| Background:There are trillions of microbial cells in the human body,and while the skin,vagina,and even respiratory tract have specific microbiomes,the density is highest in the gut,particularly the colon,known as the gut microbiome.The intestinal flora is initially unstable in infant colonization,but it is a very critical window period.The specific node of microbial maturation is still not very clear,but the first 1000 days,especially the period from birth to 2 years of age,are generally considered to be an important period for the healthy development of infant intestinal microbiota.The neonatal period is the first period after birth when intestinal microbiota is established,and there is an interaction with the mother,which is particularly important.And if the intestinal microbial balance is disrupted during this critical period it may have long-term effects on immunopathology and be associated with obesity,atopic eczema,asthma,inflammatory bowel disease,irritable bowel syndrome and type I diabetes in later life.More and more studies have confirmed the existence of vertical transmission of microflora from mother to child,so the status of maternal microflora during pregnancy is very important to the neonatal microbiome,especially the vaginal microflora,which determines not only the number of microorganisms exposed to the newborn through the way of delivery,but also the type of microorganisms.The colonization of neonatal intestinal tract by vaginal lactobacillus is of great significance,so the influencing factors of perinatal flora have attracted attention,especially the medical intervention measures.The 9th edition of Obstetrics and Gynecology textbook People’s Medical Publishing House clearly stipulates that disinfection is required before each vaginal examination.At present,the commonly used disinfectant in China is povidone iodine solution.However,the previous study of the research group found that compared with the unsterilized group(control group),the content of lactobacillus in the oral microflora of neonates after exposure to povidone iodine during vaginal delivery was significantly reduced,and the number of potentially harmful bacteria was increased.The structure of microflora was similar to that of neonates delivered by cesarean section,and the changes of vaginal microflora were different after exposure to povidone iodine at different concentrations(0.1%and 0.5%),and the high concentration group was more damaged by lactobacillus.Objective:To further verify the effect of povidone iodine exposure on vaginal microflora in healthy mothers based on the previous research group.Povidone iodine is a broad-spectrum disinfectant.If exposure to povidone iodine during natural delivery(in hospital and during vaginal delivery)disrupts the balance of lactobacillus and has adverse effects on healthy pregnant women,is there any difference after exposure to povidone iodine for pregnant women with bacterial flora disorders caused by infectious diseases(vulvovaginal candidiasis,VVC)?Based on the theory of vertical transmission of microflora from mother to child,will the initial intestinal microflora of corresponding neonates of povidone iodine exposed mothers also be affected?In addition,whether the imbalance of microflora in this window period will have an impact on the health status of infants?This paper is divided into three parts to study the above three problems in turn.In the third part of the study,the situation of 6-month-old infants after their mothers were exposed to different concentrations of povidone iodine(0.1%and 0.5%)was also explored.Methods:Part Ⅰ:Effects of povidone iodine exposure during natural delivery on vaginal flora of mothers who be healthy and with vulvovaginal candidiasis1.Case selection and sample collection:Pregnant women being Healthy and with vulvovaginal candidiasis(VVC)admitted to obstetrics department of Nanshan District Maternal and Child Health Hospital,Shenzhen from June to November 2021 were obstained.With the approval of the ethics committee and the signed informed consent,the patients were strictly enrolled according to the inclusion and exclusion criteria and divided into the healthy group(27 cases)and the VVC group(18 cases)according to the VVC screening results of the third trimester(35-36 weeks).According to whether the vagina was exposed to povidone iodine,the pregnant women in each group were divided into pre-disinfection and post-disinfection groups.The healthy group and the VVC group were all collected vaginal secretions(2 samples)at the time of admission for delivery(before disinfection)and after the last vaginal disinfection(after disinfection)at the time of natural delivery.All samples were stored in ultra-low temperature refrigerator.2.16S-rRNA high-throughput sequencing analysis:DNA was extracted firstly,and the DNA quality control test was conducted,followed by biological information analysis,and the a diversity(Shannon index,Observed feature number index and Pielou index)andβ diversity[Principal Coordinate Sorting Analysis based on UniFrac distance(PCoA)]was observed.Analyze the microorganisms with significant differences between the two groups using LEfSe software based on the standard of LDA>3.0 and P<0.05.The software PICRUSt2.0 was used to analyze the differential metabolic pathways between the two groups based on KEGG or MetaCyc databases.Part Ⅱ:Effects of maternal povidone iodine exposure during Natural delivery on initial intestinal flora of neonates1.Case selection and sample collection::Healthy and full-term neonates of the mother in Part Ⅰ.Approved by the Ethics Committee and signed the informed consent,patients were enrolled in strict accordance with