been reported using gene chip,RNA sequencing technology and public databases such as GEO.However,there has been little coverage of PE expression profiles.Changes in the composition of microflora is not only involved in the occurrence and development of many diseases,but also has a multi-factor,multi-pathway and multi-site impact on PE,both of which are the focus in research.Therefore,this project explored the characteristics of microflora in specific parts in patients with PE,annotated their functional genes,analyzed their gene metabolic pathways,identified the biological marker of Hub gene with PE and explored the potential impact of Hub gene on PE.This series of research plays an important basic theoretical role in early recognition and diagnosis of pre-eclampsia.Research methods and contentsOur case-control study was conducted between PE group and N group of normal pregnancy.12 cases in PE group and 12 cases in N group were randomly selected and matched.Confounding factors were controlled by strict inclusion and exclusion criteria for microbiological studies.The specimen and clinical data of 6 pregnant women in PE group and 6 pregnant women in N group were obtained.All of samples were obtained from placental tissue,feces,posterior fornival vaginal secretions and gingival plaque.Then the placenta and intestinal flora of the two groups were sequenced using 16SrRNA and metagenomic sequencing,but vaginal and dental plaque flora were only sequenced by 16SrRNA.Bioinformatics analysis was performed on all metadata to explore the characteristic microorganism related to the development of PE and to carried out the correlation analysis of significant differences in microflora,functional genes and clinical biochemical indicators.Meanwhile,gene expression profile of GEO dataset GSE60438 was used to screen DEGs in preeclampsia and normal pregnancy.Then GO and KEGG enrichment analyses were performed on DEGs,and a protein interaction(PPI)network based on the above DEGs was established using STRING database.17 significant Hub genes were screened out by module analysis and identify.Support vector machine(SVM)model was used to predict the potential application of biomarkers in PE diagnosis.Research results and conclusions1.Standardization of clinical microbiological studies.After strict confounder matching,there were no significant differences in age,gestational age,BMI,diet,oral health and regional factors between PE and N groups(P>0.05),indicating good comparability between the two groups.The success of this study was due to standardized sample collection,optimized DNA extraction and amplification database,setting of negative control group and obtaining effective study sample size by using Alpha diversity dilution curve to evaluate microbial sequencing data from many public platforms.All of these made our results accurate and valid.The key of this project is to pay enough attention to the whole microbiological research criteria and confounding factor control.2.Characteristics composition and functional genes of placental and intestinal microbiota in preeclampsiaThe LEfSe method was used to screen the placental and intestinal microflora.The results showed that there were significant differences in phylum,class,order,family,genus and species between normal pregnant women and preeclampsia placental microflora in 16SrRNA and metagomenomics sequencing(P<0.05).(1)Placental microflora:At 16S level,Firmicutes were dominant in PE and verrucomicrophyla was dominant in N.At metagenomic level,the PE group was dominated by Bacteroidetes,while the N group was dominated by Proteobacteria.Fifteen different species were detected at 16S level,most of which were found at genus level.There were 37 metagenomic species at species level.There were 11 kinds of bacteria in placenta that were closely related to age phenotype.The abundance changes of Firmicutes and Proteobacteria were correlated with the markers of abnormal function such as blood pressure,blood glucose and liver function.In KEGG database,the biosynthesis of primary bile acids in PE was lower than that in N(P<0.05),and the prediction of lipid transport and metabolism of functional genes in gene annotation of metagenomic sequencing was verified.Our results suggest that there may be a correlation between placental microflora and age,and age is clinically correlated with preeclampsia.The placental microbiota-agepreeclampsia axis can be used as the direction of next further research.It is not clear whether the composition of placental microorganisms in preeclampsia has a common pathway in blood glucose,blood pressure and lipid metabolism,which requires further study.