A4心 B SI Appendix, Fig S8). However, this increase was not universal, and some individual hibited a variable even a overall decrease (S/ Appendix, Fig. ry mode e1(i.e,vaginal n section) did not he non(SI Appendix, The role of other potential factors, such o4 as active breast feeding and the use of contraceptives or antibiotics, 8/05 could not be evaluated because those data were not collected after delivery. Stool and oral communities did not demonstrate similar degrees of change upon deliver The postdelivery shift in vaginal community diversity appeared to be particularly associated with significant declines in abun- dances of the most prevalent Lactobacillus species(SI Appendi Fig 4. The high sity vaginal cT4 was associated with earlier deliveries Fig S10 and Table S6). Lactobacilli were replaced by a diverse signed to the high-diversity cst 4 for the 33 subjects for whom at least 10 Angerococcus, which are typically anaerobic (S/Appendix, Fig SII reased prevalence of the diverse vaginal CST was significantly correlated with nities caused them to become significantly more similar to stool earlier delivery(P=1.1*10-4, Pearson; P=0.0147, Spearman) .(8) The fraction communities(P< 10-3, paired Wilcoxon test; SI Appendix, Fig. of specimens collected from subjects who delivered pre is shown by S12). Analogous changes in similarity were not observed when pecimen CST and the gestational period during which the specimens were comparing the vaginal communities with either of the oral co collected CST 4 specimens collected at any time during pregnancy were as. munities. We found no evidence for systematic interactions be- The validation dataset from the second group of subiects cor Ewalwation of a Second Group of Nine Women for Putative Preterm shift in vaginal community diversity (Si Appendi, F1g Delivery borated the finding from our discovery dataset of a poste Risk Factors Identified in Vaginal Communities from the First Group of 40 Women. We used the data from an additional group of nine Discussion women as a validation set for three preterm risk factors identi- Pregnancy is a time of dramatic host environmental remodeling tobacillus was used as a proxy);()CSt 4 with abundant Although it is believed that the indigenous microbiota plays a Gardnerella; and (ii) CST 4 with abundant Ureaplasma. In this fundamental role in both sh women had 4 samples environment, there are relatively few data on human microbiota collected during pregnancy, two of these women delivered pre- structure at m le body sites with high temporal resolution term.Given that four of nine women delivered preterm, this is a during this critical time. The importance of the microbiota fo neutral result for cst 4 alone when used as a sin for classification. However. when women with binary trait maternal and fetal health is suggested by links between conditions of altered microbial community structure in the vagina(e.g, bac- communities were evaluated for high abundance of either terial vaginosis)(12, 13)or in the oral cavity(e.g, periodontitis) Gardnerella or Ureaplasma, both community profiles were asso-(26)and preterm birth. In addition, studies of amniotic fluid from ciated with preterm birth. Two women with multiple Cst 4 les had abundant Ureaplasma, and both deliv Saliva with discovery dataset as exhibiting the sty features identified findings corroborate the microbial commu 谷4- Effect of Sampling Frequency on Study Findings We assessed whether sampling less frequently than weekly would have affected overall 40.5 wk: CST 3, 7. 5 wk: CST 5. 5.4 wk; however, CST 4 had a 2002040 much shorter persistence time, 2.6 wk. Therefore, a longer Time Vs Delivery(Weeks) Time vs Delivery(Weeks) interval would have disproportionately impacted the ability to de- Tooth/Gum tect excursions into CST 4 and to estimate the proportion of time that a woman spent in CST 4, the state most strongly associated 3 4 with preterm birth(Figs. 3 and 4A and SI Appendi, Ta The communities at other body sites did not cluster CSTs 8 rsistence times could not be extracted in the same manner However, the average distance between communities was assessed 2 1 s a function of their separation in time, or lag(SI Appendix, Fi S7). In the oral communities the increased similarity betweer samples collected closely in time decayed on a roughly monthly time scale. The results for the stool community were less clear Time vs Delivery(Weeks) Delivery Was Accompanied by a Strong Disruption of Vaginal Community Fig. 5. Alpha diversity pre-and postdelivery at each body site. The Shannon Structure in Most Women. Twenty-five of the 40 women in the first tive to delivery for the subjects in the first group of 40 subjects who provided subject group provided at least one postpartum sample, and sever Fig. SI). We found that delivery typically was accompanied by a shifts to higher diversity were observed postdelivery in vaginal communities ignificant change in vas mmunity structure, the clearest (P=5.2 x 10-paired Wilcoxon test)and, to a lesser extent, in the tooth/gur feature being a sharp increase in diversity after delivery(Fig. 5 and communities(P=0.014)but not in saliva or stool PNAS I September 1, 2015 I voL. 112 I no 35 I 11063Evaluation of a Second Group of Nine Women for Putative Preterm Risk Factors Identified in Vaginal Communities from the First Group of 40 Women. We used the data from an additional group of nine women as a validation set for three preterm risk factors identi￾fied in our discovery dataset: (i) CST 4 (for which <50% Lac￾tobacillus was used as a proxy); (ii) CST 4 with abundant Gardnerella; and (iii) CST 4 with abundant Ureaplasma. In this group of subjects, four women had multiple CST 4 samples collected during pregnancy; two of these women delivered pre￾term. Given that four of nine women delivered preterm, this is a neutral result for CST 4 alone when used as a simple binary trait for