the inclusion and exclusion criteria.According to the VVC screening results of their mothers in third trimester(35-36 weeks),the patients were divided into the healthy group(27 cases)and the VVC group(18 cases).Within 24 hours after birth,1 piece of the newborn’s first fetal stool was kept and frozen in ultra-low temperature refrigerator for further detection.2.16S-rRNA high-throughput sequencing analysis:DNA was extracted,and DNA quality detection was carried out,samples that did not meet the standards were removed,and qualified specimens were analyzed for subsequent biological information.The same three indices as the first part of this paper were used for α diversity analysis and β diversity analysis is also done.And analyze the different bacterial genera and metabolic pathways between the two groups.The SourceTracker analysis was also used to analyze the genetic relationships between maternal reproductive tract microorganisms and neonatal meconium microorganismsPart Ⅲ:Effects of maternal povidone iodine exposure during natural delivery on obesity and anemia in 6-month-old infants1.Incidence of obesity and anemia in 6-month-old infants after povidone iodine exposure of VVC mothersHealthy and full-term newborns with natural delivery only to mothers in Part I above were followed up to 6 months of age,including the weight,length,head circumference hemoglobin(Hb),mean red blood cell volume(MCV),Hematocrit(HCT)at 6 months of age,feeding patterns and health status in 6 months after birth(approved by ethical review).The above infants were divided into healthy group and VVC group according to whether the mother combined VVC during late pregnancy.2.The incidence of obesity and anemia in infants at 6 months of age after maternal exposure to povidone iodine at different concentrations2.1 Source and content of information:Information of pregnant women from November 2019 to March 2022 and physical examination information of children aged 6 months from May 2020 to September 2022 from Nanshan District Maternal and Child Health Hospital of Shenzhen;Maternal and childbearing information from March 2021 to March 2022 and physical examination information of children aged 6 months from September 2021 to September 2022 from Baoan District Maternal and Child Health Hospital of Shenzhen.This study was approved by the Ethics Committee of Shenzhen Nanshan District Maternal and Child Health Hospital and Shenzhen Baoan District Maternal and Child Health Hospital.Clinical data of pregnant women include age,the number of pregnancies and deliverie,pregnancy complications and weight gain during pregnancy,delivery mode,gestational age,weight,length of newborn;physical examination data of children aged 6 months,including age,sex,weight,height,feeding,health status,stool,Hb,MCV and HCT.And match maternal and infant information by the unique health care number.2.2 Information screening and grouping:Information is screened according to inclusion and exclusion criteria,the number of enrolled cases in each hospital was obtained(larger than 1636 patients estimated by sample size),and the patients were divided into low concentration group(0.1%povidone iodine)and high concentration(0.5%povidone iodine)group according to different concentrations of povidone iodine used in disinfection during vaginal examination in the hospital.3.According to the formula BMI=body weight(kg)/[body length(m)]2,the body mass index(BMI)was calculated.According to the diagnostic criteria of overweight,obesity and pine of 6-month-old male and female infants in the growth standard for children under 7 years old of the People’s Republic of China Health Industry Standard(WS/T423-2022)in 2022,the number of overweight,obese and emaciated male and female infants in each group was calculated,then the number above in each group was counted,and the corresponding incidence was calculated according to the total number of groups.According to the diagnostic criteria of anemia in Pediatrics,the 9th Edition of People’s Medical Publishing House,the number of anemia in male and female infants in each group was calculated,and the total number of anemia in each group was calculated,and the corresponding incidence rate was calculated according to the total number of anemia in each group.Results:Part Ⅰ:Effects of povidone iodine exposure during natural delivery on vaginal flora of mothers who be healthy and with vulvovaginal candidiasis1.Effect of povidone iodine exposure during natural delivery on vaginal microflora in healthy mothersThe changes of the structure and function of the vaginal flora in the healthy group before and after disinfection:The results of α and β diversity analysis showed that the diversity and abundance of the vaginal flora in the healthy group were increased significantly after disinfection,and average degree was decreased.The relative proportion of Firmicutes and its important bacteria genus Lactobacillus decreased significantly after disinfection,accompanied by the rise of potential pathogenic bacteria including Prevotella,Enterococcus,Corynebacterium and Staphylococcusj.The results of differential microbial analysis showed that there were nearly 50 different bacteria genera,in addition to the above conditional pathogenic bacteria,there were the increase of anaerobe genera such as Anaerococcus,Vellonella,Mobiluncus,Ruminococcus,etc.The abundance of Gardnerella did not change much.It is also differences in metabolic pathways related to amino acid and sugar metabolism.It is suggested that the structure of vaginal flora in healthy mothers after disinfection is in a relatively unbalanced state.2.Effect of povidone iodine exposure during natural delivery on vaginal