| BackgroundThe human intestine is home to a wide variety of microbial communities,mainly composed of bacteria,the density of which reaches a maximum in the distal colon.Approximately 1011～1012 bacteria are present in every gram of feces.Studies have shown that the intestinal flora is closely related to the human development,nutrient metabolism,immunity and disease occurrence.The symbiosis of host microorganisms is crucial to intestinal homeostasis.Once this symbiotic balance is broken,it will cause micro dysbiosis,which will further cause various diseases.Increasing evidence shows that intestinal flora dysbiosis are closely related to gastrointestinal disorders such as constipation,gastric cancer,irritable bowel syndrome(IBS)and inflammatory bowel disease(IBD),however,most of these studies have been limited to non-pregnant populations.Studies have shown that gastrointestinal dysfunction such as nausea,vomiting,gastroesophageal reflux,and constipation are common during pregnancy and the incidence is increasing.Some surveys show that the incidence of constipation during pregnancy is about 20-39%,while the incidence of nausea and vomiting is 70%-80%,among which 0.3%-3.6%can progress into hyperemesis gravidarum(HG).Studies have shown that HG and constipation during pregnancy often cause physical discomfort and mental burden for pregnant women,sometimes can even cause adverse perinatal outcomes such as maternal malnutrition,electrolyte disturbances,liver failure,premature birth,and fetal malformations,which will threaten the life of mothers and babies.At present,the etiology of HG and constipation during pregnancy has not been fully understood,and its pathogenesis has become one of the research highlights in recent years.Studies have shown that changes in the intestinal flora during pregnancy may affect the mother’s material synthesis and metabolism,cause changes of blood pressure,blood glucose,blood lipids and other physiological indicators,which will further lead to pregnancy complications.However,the changes of intestinal flora in HG and constipation during pregnancy have not yet been reported.Due to teratogenicity of drugs,physicians have to balance the benefits and risks of both mother and child when treating patients with HG and constipation during pregnancy,so that the disease can’t be controlled immediately and the symptoms won’t be alleviated effectively.Therefore,a safe and effective intervention has become an urgent need for the treatment of HG and constipation during pregnancy.Probiotics are a biological agent that is beneficial to human health,providing barrier protection,enhancing immune response and eliminating gastrointestinal pathogens.A large number of studies have shown that probiotics have significant effects on the prevention and treatment of gastrointestinal diseases such as IBS,Helicobacter pylori infection,necrotizing enterocolitis,and antibiotic-related diarrhea.At the same time,some studies have reported that the use of probiotics to regulate the intestinal flora during pregnancy is safe,and may play a potential role in preventing complications during pregnancy through affecting pregnant women’s blood pressure,oxidative stress,and glucose metabolism.To safely treat HG and constipation during pregnancy,we urgently need to study the changes in the intestinal flora of these gastrointestinal dysfunction diseases,which will provide theoretical basis for future intestinal flora intervention.ObjectiveThe purpose of this research is to study the composition and structural changes of intestinal flora in pregnant women with HG and constipation,explore the possible mechanism of intestinal flora in the occurrence and development of these two diseases,which may tap the potential of intestinal flora as a biomarker to guide the treatment of HG and constipation during pregnancy.Part I:Study on the relationship between intestinal flora composition and hyperemesis gravidarumMethodsFrom 2017 to 2018,pregnant women with hyperemesis gravidarum who met the research criteria in the outpatient Department of Gynecology and Obstetrics in Qilu Hospital of Shandong University were enrolled in this research as the experimental group,healthy pregnant women and non-pregnant women were also enrolled as two control groups.The fecal samples and blood samples of the subjects were collected and the fecal flora was analyzed through high throughput sequencing of 16SrDNA amplicon.Comparison of intestinal microflora differences and clinical indicators with software such as LEfSe and SPSS.Spearman analyze was performed to test