Study On Association Between Poly-and Perfluoroalkyl Exposure Levels During Pregnancy,Intrauterine Flora,and Intrauterine Growth Restriction Based On Birth Cohort

Objectives:To explore the relationship between Poly-and perfluoroalkyl substances（PFASs）exposure during pregnancy and the risk of Intrauterine Growth Restriction（IUGR）,as well as the association of PFASs exposure and IUGR with intrauterine flora,and to analyze the intermediation role of intrauterine flora in linking PFASs exposure and IUGR.Method:The subjects are mother-son pairs in the birth cohort of Guangxi Zhuang population from 2015 to 2018.Data were collected from the subjects through questionnaires and Guangxi Women and Children Health Service Information Management System.Peripheral intravenous blood samples were collected from the pregnant women during the initial check-up.Oral and oropharyngeal extracts were obtained from the newborns immediately after birth.The oropharyngeal extracts from the newborns are highly consistent with amniotic fluid flora and are prenatal intrauterine flora or fetal flora[1].The concentrations of 11 PFASs,including perfluorooctanoatic acid（PFOA）,perfluorononanoic acid（PFNA）,perfluoroundecanoic acid（PFUnA）,perfluorohexane sulfonic（PFHxS）,and perfluorooctane sulfonic（PFOS）,in pregnant women serum were determined by ultra performance liquid chromatography-mass spectrometry（UPLC-MS）.The structure and diversity of the flora of the oropharyngeal extracts of newborns immediately after birth were detected by the 16S rRNA high-throughput sequencing method.A bioinformatics analysis was conducted.Conditions for screening differential flora were as follows:P<0.05;the difference in abundance between high and low exposure groups or between IUGR and Non-IUGR≥2 times（ie |logFC|≥1）;and the detection rate of flora species>25%.The relationship between PFASs exposure during pregnancy and the risk of IUGR occurrence,as well as the relationship between PFASs exposure and intrauterine flora,were studied by using the cohort study method.The association between intrauterine flora and IUGR was studied by using the nested case-control study.The PFASs were screened for LASSO regression analysis.The relationship between PFASs exposures and the risk of intrauterine growth restriction was explored using the binary Logistic regression model.The association between PFASs exposure and intrauterine flora in pregnant women and the association between intrauterine flora with IUGR were analyzed using the binary Logistic regression model.The mediation effect was used to determine the role of intrauterine flora in linking the association of PFASs with IUGR during pregnancy.Results:1.Of the 1,545 subjects,230 cases had IUGR,reflecting an incidence rate of 14.9%.LASSO regression analysis found that all nine PFASs had an impact on the occurrence of IUGR.Among the nine PFASs models,five PFASs,namely,PFOA,PFNA,PFUnA,PFHxS,and PFOS,were found to be related to the risk of IUGR occurrence after adjusting for covariates.With increasing concentration of 1g PFOA in pregnant women serum,and the risk of IUGR decreased（OR=0.31;95%CI[0.12,0.81];P=0.016）.Compared with the low exposure group,the risk of IUGR increased in the serum of pregnant women in the PFNA high-exposure group（OR=1.75;95%CI[1.01,3.03];P=0.047）.With increasing concentration of 1g PFHxS,the risk of IUGR increased（OR=2.31;95%CI[1.07,5.01];P=0.033）.Gender stratification analysis results showed that PFUnA and PFOS were associated with IUGR risk in female newborns.With increasing concentration of 1g PFUnA in the serum of pregnant women,the risk of IUGR increased in female newborns（OR=22.79;95%CI[3.59,144.46];P=0.001）.The risk of IUGR in the PFOS exposure group in the serums of pregnant women increased compared with the relatively low exposure group（OR=2.35,95%CI[1.06,5.25],P=0.036）.2.The species composition and distribution of the intrauterine flora of 212 subjects were as follows.At the phylum level,the top three bacteria were p_Proteobacteria（38.03%）,p_Firmicutes（27.55%）,and p_Actinobacteria（16.82%）.At the genus level,the top three bacteria were g_Lactobacillus（19.12%）,g_Ralstonia（17.81%）,and g_unclassified_knorank（6.42%）.Three types of bacteria were screened at the phylum level,and 30 types of bacteria were screened at the genus level.Binary logistic regression results showed that PFOS was associated with the first three axes of PCoA of β diversity,and the association was found only in female newborns after gender stratification.At the phylum level,the bacteria associated with PFUnA exposure were members of p_Spirochaetae.At the genus level,the bacteria associated with the PFOA exposure were members of the g_[Ruminococcus]_gnavus_group;the bacteria associated with PFNA were g_Dokdonella,g_Roseburia,g_Enterococcus;the bacteria associated with PFUnA exposure were g_Enhydrobacter,g_Senegalimassilia,and g_Adlercreutzia;the bacteria associated with PFHxS were g_Fusicatenibacter,g_Coprococcus₁,g_Anaerostipes,g_Dorea,g_Lachnospira,g_Leptothrix,g_Tsukamurella,g_Blautia,g_Candidatus_Odyssella,and g_Arthrobacter;and the bacteria associated with PFOS were g_Microbacterium,g_Afipia,g_Granulicatella,g_Enorma,g_Exiguobacterium,g_Cellvibrio,and g_Skermanella.Of the above 24 types of bacteria,11 were from p_Firmicutes at the phylum level,seven were from p_Proteobacteria,and six were from p_Actinobacteria.3.The species composition and distribution of the intrauterine flora of 222 subjects were as follows.At the phylum level,the top three bacteria were p_Proteobacteria（39.81%）,p_Firmicutes（26.89%）,and p_Actinobacteria（15.83%）.At the genus level,the top three bacteria were g_Lactobacillus（18.84%）,g_Ralstonia（17.21%）,and g_Pseudomonas（8.17%）.One type of bacteria was screened at the family level,and 10 types of bacteria were screened at the genus level.The bacteria associated with IUGR at the genus level were g_Microbacterium,g_Tsukamurella,g_Sphingobium,g_Thermomonas,and g_Hydrogeno phaga;three of these were from p_Proteobacteria,and two were from p_Actinobacteria.4.The common different bacteria in the uterus which were associated with the exposure levels of PFOA,PFNA,PFUnA,PFHxS,and PFOS during pregnancy and associated with whether IUGR were g_Afipia,g_Microbacterium,and g_<sub>Tsukamurella.Mediation analysis results showed that the intrauterine flora g_Microbacterium had a statistical association with PFOS exposure and IUGR during pregnancy.Intrauterine flora g_Microbac terium played a negative intermediation role in PFOS exposure and IUGR during pregnancy,and the proportion of the mediation effect in the total effect was28.6%.Conclusions:1.The incidence rate of IUGR was 14.9%in the Zhuang population,and exposure of PFASs during pregnancy was closely related to the risk of IUGR.PFNA,PFUnA,PFHxS,and PFOS were risk factors,and PFUnA and PFOS were found only in female newborns.2.PFASs exposure during pregnancy was associated with the changes in the intrauterine flora.3.Changes in abundance of some species in the intrauterine flora were associated with IUGR.4.The intrauterine flora g_<sub>Microbacterium had a statistical association with PFOS exposure and IUGR during pregnancy.These results provided a reference for exploring the etiology and mechanism of IUGR and for preventing and controlling IUGR occurrence.