microflora in mothers with vulvovaginal candida disease2.1 Differences in the structure and function of vaginal microflora in the VVC group compared with the healthy group before disinfectionCompared with the healthy group,the diversity and richness of vaginal microflora in the VVC group before disinfection were increased(α diversity),and the overall bacterial structure was different(β diversity).In addition,the relative proportion of Firmicutes in the VVC group before disinfection was decreased,and the proportion of Actinobacteria and Proteobacteria was increased.While the proportion of Lactobacillus decreased,Gardnerella and Delftia increased,and Dennerella increased more obviously.In the aspect of functional annotation,it was found that there were obvious differences in the metabolic pathways related to cyclic aromatic compounds,and there was an abnormal metabolic pathway,namely mycolate biosynthesis,which suggested the imbalance of vaginal flora caused by VVC in late pregnancy,but did not return to normal before delivery.2.2 After disinfection,the VVC group showed no difference in the diversity of vaginal microflora structure compared with the healthy groupAfter disinfection,although the overall bacterial structure(β diversity)of the VVC group was slightly different from that of the healthy group,statistical analysis of α diversity showed no difference among the three indexes,suggesting that the richness and degree of vaginal bacterial diversity of the VVC group had no change compared with the healthy group.In addition,compared with the healthy group,after disinfection,the relative proportion of Firmicutes in the VVC group decreased,and the proportion of Actinomycetes and Proteobacteria increased,and the increase of Proteobacteria was more obvious.Compared with the healthy group,while the proportion of Lactobacillus decreased,the relative abundance of Gardnerella also increased slightly,but the proportion of Delftia increased more obviously(relative abundance ratio of about 50%).Due to the existence of disinfection and VVC,there were nearly 100 different metabolic pathways,and the VVC group had enrichment of pathways related to lipopolysaccharide metabolism,and it was suggested that although the proportion of Lactobacillus was still relatively low in the VVC group after disinfection,Delftia,which was less likely to cause disease to humans,instead of Gardnerella,which was the pathogenic bacteria,was dominant in the VVC group.2.3 The differences in the structure and function of vaginal microflora in the VVC group before and after disinfectionSimilar to the healthy group before and after disinfection,after disinfection,the diversity and richness of microflora in the VVC group increased,and the overall microflora structure was also different.However,the relative proportion of bacteria decreased significantly after disinfection were Firmicutes and Actinomyces.In addition,when the proportion of Lactobacillus decreased,the abundance of Gardnerella also decreased significantly,and the abundance of Atopotella also decreased relatively,with the increase of Delftia,there were dozens of different metabolic pathways.Further,after disinfection(povidone iodine exposure)in the VVC group,not only Lactobacillus was affected,but the relative abundance of Gardnerella was also significantly changed.Part Ⅱ:Effects of maternal povidone iodine exposure during Natural delivery on initial intestinal flora of neonates1.SourceTracker was used to analyze the genetic relationship between reproductive tract microflora and meconium microflora,and the results showed that the initial intestinal microflora of newborns both was more similar to the structure of the vaginal microflora of their mothers after disinfection in two group.These results suggest that maternal povidone-iodine exposure during natural delivery may affect the initial intestinal flora of neonates through the influence of maternal vaginal flora.2.Comparison of the structure and function of the initial intestinal flora between the VVC group and the healthy groupThe α diversity and β diversity analysis results showed that compared with the healthy group,there was no significant change in the richness and average degree of the initial intestinal flora in the VVC group.In addition,compared with the healthy group,the proportion of Proteobacteria decreased significantly in the VVC group,while the abundance of the Actinobacteria belonging to Bifidobacterium increased,while the Firmicutes belonging to Lactobacillus had no obvious change,but slightly increased.The changes of Bifidobacteriaceae and Lactobacillaceae were not obvious,while the proportion of Enterococcaceae,Rhizobiaceae,Clostridiaceae and Enterococcaceae decreased significantly,while the proportion of Rumenococcaceae increased.Streptococcus,Rhizobium,Enterococcus and Bacteroides decreased significantly,but Lactobacillus did not decrease significantly,but slightly increased.In addition,Ackermannia was only enriched in the healthy group.Functional annotation analysis showed that the healthy group and the VVC group had different enrichment of metabolic pathways,but the VVC group mainly increased the number of synthetic pathways.Part Ⅲ:Effects of maternal povidone iodine exposure during natural delivery on obesity and anemia in 6-month-old infants1.Incidence of obesity and anemia in infants aged 6 months after povidone iodine exposure in VVC mothers.There was no significant difference in BMI overweight rate and anemia rate of 6-month-old infants between VVC group and healthy group,but the overall incidence of VVC group was higher than that of healthy group.2.Incidence of obesity and anemia in infants aged 6 months after