the correlation between intestinal flora and clinical indicators.Results1.The weight of pregnant women with HG was significantly lower than that of healthy women,while the values of ketonuria,ALT,NLR,PLR and PCT were significantly higher than those of healthy pregnant women70 subjects were recruited in this study,including 24 pregnant women with HG,20 healthy pregnant women and 26 healthy non-pregnant women.No significant difference was observed in mean age and height among the groups(P>0.05).Gestational age was no significant difference between HG and healthy pregnant women(P>0.05).Compared with healthy women,the weight of pregnant women with HG decreased significantly(P<0.05).There was no significant difference in the values of Na,K,Cl,TCHO,HDL,LDL and AST among the three groups(P>0.05),while the values of ketonuria,ALT,NLR,PLR and PCT in pregnant women with HG were significantly higher than those in healthy pregnant women(P<0.05).2.There was no significant correlation between the degree of ketonuria and the severity of HGAccording to the degree of ketonuria in HG pregnant women,they were divided into two groups:mild ketonuria group(Neg - 2+)and severe ketonuria group(3+～4+).There was no significant difference between the two groups in basic information and clinical indicators(P>0.05).What’s more,no significant correlation was observed between the clinical data and ketonuria grading.There was no correlation between the severity of HG and ketonuria.3.The results of a diversity and β diversity analysis showed that the intestinal flora diversity,richness and structure of pregnant women with HG had significant changesWith the increase of sample size,the location of the species accumulation boxplot in this study tends to be flat,indicating that the sampling is sufficient,and the increase of samples will not help to find more new OTUs,which can be used for subsequent data analysis.Through the high throughput sequencing analysis of V4 hypervariable region of 16SrRNA gene,the microbial a diversity of each group of samples was determined to evaluate whether the intestinal flora diversity and richness of pregnant women with HG had significant changes.Further comparative analysis showed that there was no significant difference in the Shannon index of intestinal flora in hyperemesis gravidarum group compared with the healthy control group,but the Simpson index(P<0.05),Chaol and ACE index(P<0.001)were higher than those in the healthy control group,reflecting that the diversity and richness of intestinal flora in pregnant women with HG were significantly higher than those in the healthy control group.In addition,there was no significant difference in Shannon.Simpson,Chaol.ACE index between the healthy pregnant group and the healthy non pregnant group(P>0.05).indicating that there was no significant difference in intestinal flora a diversity between the pregnant women in the first and second trimester of pregnancy and the healthy non pregnant women.We also analyzed the microflora composition of the three groups through PCoA which based on unweighted_unifrac and bray_curtis distance,the subjects were clustered into three groups.We further analyzed the data though Adonis which revealed the microflora composition of HG pregnant group was significant different from that of the healthy pregnant group(unweighted_unifrac,R=0.21 7,P=0.001;bray_curtis,R=0.129,P=0.001).and that of the healthy non-pregnant women(unweighted_unifrac,R=0.201,P=0.001;bray_curtis,R=0.215.P=0.001).The flora structure of healthy pregnant women is also different from non-pregnant women(unweighted_unifrac,R=0.106,P=0.003;bray_curtis,R=0.065.P=0.001).indicating pregnancy also affects the structure of the flora.4.There was significant difference in the abundance of intestinal flora at the level of phylum and genus between pregnant women with HG and healthy controlsThe above analysis revealed significant changes in the diversity of HG pregnant women.so we further obtained the intestinal flora composition of each group based on the cluster analysis of the OTU.The dominant phyla of the three groups were Bacteroidetes,Firmicutes.Proteobacteria and Actinobacteria,while the dominant genera of the groups were Bacteroides,Bifidobacterium,Enterococcus Faecalibacterium and Blautia etc.Relative abundance column accumulation chart showed the abundance of intestinal flora in HG pregnant women was significantly different from that in healthy controls.Further analysis revealed the abundance of Proteobacteria,Enterococus,Acinetobacterium(P<0.01),Lactobacillus(P<0.05)was