Relationship Between Gut Microbiota And Serum Inflammatory Factors In Preeclampsia

Preeclampsia(PE)manifests new-onset hypertension during pregnancy,which usually occurs after 20 weeks of gestation and affects about 3-5% of all pregnancies.Compared with healthy pregnancy,PE patients have increased risk for cardiovascular and metabolic diseases later in life.Although extensive and in-depth researches have been done,the exact pathogenesis is unclear.The gut microbiome is a vast and complex collection of microorganisms.Previous studies have shown that gut microbiota dysbiosis is associated with abnormal host metabolism and systemic inflammation,which may contribute to the development of obesity,hypertension,diabetes,metabolic diseases,nonalcoholic liver disease and malnutrition.Moreover,recent evidence has reported that the gut microbiome significantly changes during pregnancy.Thus,the present study was conducted to further investigate the relationship between gut microbiota and serum inflammatory factors to provide theoretical basis for PE mechanism from gut microbiota view.The difference in the gut microbiota composition between normal pregnant women and pregnant women with PE was investigated,as well as their functional pathways.Furthermore,the association of differential gut microbiota composition,functional pathways and inflammatory markers were identified.Finally,the association of gut microbiota and inflammatory factors on patients with preeclampsia by fecal microbiota transplantation.The study will provide theoretical basis for PE mechanism and precise intervention.Part One The Correlation between Preeclampsia and Gut MicrobiotaObjective: To compare the differences of gut microbiota and related functional pathways between preeclampsia and healthy control pregnancy.Methods: The pregnant women were registered from November 1,2021 to May 1,2022 in First Hospital of Hebei Medical University and Affiliated Hospital of Hebei University Xingtai People’ Hospital.A case-control study to compare the clinical characteristics,the fecal microbiota detected by 16 S r RNAV3-V4 gene sequencing between 41 preeclampsia patients and with 45age-,pre-pregnancy body mass index-,and gestational age-matched healthy control pregnancies between the two groups.The patients with preeclampsia were further divided into 27 patients with severe symptomatic preeclampsia(SPE)and 14 patients with non-severe symptomatic preeclampsia(NSPE).The clinical characteristics and gut microbiota composition of the three groups were compared.Then the related functional pathways were compared between preeclampsia and healthy women.Results:1.Comparison of clinical data and gut microbiota between PE group and healthy control group1)There was no difference in maternal age,pre-pregnancy body mass index(BMI),gestational age,gravidity and parity between PE group and N group(P>0.05).The weight gain during pregnancy,birth weight of newborns and delivery weeks in the PE group were lower than those in N group while systolic and diastolic blood pressure were higher than those in N group(P<0.05).In terms of pregnancy outcomes,cesarean section and iatrogenic preterm labor less than 34 weeks in the PE group were higher than those in N group(P<0.05),while there was no difference between the two groups in placental abruption,postpartum hemorrhage,fetal growth restriction,perinatal death,fetal distress,cardiac insufficiency and oligohydramnios(P>0.05).2)There was no significant difference in hemoglobin(Hb),white blood cell(WBC),percentage of neutrophil granulocyte(NEUT%),percentage of lymphocytes(LYMPH%)and the ratio of neutrophil/lymphocyte(N/L%)between the two groups(P>0.05).3)There was no significant difference in alpha diversity while exists significant difference in beta diversity between the two groups.LEf Se analysis showed that there were 24 differently abundant taxa between the two groups.The abundances of Bacteroidetes,Bacteroidia,Bacteroidales,Ruminococcaceae,Bacteroidaceae,Bacteroides,Ruminococcus,Oscillospira in PE group were lower and while the abundances of Proteobacteria,Fusobacteriales,Bacilli,Erysipelotrichi,Gammaproteobacteria,Fusobacteriia,Lactobacillales,Erysipelotrichales,Enterobacteriales,Fusobacteriales,Erysipelotrichaceae,Enterobacteriaceae,Fusobacteriaceae,Streptococcus,Shigella and Fusobacterium were higher in PE group than those in N group.2.Comparison of clinical data and gut microbiota among 3 groups1)There was no difference in maternal age,pre-pregnancy BMI,gestational age,gravidity,parity and weight gain during pregnancy