The Study On The Structure Of Intestinal Flora And Tongue Coating Flora In Early Pregnant Women Based On 16S RDNA Sequencing

ObjectiveBased on 16 S r DNA high-throughput sequencing,this study analyzed the types and structural characteristics of intestinal flora and tongue coating flora in women with ectopic pregnancy and threatened miscarriage,in order to explore whether the intestinal flora and tongue coating flora are related to the occurrence of ectopic pregnancy and threatened miscarriage,and the relationship between intestinal flora and tongue coating flora with TCM Syndrome Types.MethodsWomen with menopause for 4-8 weeks,clinically diagnosed as ectopic pregnancy,threatened miscarriage and normal pregnancy were included in the study.The demographic characteristics,clinical information,feces and tongue coating samples were collected and classified according to traditional Chinese medicine.The tongue coating samples of pregnant women were collected by e Swab microbial collection and transportation tube,and their feces were collected by fecal sample collection tube with preservation solution.The collected feces and tongue coating samples were sequenced based on Illumina Mi Seq platform,and the sequencing data were analyzed by QIIME process combined with Yi Er statistical software.The structural characteristics of the intestinal flora and tongue coating flora were described,and the differences of intestinal flora and tongue coating flora in women with ectopic pregnancy and threatened miscarriage,and the structures of different TCM syndromes were compared.Results1.QueuesFifty-nine naturally eligible subjects were enrolled in the study cohort,including ectopic pregnancy(EP),threatened miscarriage(TM),and normal pregnancy(NP),of which the latter two could be classified as intrauterine pregnancy(IUP).A total of 53 intestinal samples were sequenced on computer in the study cohort,including 15 cases in EP group,30 cases in TM group,and 8 cases in NP group.EP and IUP were divided into two groups according to the location of pregnancy,and 15 cases in EP group,38 cases in IUP group.A total of 50 tongue coating samples were sequenced in the whole study cohort,including 14 cases in EP group and 26 cases in TM group.According to TCM syndromes,it can be divided into two groups: Empirical Syndrome(E)and Deficiency Syndrome(D).There are 23 cases in Group E and 27 cases in Group D.2.Sequencing information of queuesIntestinal tract: 53 fecal samples were sequenced based on Illumina Mi Seq platform.After quality control,the data volume of clean tags ranged from68077 to 78125.After removing the chimera,the data volume of valid tags(the final data for analysis)ranged from 61353 to 72989.The average length of valid tags ranged from 407.28 to 417.78 BP,and the number of OTUs in each sample ranged from 764 to 1817 between.ANOVA algorithm: 418 OTUs,71 genus and 3 phylum;Kruskal Wallis algorithm: 551 OTUs,127 genus and 8 phylum.Tongue coating: 50 tongue coating samples were sequenced based on Illumina miseq platform.After quality control,the data volume of clean tags ranged from 61970 to 78022,and the data volume of valid tags(the final data for analysis)was 49878 to 74997.The average length of valid tags ranged from407.83 to 425.4 BP,and the number of OTUs in each sample ranged from 1.5 to1.5 BP 760 ～ 2291.The differences of project samples were statistically analyzed＿Test algorithm: 198 OTUs,42 genus and 1 phylum;Wilcoxon algorithm:544 OTUs,98 genus and 5 phylum.3.Comparing the intestinal flora structure at different pregnancy locations(EP,IUP):Alpha-diversity: Chao1 index(1739.47 & 1543.40,P=0.0022)in EP group was higher than that in IUP group,observed species(1129.8 & 1013.7,P=0.03)in EP group was higher than that in IUP group,suggesting that the diversity of intestinal flora in EP group was higher than that in IUP group.Beta-diversity: Unweighted Anosim: R=0.1654,P=0.0089.Combined with Alpha diversity analysis,it was found that the intestinal flora structure of women with ectopic pregnancy was more diverse than that of women with intrauterine pregnancy.Phylum level: It is mainly composed of Firmicutes and Bacteroides.The relative abundance of trendy bacteria in EP group was lower than