findings support the need for longitudinal investigations of the any of the four body sites during the course of gestation(P> A0(4m2 asures(si Apps dn nig s r iust tr oth间 microbiota at multiple body sites during pregnan cluded controls(term deliveries) and cases(preterm deliveries). UniFrac distance between same-subject communities sampled in We characterized the te ral dyna tion based on prospective weekly sampling during pre body sites(P>0.05, t test)(Fig. 1B). This finding also was robust from four body sites: vagina, distal gut(stool), saliva, and to the choice of distance measure (SI Ap 1- was no particular week(or series of weeks)of pregnane itional stability during pregnancy at all body sites, a diverse large, coordinated shifts occurred nal community state early during pregnancy in women who The average pairwise-weighted UniFrac distance betwee ibsequently delivered prematurely, and a dramatic shift in munities in different subjects was used as an estimate of vaginal microbiota composition at the time of delivery that in versity. We evaluated the significance of beta diversity trend ome cases persisted for the maximum duration of postpartum permutation test in which the null-distribution of our test statistic (1y) was estimated from the ensemble of rar ects(Sl Appendix. SI Methods). We found no significant trend with Results time at any of the four body sites(P>0.05, permutation test)(Fig. IC). Similarly, this finding was robust to the choice of distance discovery dataset(11 of these 40 women delivered preterm)and measure for quantifying beta diversity (SI Appendix, Fig. S4) ne ot whom contributed samples tor a validation dataset(tour then microbiota composition near the end of pregnancy should be distinct from that near the start, although other types of dy- SI Appendix, Table S1. The first group of 40 subiects provided However, as did the statistical summaries of these communities, quencing reads per sample(19 306, 851 total reads). This discovery gestational taxonomic composition remained constant over dataset was used to characterize temporal dynam distances based on relative operational taxonomic unit (OTU) preterm birth. The second group of nine subjects provided munities sampled ear. ealed significant overlap between com- pregnancy at all body sites(/ Appeyvus(those present in >25% Most none of the most prevalent O 203,391 filtered, high-quality Illumina reads per sample of samples)exhibited sig nificant shifts in relative abundance 50.034, 186 total reads); this validation dataset was used to test between early and late in pregnancy(SI Ap Table s3 dentified in the di dataset as being associated with preterm birth. SI Appendix, pregnancy is not associated with a dramatic remodeling of the who delivered preterm(before gestational week 37) iversity and Composition of Bacterial Communities of the Vagina, the 40 women Vaginal Community State Types of Pregnancy. With the data from he first subject group, we applied de novo Pregnancy. We evaluated spatial and temporal trends in the from each body site to explore community structure and to 需 LME) model to regress alpha diversity measures against ges- uneven communities dominated by different species of Lactobacil tational time while accounting for the subject-structure of these hus and a fifth CSt that was characterized by much greater evenness longitudinal data by treating the subject as a random effect. No and taxonomic diversity(Fig. 2). These CSTs corresponded well to trend in the Shannon diversity index was found for those described by Ravel et al.(11, 25)and thus Fig. 1. Human-associated bacterial communities A Tooth/Gum rom the first group of 40 women, the estimated trends of alpha diversity weekly instability(week o-week variation within subjects), and beta di- with gestational time are insignificant (p> 褴H" umtt all body sites. (A)Shannon diversity is against gestational time for specimens B ken from the vagina, stool, saliva, and tooth/gum pm0.942 pm0099 Blue lines indicate the linear mixed-effects re 时8时 bject Shading indicates the 95% confidence in- erval (CI). Because vaginal diversity and stability 020 C p=0314 p=04890.5 o Weighted-UniFrac distance between same-subject mples taken 1 wk apart is plotted against gesta CL(o) nal time. The green lines indicate the linear fit, and the shading indicates the 95% Cl as estimated by a permutation bootstrap (S/ Appendix, S/ Methods PNAS I September 1, 2015 I voL. 112 I no 35 I 11061findings support the need for longitudinal investigations of the microbiota at multiple body sites during pregnancy. As part of a larger ongoing study, we examined a total of 49 women who were divided into two groups, each of which in￾cluded controls (term deliveries) and cases (preterm deliveries). We characterized the temporal dynamics of microbiota compo￾sition based on prospective weekly sampling during pregnancy from four body sites: vagina, distal gut (stool), saliva, and tooth/ gum, as well as after delivery. Our data reveal microbiota com￾positional stability during pregnancy at all body sites, a diverse vaginal community state early during pregnancy in women who subsequently delivered prematurely, and a dramatic shift in vaginal microbiota composition at the time of delivery that in some cases persisted for the maximum duration of postpartum sampling (1 y). Results We studied 49 women, 40 of whom contributed samples for a discovery dataset (11 of these 40 women delivered preterm) and nine of whom contributed samples for a validation dataset (four of these nine women delivered preterm). Demographic and baseline clinical characteristics of the study population appear in SI Appendix, Table S1. The first group of 40 subjects