among 3groups(P>0.05).The birth weight of newborn and delivery week in the SPE were lower than those in the NSPE and N groups(P<0.05);In the systolic and diastolic blood pressure level,SPE was higher than those in the NSPE and N groups,NSPE was higher than those in N group(P<0.05).The 24-hour urine protein quantity in SPE group was higher than those in NSPE group.In terms of pregnancy outcome,iatrogenic preterm labor less than 34 weeks in the SPE group was higher than those in the NSPE and N groups(P<0.05).Cesarean section in SPE group was higher than those in the N group(P<0.05),while there was no difference between the 3 groups in placental abruption,postpartum hemorrhage,fetal growth restriction,perinatal death,fetal distress,cardiac insufficiency and oligohydramnios(P>0.05).2)There was no significant difference in Hb,WBC,NEUT%,LYMPH%and N/L% among 3 groups(P>0.05).3)There was no significant difference in alpha diversity while exists significant difference in beta diversity among the 3 groups.The result demonstrated that the difference between SPE group and N group was statistically significant,but the difference between NSPE group and N group was not statistically significant.LEf Se analysis showed that there were 18 differently abundant taxa.The abundances of Proteobacteria,Gammaproteobacteria,Enterobacteriaceae,Enterobacteriales,Bacilli,Lactobacillales,Streptococcus and Fusobacterium were rich in SPE group,the abundances of Erysipelotrichi,Erysiplotrichaceae,Erysipelotrichales and Gemella were rich in NSPE group,while the abundances of Bacteroidetes,Bacteroidia,Bacteroidales,Bacteroidaceae,Bacteroides,Ruminococcus were rich in N group.Compared with N group on gut microbiota β diversity,the abundance of Bacteroides,Bacteroides and Ruminococcus in SPE group was lower(P<0.05),while the bacterial abundance of Proteobacteria,Streptococcus,Blautia,Shigella and Fusobacterium were higher in SPE group from the bacterial level of phylum and genus(P<0.05).3.Functional prediction of gut microbiota by PICRUSt2The difference in related functional pathways between the two groups showed that PE and SPE groups were enriched in Human Diseases-Infections Diseases-Bacteria and Xenobiotics biodegradation and metabolism,while the healthy control group was enriched in Metabolism-Carbohydrate metabolism and Genetic Information Processing-Folding,sorting and degradation.Summary: There are differences in the gut microbiota and functional pathways between the two groups.Part Two The Correlation between Gut Microbiota and Serum Inflam-matory Factors in PreeclampsiaObjective: To explore the correlation with differential bacteria of gut microbiota,functional pathway and inflammatory factor between preeclampsia and healthy control pregnancy.Methods: The pregnant women were registered from November 1,2021 to May 1,2022 in First Hospital of Hebei Medical University and Affiliated Hospital of Hebei University Xingtai People’ Hospital.A case-control study to compare the serum inflammatory factors detected by ELISA between 41 preeclampsia patients and with 45 age-,pre-pregnancy body mass index-,and gestational age-matched healthy normal pregnancies between the two groups,then to explore the correlation analysis with differential bacteria,functional pathway of gut microbiota and inflammatory factor between preeclampsia and healthy control pregnancy.The patients with preeclampsia were further divided into 27 patients with severe symptomatic preeclampsia(SPE)and 14 patients with non-severe symptomatic preeclampsia(NSPE),then to explore the correlation analysis with differential bacteria and functional pathway of gut microbiota and inflammatory factor between SPE and healthy control pregnancy.Results:1.Comparison of serum inflammatory factors and the correlation with differential bacteria and functional pathway of gut microbiota and inflammatory factor between PE group and healthy control group.There was no difference in maternal age,pre-pregnancy BMI,gestational age,gravidity and parity between PE and N group(P>0.05).Spearman association was performed between differential bacterial genera and inflammatory factor levels found that Streptococcus was positively correlated with TNF-α and IL-6,Shigella was positively correlated with IL-6,while Bacteroidetes and Bacteroides were negatively correlated with TNF-α and IL-6and Rumminococcus was negatively correlated with IL-6.There is a correlation between functional pathway and serum inflammatory factors.The levels of proinflammatory factors tumor necrosis factor-α(TNF-α)and Interleukin-6(IL-6)were