women with preterm labor have revealed bacterial species in- and studies. The relati of each lactobacillus-dominated digenous to the vagina, oral cavity, and gut(6-9 CST in dy wa Using prospectively collected samples, we analyzed the micro- women(25). We also rispatus to be associated with biota from a total of 49 women in two case-control analyses of increased stability of the nonoverlapping subject groups. First, we characterized the temporal dynamics of microbiota structure and composition during pregnancy Vaginal Community Signatures of Preterm Birth. We found the high- in 40 women based on weekly sampling from four relevant body diversity vaginal CST 4 to be associated with an increased in- sites: vagina, distal gut(stool), saliva, and tooth/gum Of note, in this cidence of preterm birth. Indeed, CST 4 exhibited both dose discovery dataset we found that the structure and composition of response(Fig. 4A) and temporal(Fig. 4B)relations he microbiota are relatively stable overall but that some site-spe reterm birth. This finding is consistent with previous associations testate transition rat aginal CSTs). Despite the small vas 12, 13)and increased risk for preterm birth or bacterial cific communities can vary considerably over time(e.g, disparate between either high-diversity vaginal communities(16) number of women with premature deliveries in this study, we we had collected specimens less frequently than weck. note, if eate f seibnsance t a term birth we s associated wnta ze e veavaitead have been hindered in associating this state with preterm birth. amples from nine additional women and corroborated the asso- that high abundance of Gardnerella combined with low abun- ciation between the high-risk community profiles and preterm dance of Lactobacillus might be particularly predictive of pre In nonpregnant women, longitudinal studies of the term birth. Although this finding was based on a relatively small microbiota have demonstrated stability when viewed at a number of subjects, it remained significant even when the most 32).Thee, punctuated by occasional daily fluctuations(25, 21- conservative approach was taken to test the independence of tudies also d nted significant variability among samples from the same subject. The finding was confirmed with a individuals with respect to the typical vaginal csT and its level of validation dataset from nine subjects in which the one woman by L. crispatus, are more stable than others. Menses can disrupt delivered preterm. Our analysis also suggested that a high vaginal community composition, albeit transiently(25). In inal microbial community dy have been less well studied. and d Lactobacillus is associated with preterm birth. Both gardnerella es at n sites have hardly been studied at all. Two recent studies by la! boay and Ureaplasma have been implicated by a large body of litera Romero and colleagues, with a sampling frequency of every 4 wk ture as having potential roles in the pathogenesis of preterm until week 24 of gestation and then every 2 wk until the last delivery (SI Appendix, SI Discussion) nt lor prenatal visit, Provide the most meaningful comparions with our found no significant difference in taxon abundances or diversity of vaginal communities between term and preterm pregnance nancies reported the vaginal microbiota to be more stable during One possible reason for the contradictory results is the differ pregnancy than in the nonpr state(18 tion of our study cohorts. Previous changes in the composition of the vaginal microbiota (e.g, an work has demonstrated significant differences between vaginal decrease in an rabic species) as a function of g station l age study by Romero et al. included only births before gestational (17). In contrast to this latter study, we found no signifi change in the taxonomic structure of the vaginal microbial cluded some women whose labor onset was nonspontaneous preclude the possibility of significant changes in the vaginal mi- between our study and previous work, especially when consid- Two longitudinal studies(16, 19) and two cross-sectional studies different PCR primers(18, 25) ginal communities sampled from one or more trimesters In ag- Vaginal Community Alteration After Delivery. We found that most gregate their findings suggested that the vaginal microbiota duri t, abrupt, and durable alteratio healthy pregnancy mostly resembles the nonpregnant microbiota, in the vaginal community beginning with the first sample col lected postdelivery. Compared with samples from other body from each of the first and third trimesters in 91 pregnant women. disturbance, as well as its presence after otr a anismses became The results of our analysis of the distal gut microbiota differ sites in the postpartum period, the vaginal communi from those from Koren et al. ( 24)who reported a"remodeling of more similar to the gut communities. The meck the gut microbiome"based on analysis of a single time point this potential convergence are unknown. The persistence of al and caesarean Although Koren et al. reported that 16 of the women in their deliveries, argues against