maternal exposure to povidone iodine at different concentrations.2.1 There was a difference in BMI of 6-month-old infants born naturally between different concentration groups,and the average BMI of the high concentration group was higher than that of the low concentration group.2.2 The overweight/obesity rates of 6-month-old infants in different concentration groups were 14.68%and 2.42%,and 13.56%and 2.51%,respectively.There was no difference in the overweight/obesity rates of boys and girls in the two groups.2.3 Comparison of overweight and obesity rates in infants at 6 months of age after exposure to povidone iodine at different concentrations:Except for the obesity rate among female infants,there were differences in the overall overweight/obesity rates、overweight/obesity rates of boys and overweight rates of girls between the two hospitals,and the incidence of the high concentration group was higher than that of the low concentration group.2.4 The anemia rates of 6-month-old infants in different concentration groups were 12.44%and 9.71%,respectively.There was no difference in the anemia rate between male and female infants within the two groups in hospital A,but there was a difference in hospital B.2.5 Comparison of the anemia rate of infants aged 6 months after exposure to povidone iodine at different concentrations:The overall anemia rate of the two hospitals is different,and the incidence in the high concentration group was higher than that in the low concentration group.Conclusion:Part Ⅰ:Effects of povidone iodine exposure during natural delivery on vaginal flora of mothers who be healthy and with vulvovaginal candidiasis1.After exposure to povidone iodine during natural delivery in healthy pregnant women,the colonization of Lactobacilli decreased,accompanied by the increase of opportunistic pathogens and anaerobic bacteria,suggesting that povidone iodine exposure during natural delivery may have adverse effects on healthy pregnant women.However,what is the degree of influence and whether it will cause adverse consequences needs further study.2.In the third trimester of pregnancy(35-36 weeks)combined with VVC,even after antibiotic treatment,the vaginal microflora before delivery still does not return to normal,and is still in a state of microflora imbalance or convalescence transition,that is,low colonization of Lactobacillus accompanied by the increase of harmful bacteria Gardnerella.The therapeutic strategy of pregnancy combined with VVC based on the mechanism of microflora imbalance remains to be further studied.3.In both healthy and VVC pregnant women,colonization of Lactobacillus decreased after povidone iodine exposure during natural delivery,but Gardnerella decreased in pregnant women with VVC.In addition,compared with healthy pregnant women,the diversity difference of vaginal microflora structure in pregnant women with VVC before disinfection disappeared after disinfection,and the abundance of Gardnerella,which was significantly increased before disinfection,was significantly decreased after disinfection.It is suggested that povidone iodine exposure during natural delivery may be beneficial to pregnant women with VVC,but whether the mechanism of action is related to the decreased abundance of Gardnerella and under what conditions can give full play to its beneficial effect still needs more research.Part Ⅱ:Effects of maternal povidone iodine exposure during Natural delivery on initial intestinal flora of neonates1.Povidone iodine exposure during natural delivery can affect the initial intestinal flora of newborns by affecting the mother’s vaginal flora.However,it remains to be further investigated whether the effects on newborns in different health status are different.2.The vaginal microbiota during the third trimester(35-36 weeks)may affect the neonatal intestinal microbiota,and the closer to the pre-delivery vaginal microbiota may affect the neonatal intestinal microbiota.Diseases or procedures that cause the greatest imbalance of the vaginal microbiota at these time points need careful management to minimize adverse effects.Part Ⅲ:Effects of maternal povidone iodine exposure during natural delivery on rate of obesity and anemia in 6-month-old infants1.Whether the combination of VVC during pregnancy will increase the overweight rate and anemia rate of 6-month-old infants,and whether there is a difference after povidone iodine exposure,still needs to be further confirmed by a large sample cohort study.2.The overweight/obesity rate of 6-month-old infants born naturally and breastfed in two areas of Shenzhen are 14.68%,2.42%and 13.56%,2.51%,respectively.In addition,there is no difference in the overweight/obesity rate between boys and girls.3.The anemia rate of infants born naturally and breastfed for 6 months in in two areas of Shenzhen were 12.44%and 9.71%respectively.4.The incidence of overweight/obesity and anemia at 6 months of age was significantly different after maternal exposure to povidone iodine at different concentrations.That is,high maternal exposure to povidone iodine increased the incidence of overweight/obesity and anemia at 6 months of age,Suggesting that maternal povidone iodine exposure during natural childbirth can affect the health status of infants by affecting the microflora in the window period,and also suggesting that that medical staff should use low concentration for obstetric disinfection as far as possible to reduce adverse effects on the condition that the incidence of perinatal infection does not increase,but the specific concentration and disinfection methods are still to be further studied investigate. |
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