that in IUP group: Firmicutes and Bacteroides.Genus level: It is mainly composed of Bacteroides,Faecalis,Prevotella-9,Collinsella,Roseburia,etc.The relative abundance of trendy bacteria decreased in EP group compared with IUP group: Bacteroides,Prevotella-9;Trendy bacteria increased in EP group: Faecalis,Collinsella,Roseburia.LEf Se analysis: The relative abundance was significantly higher in EP group: uncultured＿bacterium,Prevotella＿1,Eubacterium ruminantium group,uncultured,Sutterella,Proteobacteria,Gammaproteobacteria,Coriobacteriaceae,Collinsella;the relative abundance was significantly higher in IUP group: Alphaproteobacteri,Lachnospiraceae ND3007 group,SAR11 clade,Clade＿III,uncultured＿bacterium,metagenome,Ruminococcaceae UCG 013.4.Comparing the Intestinal Microflora Structure of EP,TM and NPAlpha-diversity: Chao1 index(1739.476 & 1454.623,P=0.0019)in EP group was higher than that in NP group,and Chao1 index(1739.476 & 1584.80,P=0.0099)in EP group was higher than that in TM group.Therefore,the diversity of intestinal flora in EP group was higher than that in TM group and NP group.Beta-diversity: Unweighted Anosim value: EP vs NP:R=0.5904,P=0.001;TM vs NP:R=0.3255,P=0.004;EP vs TM:R=0.3761,P=0.001.The results showed that the structure of intestinal flora in women with ectopic pregnancy was significantly different from that in women with threatened miscarriage and those in women with normal pregnancy.The structure of intestinal flora in women with threatened miscarriage was significantly different from that in women with normal pregnancy.Phylum level: Compared with NP group,trendy bacteria with increasing average relative abundance in EP group were Actinobacteria;Trendy bacteria with decreasing average relative abundance were Firmicutes and Bacteroidetes.The average relative abundance of trendy bacteria in TM group increased as follows: Pseudomonas,Proteus;and the average relative abundance decreased as follows: Actinomycetes.Genus level: Compared with NP group,trendy bacteria with increasing average relative abundance in EP group were: Faecalibacterium,Roseburia,Lachnospira;and decreased trendy bacteria were: Bacteroidetes,Prevotella-9,Agathobacter,Bifidobacterium.The relative abundance of trendy bacteria in TM group increased as follows: Bacteroidetes,Faecalibacterium,Roseburia,Lachnospira,and decreased as follows: Prevotella-9,Agathobacter,Bifidobacterium.LEf Se analysis: In EP group,the relative abundances increased significantly:Collinsella,Coriobacteriaceae、Gammaproteobacteria.In TM group,relative abundances increased significantly: Alphaproteobacter.In NP group,relative abundances increased significantly: Muribaculaceae,metagenome.5.Comparing the intestinal flora structure of different TCM syndromes in the whole cohort:Alpha-diversity: Alpha diversity index did not differ between the two groups,P > 0.05,indicating no significant difference in intestinal flora richness and diversity between the two groups.Beta-diversity: Unweighted,Weighted Anosim analysis P value > 0.05,there was no significant difference in the structure of intestinal flora between the two groups.Phylum level: Compared with the E group,the relative abundance mean of the deficiency syndrome group increased trend bacteria was: Firmicutes,the relative abundance mean decreased trend is: Bacteroides.Genus level: Compared with the E group,the relative abundance mean of the D group increased trend bacteria was Prevotella-9;the relative abundance mean decreased trend bacteria were: Bacteroidetes,Faecalibacterium.LEf Se analysis: The relativea bundances of the E group increased significantly: Bacteroidaceae,Bacteroides,Actinobacteria,Parabacteroides,tannerellaceae,Alphaproteobacteria,Odoribacter,Tyzzerella＿4.The relative abundance of deficiency syndrome group increased significantly: uncultured bacterium,SAR11＿Clade,Clade III,NK4A136,Agathobacte.6.Comparing the structure of intestinal flora of different TCM syndromes of threatened miscarriageAlpha-diversity: There was no difference in alpha diversity index between the two groups,P > 0.05,indicating no significant difference in intestinal flora richness and diversity between the two groups.Beta-diversity: The Unweighted,Weighted Anosim analysis P