simple translocation of stool communities udy took probiotic supplements during pregnancy, and that to the vagina as the primary mechanism. seven used antibiotics, the authors could not relate changes in The potential impact on maternal health of the postdelivery beta diversity in individual women to their use of either sup- shift of the vaginal community also is unknown but includes the plements or antibiotics(24). The differences in study findings possibility that a persistently altered vaginal community might lay reflect the fact that many of their subjects received a dietary affect the outcome of a subsequent preg intervention between those two time points occurs too soon after delivery. A short interpregnancy interval (e. g, <12 mo) is associated with an increased risk of preterm Vaginal Community Structure. We found, as have others(11, 17, birth. Whether an altered postdelivery vaginal community plays a 18), that vaginal nities exhibited distinct patterns of as- contributing role warrants further study. mbly distinguished by the dominance of a particular Lactobo cillus species(i. e, CST 1, 2, 3, and 5)or by a non-Lactobacillus- Materials and Methods munities that cape vae erep ity(CST 4). The robustness of CST To enhance the reproducibility of our results, the reader will find studies using various methodologies necessary to reproduce the analyses and figures presented in supports CSTs descriptors of vaginal microbial com- available at statwebstanfordedw/susan/papers/PNASRRhtml acilitate comparisons across pregnancy status includes pointers to the R markdown files, the output in html, Thi w ets 11064Iwww.pnas.org/cgi/doi/10.1073/pnas.1502875112 DiGiulio et alwomen with preterm labor have revealed bacterial species in￾digenous to the vagina, oral cavity, and gut (6–9). Using prospectively collected samples, we analyzed the micro￾biota from a total of 49 women in two case-control analyses of nonoverlapping subject groups. First, we characterized the temporal dynamics of microbiota structure and composition during pregnancy in 40 women based on weekly sampling from four relevant body sites: vagina, distal gut (stool), saliva, and tooth/gum. Of note, in this discovery dataset we found that the structure and composition of the microbiota are relatively stable overall but that some site-spe￾cific communities can vary considerably over time (e.g., disparate interstate transition rates of vaginal CSTs). Despite the small number of women with premature deliveries in this study, we identified candidate vaginal community compositional profiles in early pregnancy that were significantly associated with an elevated rate of subsequent preterm birth. We subsequently analyzed vaginal samples from nine additional women and corroborated the asso￾ciation between the high-risk community profiles and preterm birth. In nonpregnant women, longitudinal studies of the vaginal microbiota have demonstrated stability when viewed at a weekly timescale, punctuated by occasional daily fluctuations (25, 27– 32). These studies also documented significant variability among individuals with respect to the typical vaginal CST and its level of stability. Certain types of communities, such as that dominated by L. crispatus, are more stable than others. Menses can disrupt vaginal community composition, albeit transiently (25). In pregnant women, vaginal microbial community dynamics have been less well studied, and dynamics at nonvaginal body sites have hardly been studied at all. Two recent studies by Romero and colleagues, with a sampling frequency of every 4 wk until week 24 of gestation and then every 2 wk until the last prenatal visit, provide the most meaningful comparisons with our findings (17, 18). One study of 22 women with healthy preg￾nancies reported the vaginal microbiota to be more stable during pregnancy than in the nonpregnant state (18). The other study, involving 72 women with healthy term pregnancies, described changes in the composition of the vaginal microbiota (e.g., an increase in the relative abundance of Lactobacillus species and a decrease in anaerobic species) as a function of gestational age (17). In contrast to this latter study, we found no significant change in the taxonomic structure of the vaginal microbial communities as pregnancy progressed. Importantly, because we obtained few samples early in the first trimester, we cannot preclude the possibility of significant changes in the vaginal mi￾crobial community at the beginning of pregnancy. Two longitudinal studies (16, 19) and two cross-sectional studies (14, 15) also used culture-independent methods to characterize vaginal communities sampled from one or more trimesters. In ag￾gregate their findings suggested that the vaginal microbiota during healthy pregnancy mostly resembles the nonpregnant microbiota, but with an even higher prevalence of Lactobacillus species. The results of our analysis of the distal gut microbiota differ from those from Koren et al. (24) who reported a “remodeling of the gut microbiome” based on analysis of a single time point from each of the first and third