classification. However, when women with CST 4 vaginal communities were evaluated for high abundance of either Gardnerella or Ureaplasma, both community profiles were asso￾ciated with preterm birth. Two women with multiple CST 4 samples had abundant Ureaplasma, and both delivered preterm. One of these two women also had abundant Gardnerella (SI Appendix, Fig. S6). Although these numbers are very small, these findings corroborate the microbial community features identified in our discovery dataset as exhibiting the strongest associations with preterm birth. Effect of Sampling Frequency on Study Findings. We assessed whether sampling less frequently than weekly would have affected overall study findings. We found that most vaginal CSTs in the discovery dataset had persistence times of many weeks: CST 1, 48.1 wk; CST 2, 40.5 wk; CST 3, 7.5 wk; CST 5, 5.4 wk; however, CST 4 had a much shorter persistence time, 2.6 wk. Therefore, a longer sampling interval would have disproportionately impacted the ability to de￾tect excursions into CST 4 and to estimate the proportion of time that a woman spent in CST 4, the state most strongly associated with preterm birth (Figs. 3 and 4A and SI Appendix, Table S5). The communities at other body sites did not cluster into CSTs, so persistence times could not be extracted in the same manner. However, the average distance between communities was assessed as a function of their separation in time, or lag (SI Appendix, Fig. S7). In the oral communities the increased similarity between samples collected closely in time decayed on a roughly monthly time scale. The results for the stool community were less clear. Delivery Was Accompanied by a Strong Disruption of Vaginal Community Structure in Most Women. Twenty-five of the 40 women in the first subject group provided at least one postpartum sample, and several women provided samples for up to 1 y after delivery (SI Appendix, Fig. S1). We found that delivery typically was accompanied by a significant change in vaginal community structure, the clearest feature being a sharp increase in diversity after delivery (Fig. 5 and SI Appendix, Fig. S8). However, this increase was not universal, and some individual women exhibited a variable course or even an overall decrease in diversity (SI Appendix, Fig. S8). Delivery mode (i.e., vaginal versus cesarean section) did not affect this phenome￾non (SI Appendix, Fig. S9). The role of other potential factors, such as active breast feeding and the use of contraceptives or antibiotics, could not be evaluated because those data were not collected after delivery. Stool and oral communities did not demonstrate similar degrees of change upon delivery. The postdelivery shift in vaginal community diversity appeared to be particularly associated with significant declines in abun￾dances of the most prevalent Lactobacillus species (SI Appendix, Fig. S10 and Table S6). Lactobacilli were replaced by a diverse mixture of bacteria, including Peptoniphilus, Prevotella, and Anaerococcus, which are typically anaerobic (SI Appendix, Fig. S11 and Table S6). The postdelivery changes in the vaginal commu￾nities caused them to become significantly more similar to stool communities (P < 10−3 , paired Wilcoxon test; SI Appendix, Fig. S12). Analogous changes in similarity were not observed when comparing the vaginal communities with either of the oral com￾munities. We found no evidence for systematic interactions be￾tween the communities at different body sites during pregnancy. The validation dataset from the second group of subjects cor￾roborated the finding from our discovery dataset of a postdelivery shift in vaginal community diversity (SI Appendix, Fig. S13). Discussion Pregnancy is a time of dramatic host environmental remodeling as a mother accommodates and nourishes her developing fetus. Although it is believed that the indigenous microbiota plays a fundamental role in both shaping and responding to the host environment, there are relatively few data on human microbiota structure at multiple body sites with high temporal resolution during this critical time. The importance of the microbiota for maternal and fetal health is suggested by links between conditions of altered microbial community structure in the vagina (e.g., bac￾terial vaginosis) (12, 13) or in the oral cavity (e.g., periodontitis) (26) and preterm birth. In addition, studies of amniotic fluid from A B Fig. 4. The high-diversity vaginal CST 4 was associated with earlier deliveries and a higher likelihood of preterm birth in the first group of 40 women. (A) Gestational age at delivery is plotted against the fraction of vaginal specimens assigned to the high-diversity CST 4 for the 33 subjects for whom at least 10 vaginal specimens were collected. The dashed line indicates the linear fit. In￾creased prevalence of the diverse vaginal CST was significantly correlated with earlier delivery (P = 1.1 × 10−4 , Pearson; P = 0.0147, Spearman). (B) The fraction of specimens collected from subjects who delivered preterm is shown by specimen CST and the gestational period during which the specimens were collected. CST 4 specimens collected at any time during pregnancy were as￾sociated with a higher proportion of preterm birth. Fig. 5. Alpha diversity pre- and postdelivery at each body site. The Shannon index is plotted versus the time at which each specimen was collected rela￾tive to delivery for the subjects in the first group of 40 subjects who provided at least one postdelivery sample. Linear fits are shown for the predelivery (red) and postdelivery (green) specimens, considered separately. Significant shifts to higher diversity were observed postdelivery in vaginal communities (P = 5.2 × 10−6 paired Wilcoxon test) and, to a lesser extent, in the tooth/gum communities (P = 0.014) but not in saliva or stool. DiGiulio et al. PNAS | September 1, 2015 | vol. 112 | no. 35 | 11063 MICROBIOLOGY