significantly increased in HG pregnant group compared with healthy pregnant group,while Blautia,Lachnospira(P<0.01)were significantly decreased.The abundance of Bacteroidetes and Firmicutes also decreased,but no significant difference was observed(P>0.05).Compared with non-pregnant group,the abundance of Faecalibacterium(P<0.01)in HG pregnant group increased significantly,while that of Actinobacteria,Blautia,Anaerostipes(P<0.01)and Bifidobacterium(P<0.05)decreased significantly,on the other hand,the decrease of Bacteroidetes was not significant(P>0.05).5.The abundance of Enterobacteriaceae,Escherichia coli and other characteristic microorganisms in intestinal flora of pregnant women with HG increased significantlyOn this basis,the LEfSe analysis was used to further analyse the difference of the enriched microorganisms among the three groups,and the key biomarker in the intestinal flora of pregnant women with HG was screened(LDA>4.0).The results showed that Proteobacteria,Gammaproteobacteria,Ruminococcaceae,Enterobacteriaceae and Faecalibacterium were significantly increased in HG pregnant women,while Actinobacteria,Prevotellaceae,Lachnospiraceae,and Blautia were more enriched in non-pregnant women.Compared with the the flora of healthy pregnant women though LEfSe analysis,Proteobacteria,Ruminococcaceae and Enterobacteriaceae were significantly increased in HG pregnant women,while Lachnospiraceae,Veillonellaceae and Blautia were more abundant in healthy pregnant women.6.Correlation analysis between NLR、PLR、AST、ALT and intestinal floraTo study the correlation of NLR,PLR,AST,ALT,etc and intestinal flora.Spearman analysis was performed.It was found that NLR was negatively correlated with Dialister,PLR was negatively correlated with Lachnospira and Megamonas,while positively correlated with unidentified Enterobacteriaceae.Ketoneuria was positively correlated with Enterococcus,while negatively correlated with Blautia,Bifidobacterium,Alloprevotella and Lachnospira.LDL is positively correlated with Collinsella,Acinetobacter and Enterococcus;HDL is positively correlated with Blautia,while negatively correlated with Enterococcus;AST is positively correlated with Enterococcus and Weissella;while negatively correlated with Dialister etc;ALT is positively correlated with Porphyrobacter,while negatively correlated with Lactobacillus,etc.Conclusion1.The inflammatory indexes in pregnant women with HG were significantly increased and ketonuria existed in different degrees,but there was no significant correlation between ketonuria and the severity of HG.2.The composition and structure of intestinal flora of HG pregnant women were significantly different from healthy controls.3.The intestinal flora is correlate with NLR,PLR,AST,ALT,etc,indicating that they may affect the occurrence of inflammation and liver function.4.Blautia,Anaerostipes,Lachnospira,Bifidobacterium and other beneficial bacteria decreased in the intestinal flora of pregnant women with HG,while pathogens and opportunistic pathogens such as Enterobacteriaceae,Parabacterorides,Enterococcus,Acinetobacter,Escherichia coli increased,suggesting that they may be involved in the occurrence of HG inflammation.Part II:Study on the dysbiosis of intestinal flora in the pathogenesis of constipation during pregnancyMethodsPregnant women with constipation who met the research criteria in the outpatient Department of Gynecology and Obstetrics in Qilu Hospital of Shandong University from 2017 to 2018 were enrolled as experimental group,healthy pregnant women and non-pregnant women were enrolled as two control groups.The fecal samples of the subjects were collected and analyzed though high throughput sequencing of 16SrDNA amplicon.The difference of intestinal microflora among groups were analyzed by LEf Se and SPSS.Results1.BMI of women with constipation during pregnancy is significantly higher than that of healthy women without pregnancy67 subjects were recruited in this study,including 21 pregnant women with constipation,20 healthy pregnant women and 26 healthy non-pregnant women.There were no significant difference in mean age and gestational weeks among the groups(P>0.05),while compared with the non-pregnant group,the BMI of pregnancy constipation and healthy pregnant group are higher(P<0.05).2.The rarefaction curve and the species accumulation boxplot show that the sample size of the study is sufficientAccording to the rarefaction curve and the species accumulation boxplot,the sample size of this study is sufficient,and the new species(OTUs)will not increase significantly with