trimesters in 91 pregnant women. Although Koren et al. reported that 16 of the women in their study took probiotic supplements during pregnancy, and that seven used antibiotics, the authors could not relate changes in beta diversity in individual women to their use of either sup￾plements or antibiotics (24). The differences in study findings may reflect the fact that many of their subjects received a dietary intervention between those two time points. Vaginal Community Structure. We found, as have others (11, 17, 18), that vaginal communities exhibited distinct patterns of as￾sembly distinguished by the dominance of a particular Lactoba￾cillus species (i.e., CST 1, 2, 3, and 5) or by a non-Lactobacillus– dominated diverse community (CST 4). The robustness of CST assignment across different studies using various methodologies supports CSTs as valid descriptors of vaginal microbial com￾munities that can facilitate comparisons across pregnancy status and studies. The relative stability of each Lactobacillus-dominated CST in our study was similar to that reported in nonpregnant women (25). We also found L. crispatus to be associated with increased stability of the vaginal community. Vaginal Community Signatures of Preterm Birth. We found the high￾diversity vaginal CST 4 to be associated with an increased in￾cidence of preterm birth. Indeed, CST 4 exhibited both dose– response (Fig. 4A) and temporal (Fig. 4B) relationships with preterm birth. This finding is consistent with previous associations between either high-diversity vaginal communities (16) or bacterial vaginosis (12, 13) and increased risk for preterm birth. Of note, if we had collected specimens less frequently than weekly, we would have missed a number of excursions to CST 4 and hence would have been hindered in associating this state with preterm birth. Further analysis of taxa abundances within CST4 suggested that high abundance of Gardnerella combined with low abun￾dance of Lactobacillus might be particularly predictive of pre￾term birth. Although this finding was based on a relatively small number of subjects, it remained significant even when the most conservative approach was taken to test the independence of samples from the same subject. The finding was confirmed with a validation dataset from nine subjects in which the one woman who had high Gardnerella in conjunction with low Lactobacillus delivered preterm. Our analysis also suggested that a high abundance of Ureaplasma combined with a low abundance of Lactobacillus is associated with preterm birth. Both Gardnerella and Ureaplasma have been implicated by a large body of litera￾ture as having potential roles in the pathogenesis of preterm delivery (SI Appendix, SI Discussion). In their recent longitudinal study, Romero and colleagues (17) found no significant difference in taxon abundances or diversity of vaginal communities between term and preterm pregnancies. One possible reason for the contradictory results is the differ￾ence in the racial composition of our study cohorts. Previous work has demonstrated significant differences between vaginal microbial communities associated with race (11). In addition, the study by Romero et al. included only births before gestational week 34. Our cohort was clinically more heterogeneous and in￾cluded some women whose labor onset was nonspontaneous (e.g., caused by preterm premature rupture of membranes) (SI Appendix, Table S2). Another potentially important difference between our study and previous work, especially when consid￾ering patterns related to Gardnerella abundance, is the use of different PCR primers (18, 25). Vaginal Community Alteration After Delivery. We found that most women experienced a significant, abrupt, and durable alteration in the vaginal community beginning with the first sample col￾lected postdelivery. Compared with samples from other body sites in the postpartum period, the vaginal communities became more similar to the gut communities. The mechanisms underlying this potential convergence are unknown. The persistence of the disturbance, as well as its presence after both vaginal and caesarean deliveries, argues against simple translocation of stool communities to the vagina as the primary mechanism. The potential impact on maternal health of the postdelivery shift of the vaginal community also is unknown but includes the possibility that a persistently altered vaginal community might affect the outcome of a subsequent pregnancy if conception occurs too soon after delivery. A short interpregnancy interval (e.g., <12 mo) is associated with an increased risk of preterm birth. Whether an altered postdelivery vaginal community plays a contributing role warrants further study. Materials and Methods To enhance the reproducibility of our results, the reader will find all the code necessary to reproduce the analyses and figures presented in this article available at statweb.stanford.edu/∼susan/papers/PNASRR.html. This website includes pointers to the R markdown files, the output in html, and to the 11064 | www.pnas.org/cgi/doi/10.1073/pnas.1502875112 DiGiulio et al