孕前女性口腔菌群与胎儿过度生长的关联

Association between preconception oral microbiome and fetal overgrowth

目的分析女性孕前口腔菌群与胎儿过度生长的关联及可能机制。方法基于孕前队列,采取巢式病例对照研究设计,选取队列自2016年10月至2021年12月于上海市嘉定区妇幼保健院招募的人群中分娩巨大儿和/或大于胎龄儿(large for gestational age,LGA)的51例产妇作为病例组,按1∶4选取同期分娩正常出生体重儿、适于胎龄儿的204例产妇作为对照组。以总人群中分娩LGA的48例产妇作为LGA亚组,并从其余分娩非LGA的产妇中按1∶4随机抽取192例作为其相应对照组,展开LGA亚组分析。采用16S rRNA基因测序技术检测孕前唾液样本,比较组间口腔菌群特征、差异菌群与差异功能通路。采用非参数Wilcoxon秩和检验、两独立样本t检验,或χ^(2)检验(或Fisher精确概率法)进行统计学分析。对女性孕前膳食数据进行因子分析,以膳食模式因子得分的最大取值确定每个研究对象的主要膳食模式。对菌群计数数据,采用R和QIIME2软件计算α和β多样性指标,并通过PICRUSt2获得相应菌群功能计数数据。结果(1)一般资料:2组对象从孕前采样至妊娠及采样至分娩的时间间隔差异无统计学意义。病例组51例中,单纯巨大儿3例,LGA 48例(94.1%)。LGA亚组的相应对照为192例。病例组与对照组膳食模式差异无统计学意义。(2)α多样性分析:病例组物种丰富度指数低于对照组[(367.27±84.57)与(408.71±93.08),多因素分析P=0.009],而2组间Shannon指数和Simpson指数的差异则无统计学意义;LGA亚组的物种丰富度指数亦低于相应对照组[(371.04±83.92)与(408.04±94.21),多因素分析P=0.033],而Shannon指数和Simpson指数差异则无统计学意义。(3)β多样性分析:①病例组与对照组口腔菌群非加权UniFrac距离差异有统计学意义(R^(2)=0.006,F=1.479,P=0.048)。LGA亚组与相应对照组口腔菌群β多样性指标差异无统计学意义。(4)差异菌群分析:①病例组与对照组从门到属共有14个差异菌群。在属水平,消化链球菌科G1菌属在病例组富集,而劳特罗普氏菌属、小杆菌属、纤毛菌属和罗斯氏菌属则在对照组富集。②LGA亚组与相应对照组从门到属共有14个差异菌群;在属水平,罗斯氏菌属、纤毛菌属、单糖菌门G6菌属和月形单胞菌属在对照组富集(LDA值均>2,P值均<0.05)。(5)差异功能分析:病例组女性口腔菌群中烟酸降解[log_(2)差异倍数(fold change,FC)=3.510,q=0.005]、嘧啶核苷酸从头合成途径(log_(2)FC=0.078,q=0.005)及L-酪氨酸降解通路(log_(2)FC=0.710,q=0.034)等相关代谢功能通路富集。LGA亚组与相应对照组比较发现,LGA亚组口腔菌群中烟酸降解相关代谢功能通路富集(log_(2)FC=3.660,q=0.012)。结论过度生长胎儿的母亲孕前口腔菌群结构较正常生长胎儿的母亲存在差异,且正常生长胎儿的母亲孕前口腔菌群多样性更高。孕前口腔菌群中部分致病菌富集与共生菌减少与胎儿过度生长有关,这种关联可能通过烟酸降解等功能通路实现。

Objective To analyze the association between the pre-pregnancy oral microbiota of women and fetal overgrowth,and the possible mechanisms involved.Methods A nested case-control study design based on a pre-pregnancy cohort was used to select 51 mothers who delivered macrosomia and/or large-for-gestational-age(LGA)infants from the population recruited at the Maternal and Child Health Care Hospital of Jiading District in Shanghai from October 2016 to December 2021 as the case group.A control group was formed by selecting 204 mothers who delivered infants with normal birth weight and appropriate for gestational age during the same period,in a 1:4 ratio.The LGA subgroup consisted of 48 mothers who delivered LGA infants from the total population,and a corresponding control group of 192 was randomly selected from the remaining mothers who delivered non-LGA infants in a 1∶4 ratio for the LGA subgroup analysis.The 16S rRNA gene sequencing technique was utilized to detect pre-pregnancy saliva samples to compare the characteristics of the oral microbiota,differential microorganisms,and differential functional pathways between groups.Nonparametric Wilcoxon rank-sum tests,two independent samples t-tests,or Chi-square(or Fisher's exact)tests were used for statistical analysis.Factor analysis was conducted on the pre-pregnancy diet data of women,and the primary dietary pattern of each study subject was identified based on the highest score of the dietary pattern factors.For microbiota count data,α and β diversity indices were calculated using R and QIIME2 software,and the corresponding microbiota functional count data were acquired through PICRUSt2.Results(1)General data:There was no significant difference in the time interval from pre-pregnancy sampling to pregnancy and from sampling to delivery between the two groups.In the case group,there were three cases of macrosomia and 48 cases(94.1%)of LGA.The corresponding control group for the LGA subgroup consisted of 192 cases.There were no significant differences in dietary patterns between the case group and the control group.(2)αdiversity analysis:The species richness index of the case group was lower than that of the control group[(367.27±84.57)vs.(408.71±93.08),multivariate analysis,P=0.009],while no significant differences were found between the two groups in the Shannon and Simpson indices;the species richness index of the LGA subgroup was also lower than that of the corresponding control group[(371.04±83.92)vs.(408.04±94.21),multivariate analysis,P=0.033],with no significant differences in the Shannon and Simpson indices.(3)βdiversity analysis:There was a statistically significant difference in the unweighted UniFrac distance of the oral microbiota between the case group and the control group(R^(2)=0.006,F=1.479,P=0.048).No significant differences were found in theβdiversity indices of the oral microbiota between the LGA subgroup and the corresponding control group.(4)Differential microbiota analysis:There were 14 differential microbiotas from phylum to genus between the case group and the control group.At the genus level,members of the G1 genus of the Streptococcaceae were enriched in the case group,while the Lautropia,Dialister,Leptotrichia,and Rothia were enriched in the control group.In the LGA subgroup and its corresponding control group,there were 14 differential microbiota from phylum to genus;at the genus level,Leptotrichia,Rothia,G6 genus of the Saccharibacteria,and Selenomonas were enriched in the control group(all LDA value>2,and all P<0.05).(5)Differential functional analysis:In the case group,metabolic pathways such as nicotinate degradation[log_(2) fold change(FC)=3.510,q=0.005],de novo synthesis of pyrimidine nucleotides(log_(2)FC=0.078,q=0.005),and L-tyrosine degradation pathway(log_(2)FC=0.710,q=0.034)were enriched in the oral microbiota of women.In the LGA subgroup,compared to the corresponding control group,metabolic pathways related to nicotinate degradation were enriched in the oral microbiota(log_(2)FC=3.660,q=0.012).Conclusions There are differences in the structure of the pre-pregnancy oral microbiota of mothers with overgrown fetuses compared to those with normally grown fetuses,and mothers of normally grown fetuses show higher diversity in their pre-pregnancy oral microbiota.The enrichment of certain pathogenic bacteria and the reduction of symbiotic bacteria in the pre-pregnancy oral microbiota are associated with fetal overgrowth,and this association may be mediated by functional pathways such as nicotinate degradation.