(2)Intestinal microflora:At 16S level,PE was concentrated on Actinomycetes while N was on Firmicutes.The PE was dominated by Bacteroidetes at metagenomic level.Firmicutes were dominant in N.At 16S level,9 species were detected,most of which were found at genus level.However,23 metagenomic species were found at species level.In the level 2 of KEGG database,endocrine system functional genes in PE group showed a downward trend,and parasite distribution functional genes were significantly higher than those in N(P<0.05).Gene functions on KEGG and eggNOG databases at the metagenomic level showed that intestinal microflora was concentrated on fat,carbohydrate,amino acid and coenzyme transport and metabolism,replication and repair,confirming the prediction of 16S functional genes.Macroomonas were closely correlated with SBP,DBP,UPR,LDH,BNP,GA and BW(P<0.05)and was negatively correlated with SBP,DBP,UPR,LDH and BNP.The abundance of Megomonas in PE was significantly lower than that in N,which suggesting that megomonas may be associated with hypertension.3.Characteristics composition and functional genes of vaginal microbiota in PreeclampsiaThe number of Collinella and Collinia(Actinomycetes)in PE was significantly increased,while the number of Prevotella black(Bacteroidetes)was significantly decreased(P<0.05).Gardnerella and Pseudomonas were closely related to coagulation indexes.Bifidobacteria,lactobacillus and Pseudomonas can regulate the immune and metabolic states of the body.PE group had significantly active genes of cell process and signal transduction function.The functional genes of RNA degradation,amino acid related enzymes and ribosomal biogenesis in eukaryotes were inferior to those in N group(P<0.05).The results of this study suggest that preeclampsia is associated with signs of vaginal microbiome dysregulation.4.Characteristics composition and functional genes of dental plaque microbiota in PreeclampsiaThe abundance of Proteobacteria,Burkholderaceae,Neisseria,porphyromonas and Actinobacillus in PE was higher than that in N(P<0.05),while the abundance of Monomonas lunate was lower than that in N(P<0.05).Porphyromonas were associated with periodontal disease.SBP,DBP,LDH and UPR are important clinical indicators of preeclampsia.These indexes were positively correlated with porphyromonas and Neisseria.SBP was negatively correlated with Monomonas lunate.In PE group,8 kinds of bacteria were closely correlated with D-dimer,while DD hypercoagulability was positively correlated with preeclampsia.There were significant differences between PE group and N group in immune system disease function genes(P<0.05).The characteristic changes of gingival plaque microorganisms in preeclampsia are closely related to periodontal disease.However,whether there is a causal relationship needs further analysis and research.5.Hub gene identification of diagnostic biomarkers for preeclampsia using integrated bioinformatics in GEO database17 significant Hub genes were screened,including of IL7R,IL18,CCL2,HLA-DRA,CD247,ITK,CD2,IRF8,CD48,GZMK,CCR7,HLA-DPA1,LEP,IL1B,CD8A,CD3D and GZMA.To sum up:In this paper,the importance of confounding factor matching,sample collection,standardization of DNA extraction and amplification,as well as how to set the sample size at the optimization cost and obtain meaningful and accurate research results in clinical microbiological research was verified again through experiments.In preeclampsia,the composition of placental,intestinal,vaginal and dental plaque flora changes significantly at phylum,class,order,family,genus and species levels.The clinical biochemical indexes of the organism were significantly correlated with the microbial structure.The possibility that Megomonas is associated with hypertension,and the close relationship between placental flora and age phenotype,both of those can be regarded as the direction of follow-up research.There may be a correlation between placental microflora and age.There is also a clinical correlation between age and preeclampsia.The placental microbiota-age-preeclampsia axis can be used as the direction of further research.The characteristically altered microbiota functional gene pathways in preeclampsia show significant differences in lipid transport and metabolism of primary bile acid biosynthesis,cellular processes and signaling,immune system diseases,endocrine system,parasite distribution,amino acid and coenzyme transport and metabolism,replication and repair.From the perspective of bioinformatics,it is suggested that the composition and function of microflora are correlated with pre-eclampsia disease.17 hub genes were identified as diagnostic biomarkers for PE.The results of this study may contribute to the development of site-specific microbial profiles as early identification factors for PE.