statistically significant higher in PE patients than those in N patients(both P<0.001),while there was no significant difference in the levels of Interleukin-4(IL-4)and Interleukin-10(IL-10)between the two groups.There also was no significant difference in the the WBC,NEUT%,LYMPH%and N/L between the two groups.2.Comparison of serum inflammatory factors among 3 groups and the correlation with differential bacteria,functional pathway and inflammatory factors between SPE group and N group.There was no difference in maternal age,pre-pregnancy body mass index(BMI),gestational age,gravidity,parity and weight gain during pregnancy among the 3 groups(P>0.05).Spearman association was performed between differential bacterial genera and inflammatory factor levels between SPE and N groups found that Shigella was positively correlated with TNF-α and IL-6,Proteobacteria was positively correlated with IL-6,while Bacteroidetes and Bacteroides were negatively correlated with TNF-α and IL-6 and Rumminococcus was negatively correlated with IL-6.There is a correlation between functional pathway and serum inflammatory factors.The levels of TNF-α and IL-6 were statistically significant higher in SPE patients than those in NSPE and N patients(both P<0.001).There was no significant difference in the levels of IL-4 and IL-10 among the three groups.There also was no significant difference in the the WBC,NEUT%,LYMPH%and N/L among the three groups.Summary: There is a correlation with differential bacterial general and functional pathway of gut microbiota and the serum levels of inflammatory factors between preeclampsia and healthy pregnant women.Compared with healthy control pregnant women,the serum pro-inflammatory factor levels increase in patients with preeclampsia,especially severe preeclampsia..Part Three Effect of Fecal Microbiota Transplantation on GutMicrobiota and Serum Inflammatory factors in MiceObjective: To compare the difference of gut microbiota,inflammatory factors and intestinal barrier function in mice after fecal microbiota transplantation.Methods: Fecal bacteria solution of preeclampsia and normal healthy pregnant women were transplanted into pseudo sterile mice.The 16 S r RNA V3-V4 gene of fecal bacteria in mice was sequentially analyzed to compare intestinal flora composition.The urinary protein content,serum lipopolysaccharide,TNF-α and IL-6 levels were determined by ELISA.The expression of ZO-1 and Occludin in colon tissue was determined by PCR,Western-Blot and immunohistochemistry.Results:1.Comparison of gut microbial compositionThe α diversity in the PET group was lower than those in other groups,but there was no statistical difference,while the β diversity was significantly different(P<0.05).LEf Se analysis showed that Coprococcus,Bacillales,Staphylococcus,cyanobacteria,Flexispira,Bifidobacterium and Streptococcus were rich in PET group,while Helicobacter,Klebsiella,Rhodoplanes,Exiguobacterium,Turicibacter and Anaerofustis were abundant in the fecal microbial communities of mice in NT group.2.Urine protein content in PET group was higher than those in Con,Neg and NT groups.3.The level of serum LPS,TNF-α and IL-6 in PET group was higher than those in Con,Neg and NT groups.4.The expression level of TPJ1 and Occludin genes and ZO-1 and Occludin proteins in colon tissue in PET group were lower than those in other groups.Summary: Compared with control group,negetive group and transplanted with fecal bacteria solution of healthy pregnant women,the gut microbiota composition transplanted with fecal bacteria solution of preeclampsia was significantly different,the intestinal barrier function was impaired,and the level of urine protein content,the serum of LPS and inflammatory factors were increased.Conclusions:1.There was difference in the gut microbial composition and functional pathway between the two groups.2.There is a correlation with differential bacterial genera and functional pathway of gut microbiota and the serum levels of inflammatory factors between PE and N groups.Compared with healthy control pregnant women,the serum pro-inflammatory factor levels increase in patients with preeclampsia,especially severe preeclampsia.3.Compared with control group,negetive group and transplanted with fecal bacteria solution of healthy pregnant women,the gut microbiota composition transplanted with fecal bacteria solution of preeclampsia was significantly different,the intestinal barrier function was impaired,and the level of urine protein contein,the serum LPS and inflammatory factors were increased.