value > 0.05,there was no significant difference in the structure of intestinal flora between the two groups.Phylum level: compared with the experimental group,the relative abundance mean of the deficiency syndrome group increased trend bacteria were:Firmicutes,Proteobacteria;the relative abundance mean decreased trend was:Bacteroidetes.Genus Level: Compared with the experimental group,the relative abundance mean of the deficiency syndrome group increased trend bacteria were:Prevotella-9,Faecalibacterium,Agathobacter,Lachnospira,Escherichia-Shigella,[Eubacterium]＿Eligens＿Group,Prevotella-2;Trend bacteria with decreasing relative abundance mean are Bacteroidetes.LEf Se analysis: The relative abundance of E group increased significantly:Actinobacteria,Lachnoclostridium,Mizugakiibacter,Azospirillaceae,SWB02,aecalitalea,Aeromonadales,uncultured Acidobacteria bacterium,Subgroup＿11,uncultured Acidobacteria bacterium,uncultured Acidobacteria bacterium;The relative abundance of group D increased significantly: NK4A136,uncultured,Niastella,uncultured bacterium,Subgroup 13,uncultured bacterium,Ambiguous taxa,UC5＿1＿2E3,Eubacterium oxidoreducens group,group Ruminococcaceae＿UCG＿010,uncultured bacterium,GCA＿The relative abundance of900066225,Dongiaceae,Dongia and Dongiales increased significantly.7.Comparing the flora structure of tongue coating of different TCM syndromes(D group,E group)in the whole cohortAlpha-diversity: E group observed species(933.5 & 1094.8,P = 0.047)was lower than that of deficiency syndrome group,and the diversity of tongue coating flora in the whole cohort of excess syndrome group was lower than that of D group.Beta-diversity: The Unweighted,Weighted Anosim analysis P > 0.05 suggested that the structure of tongue coating flora in the whole cohort of E group and D group was similar.Phylum level: Comparing with D group,the increasing trend of relative abundance in E group were Bacteroidetes and Firmicutes,while the decreasing trend was Proteobacteri.Genus level: Comparing with D group,the increasing trend of relative abundance in E group were prevotella-7,Streptococcus and Porphyromonas;the decreasing trend were Neisseria and Haemophilus.LEf Se analysis: The relative abundance of the E group increased significantly as follows: Prevotellaceae,Prevotella＿7,Prevotella,Enterobacteriaceae,Enterobacteriales.The relative abundance of group D increased significantly: Alphaproteobacteria,Muribaculaceae,uncultured bacterium,Rhizobiales,Burkholderiaceae.8.Comparing the flora structure of tongue coating in different TCM syndromes(D group and E group)of threatened miscarriageAlpha-diversity: The difference of alpha diversity index(P > 0.05)was not significant between the two groups.Beta-diversity: There was no significant difference in PCA and Unweighted,Weighted Anosim analysis between the two groups(P > 0.05).Phylum level: Comparing with D group,the increasing trend of relative abundance in E group was Bacteroidetes and Firmicutes,while the decreasing trend was Proteobacteri.Genus level: Comparing with D group,the increasing trend of relative abundance in E group were Prevotella-7,Streptococcus and Porphyromonas;the decreasing trend were Neisseria and Haemophilus.LEf Se analysis: The relative abundance of the E group increased significantly as follows: Prevotellaceae,Prevotella＿7,Actinomyces,Actinomycetales,Actinomycetaceae,Prevotella,Oribacterium,Lachnoanaerobaculum,Atopobium,Coriobacteriales,Atopobiaceae,Coriobacteriia.The relative abundance of group D increased significantly:Asticcacaulis,Proteobacteria.Conclusion1.The diversity of intestinal flora in ectopic pregnancy women was higher than that in intrauterine pregnancy women.2.Ectopic pregnancy and threatened miscarriage women have unique intestinal bacterial markers.Collins,red stink bug γ-Proteus is associated with ectopic pregnancy,α-Proteus is associated with threatened miscarriage.3.Bacteroides may be the intestinal bacterial marker to distinguish excess syndrome from deficiency syndrome in women with early pregnancy disorders(ectopic pregnancy,threatened miscarriage).Prevotellaceae,Prevotella 7 andα Proteus may be the bacterial marker of tongue coating to distinguish excess syndrome from deficiency syndrome.