provided 3,767 clinical specimens (SI Appendix, Fig. S1) that were ana￾lyzed at a mean depth of 5,125 filtered, high-quality pyrose￾quencing reads per sample (19,306,851 total reads). This discovery dataset was used to characterize temporal dynamics of the microbiota at four body sites (vaginal, stool, saliva, tooth/gum) and to identify stereotypic community features associated with preterm birth. The second group of nine subjects provided 246 vaginal specimens that were analyzed at a mean depth of 203,391 filtered, high-quality Illumina reads per sample (50,034,186 total reads); this validation dataset was used to test features of the vaginal community identified in the discovery dataset as being associated with preterm birth. SI Appendix, Table S2 presents additional details of the 15 women in total who delivered preterm (before gestational week 37). Diversity and Composition of Bacterial Communities of the Vagina, Distal Gut, Saliva, and Tooth/Gum Are Relatively Stable During Pregnancy. We evaluated spatial and temporal trends in the structure of the bacterial communities of each body site during pregnancy in a group of 40 women by using a linear mixed-effects (LME) model to regress alpha diversity measures against ges￾tational time while accounting for the subject-structure of these longitudinal data by treating the subject as a random effect. No significant trend in the Shannon diversity index was found for any of the four body sites during the course of gestation (P > 0.05, t test) (Fig. 1A). This finding was robust to other alpha diversity measures (SI Appendix, Fig. S2). Similarly, no signifi￾cant trends over gestational time were observed in the weighted UniFrac distance between same-subject communities sampled in consecutive weeks (i.e., weekly instability) for any of the four body sites (P > 0.05, t test) (Fig. 1B). This finding also was robust to the choice of distance measure (SI Appendix, Fig. S3). There was no particular week (or series of weeks) of pregnancy in which large, coordinated shifts occurred. The average pairwise-weighted UniFrac distance between com￾munities in different subjects was used as an estimate of beta di￾versity. We evaluated the significance of beta diversity trends with a permutation test in which the null-distribution of our test statistic was estimated from the ensemble of randomly time-reversed sub￾jects (SI Appendix, SI Methods). We found no significant trend with time at any of the four body sites (P > 0.05, permutation test) (Fig. 1C). Similarly, this finding was robust to the choice of distance measure for quantifying beta diversity (SI Appendix, Fig. S4). If consecutive weekly instability were gradual and consistent, then microbiota composition near the end of pregnancy should be distinct from that near the start, although other types of dy￾namics (e.g., oscillations) might not give rise to this distinction. However, as did the statistical summaries of these communities, the average taxonomic composition remained constant over gestational time. This compositional stability was illustrated by nonmetric multidimensional scaling ordination of Bray–Curtis distances based on relative operational taxonomic unit (OTU) abundances, which revealed significant overlap between com￾munities sampled early in pregnancy and those sampled late in pregnancy at all body sites (SI Appendix, Fig. S5). Furthermore, almost none of the most prevalent OTUs (those present in >25% of samples) exhibited significant shifts in relative abundance between early and late in pregnancy (SI Appendix, Table S3). Taken together, these results suggest that the progression of pregnancy is not associated with a dramatic remodeling of the diversity and composition of a woman’s indigenous microbiota. Vaginal Community State Types of Pregnancy. With the data from the 40 women in the first subject group, we applied de novo clustering, based on relative OTU abundances, to the samples from each body site to explore community structure and to reduce dimensionality. For the vaginal communities, this analysis yielded a set of four community state types (CSTs) comprising highly uneven communities dominated by different species of Lactobacil￾lus and a fifth CST that was characterized by much greater evenness and taxonomic diversity (Fig. 2). These CSTs corresponded well to those described by Ravel et al. (11, 25) and thus were numbered A B C Fig. 1. Human-associated bacterial communities are stable during pregnancy. Based on the data from the first group of 40 women, the estimated trends of alpha diversity, weekly instability (week￾to-week variation within subjects), and beta di￾versity with gestational time are insignificant (P > 0.05) at all body sites. (A) Shannon diversity is plotted against gestational time for specimens taken from the vagina, stool, saliva, and tooth/gum. Blue lines indicate the linear mixed-effects re￾gression of diversity on time with grouping by subject. Shading indicates the 95% confidence in￾terval (CI). Because vaginal diversity and stability data were highly skewed, they were log-trans￾formed before fitting to improve normality. (B) Weighted-UniFrac distance between same-subject samples taken 1 wk apart is plotted against gesta￾tional time. Red lines indicate the lme regression, and the shaded area indicates the 95% CI. (C) Av￾erage weighted-UniFrac distance between differ￾ent-subject samples taken within the same gestational week is plotted against gestational time. The green lines indicate the linear fit, and the shading indicates the 95% CI as estimated by a permutation bootstrap (SI Appendix, SI Methods). DiGiulio et al. PNAS | September 1, 2015 | vol. 112 | no. 35 | 11061 MICROBIOLOGY