the increase of sample size,which can be used for subsequent analysis.At the same time,the dilution curve of the pregnant constipation group was lower than that of the healthy pregnant group and the healthy non pregnant group,which indirectly indicated that the intestinal flora richness of the constipation pregnant women was lower than that of the healthy control.3.The results of α diversity of intestinal rflora showed that the richness of intestinal flora of women with constipation during pregnancy was significantly lower than that of healthy controls,but there was no significant difference in the diversityBased on these data,the alpha diversity of intestinal flora among groups was analyzed.No significant difference was observed between healthy control and pregnancy constipation group in Shannon and Simpson index of the intestinal flora(P>0.05),while Chaol and ACE index of the pregnancy constipation group were significantly lower than those in non-pregnant group(P<0.001)and healthy pregnancy group(P<0.05),indicating the intestinal flora richness of constipation during pregnancy was significantly lower than that of healthy control,while no significant difference was observed between the two groups.4.The results of β diversity of intestinal flora showed that the intestinal flora structure of women with constipation was significantly different from that of healthy controlsWe further analyzed the flora composition of each group by unweighted PCoA and found the subjects were clustered into three groups.Constipation affected the flora composition of pregnant women,and the flora composition of pregnancy constipation group was significantly different from that of the non-pregnant group(Adonis,R=0.248,P=0.001),and that of the healthy pregnancy group(Adonis,R=0.206 P=0.001).5.There are significant differences in the phylum,genera,family level of intestinal flora between women with constipation during pregnancy and healthy controlsAs shown in flora composition by OTU analysis,the abundance of intestinal flora in pregnancy constipation was significantly different from that of the healthy controls.Further analysis showed that compared with the healthy pregnant group,the abundance of Lachnospiraceae,Veillonellaceae(P<0.001),Bloutia(P<0.01),Lachnospira,Roseburia,(P<0.05)decreased significantly in pregnancy constipation group.The abundance of Bacteroidetes also decreased,but not significantly(P>0.05).While the abundance of Proteobacteria,Ruminococcacea(P<0.05),Enterococcus,Acinetobacter(P<0.01)increased significantly in the pregnancy constipation group compared with healthy pregnant group.Compared with the non-pregnant group,the abundance of Lachnospiraceae,(P<0.001),Blautia,Anaerostipes,Porphyrobacter(P<0.01),Prevotellaceae(P<0.05)in pregnancy constipation group decreased significantly.The abundance of Bacteroidetes also decreased,but not significantly(P>0.05).The abundance of Firmicutes,Ruminococcacea(P<0.05)in pregnancy constipation group increased significantly.6.The characteristic microorganisms such as Ruminococcaceae,Enterobacteriaceae,etc increased significantly in the intestinal flora of women with constipation during pregnancyBased on these analysis,we found there were great differences among groups in the composition of intestinal flora,so we further used LEfSe technology to analyze the intestinal flora and screen out the key Biomarker among groups(LDA>4.0).which revealed more Firmicutes,Ruminococcaceae,Faecalibacterium,Subdoligranulum,Rummeliibacillus,Lactobacillus were observed in pregnancy constipation group,while in non-pregnant group,the most abundant flora are Prevotellaceae,Lachnospiraceae,Veillonellaceae and Blautia.Further more,we also found Ruminococcaceae,Enterobacteriaceae and Subdoligranulum increased significantly in constipation pregnant group,whereas the Lachnospiraceae,Veillonellaceae and Blautia were more prominent in healthy pregnant women.Conclusions1.Compared with the healthy control,the richness of intestinal flora in constipated pregnant women was significantly reduced.2.The intestinal flora structure of constipated pregnant women changed significantly.3.Prevotella,Roseburia,Blautia,Anaerostipes,Lachnospira and other beneficial bacteria in the intestinal flora of constipated pregnant women were significantly reduced,while harmful bacteria such as Enterobacteriaceae,Enterococcus,Acinetobacter were significantly increased,suggesting that the dysbiosis of intestinal flora may be involved in the pathogenesis of constipation in pregnancy. |
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