Temporal and spatial variation of the human microbiota during pregnancy Daniel B. DiGiulioabc 1. Benjamin J. Callahan.d 1, Paul J. McMurdie. d, Elizabeth K Costelloe, Deirdre J. Lyell. f nna Robaczewska, b, Christine L Sun.e, Daniela S. A. Goltsman. Ronald J. Wong. 9, Gary Shaw.g, David K. Stevenson a 9, Susan P. Holmes,d and David A. Relman, b, c e. 2 March of Dimes Prematurity Research Center, Stanford U School of Medicine, Stanford, CA 94305; Department of Medicine, Stanford Universi tanford, CA 94305; Department of Microbiology and Imm tanford University School of Medicine, Stanford, CA 94305: Department of Obstetrics and Gynecology, Stanford University School of Medicin CA 94305; and9Department of Pediatrics, Stanford University School of Medicine Edited by Jeffrey L. Gordon, Washington University School of Medicine in St Louis, St Louis, MO, and approved July 17, 2015(received for review February Despite the critical role of the human microbiota in health, our with markers of inflammation and inversely with time to delivery Inderstanding of microbiota compositional dynamics during and (6-9). Preterm birth also is associated with bacterial vaginosis, a after pregnancy is incomplete We conducted a case-control study of community-wide alteration of the vaginal microbiota (10, 11) ese women, we analyzed bacterial taxonomic compo specimens collected prospectively and weekly during gestation and monthly after delivery from the vagina, distal gut, saliva, and tooth/ pregnancy using cultivation-independent techniques(14-19) um Linear mixed-effects modeling, medoid-based clustering, and collectively. these stu found the vaginal communities of Markov chain modeling were used to analyze community temporal pregnant women to be dominated by Lactobacillus species and trends, community structure, and vaginal community state transi. characterized by lower richness and diversity than in nonpregnarea remained remarkably stable at all four body sites during pr yy Pregnancy outcomes, one found preterm birth to be linked with (P>0.05 for trends over time). Prevalence of a Lactobacillus-pool ther intracommunity (alpha) diversity in the vagina(16), but the any specific community type or microbial taxon(17) more pronounced for subjects with st acompanied by elevated in the setting of pregnancy. The subgingival crevice has been with a set of 246 vaginal specimens from nine women(four of whom molecular approaches(22). Two studies of the fecal microbiota delivered preterm). Most women experienced a postdelivery distur reported differences in bacterial community structure betweer bance in the vaginal community characterized by a decrease in Lac- the first and third trimesters(23, 24); in each study, however, tobacillus species and an increase in diverse anaerobes such as Peptoniphilus,Prevotella,and Anaerococcus species. This disturbance samples were collected at only two time points. These limited was unrelated to gestational age at delivery and persisted for up to 1 y. These findings have important implications for predicting pre- Signifi mature labor, a major global health problem, and for understanding the potential impact of a persistent, altered postpartum microbiota The human indigenous microbial communities (microbiota) on maternal health, including outcomes of pregnancies following play critical roles in health and may be especially important for short interpregnancy intervals. mother and fetus during pregnancy. Using a case-control co- hort of 40 women, we characterized weekly variation in the 16S rRNA gene pregnancy I preterm birth I microbiom vaginal, gut, and oral microbiota during and after pregnancy premature labor Microbiota membership remained relatively stable at each body site during pregnancy. An altered vaginal microbial community human body harbors diverse, complex, and abundant rated by an analysis of samples from an aditional cohort of nine microbiota whose composition is determined largely by body women. We also discovered an abrupt change in the vaginal 2), The microbiota plays critical roles in health and in disease, 1y. our findings suggest that pregnancy outcomes might be ection from pathogens(3). Normal pregnancy represents a unique predicted by features of the transient, and dynamic state of altered anatomy, physiology, and authe nmune function. Preterm birth, i.e., before 37 wk of gestation d new reagents/ ccurs in 11%o of pregnancies and is the leading cause of neonatal D.A.R. analyzed data; death(4). In both term and preterm pregnancies, the interplay and DBD,BC, PJ.M,E.KC, CLS DSAG, SPH, and DAR wrote the pape between the microbiota and the host remains poorly understood. The Approximately 25%o of preterm births are associated with od cult microbial invasion of the amniotic cavity (5). Evidence Freely available online through the PNAS open access option. suggests that the most common source of invading microbes is Data deposition: Raw sequence data have been deposited at the Sequence Read Archive the host microbiota. In studies of amniotic fluid from women with preterm labor and either intact or ruptured branes. 16s'DBD and B.C. contributed equally to this work. ribosomal RNA (rRNA) sequences of known vaginal and To whom correspondence should be addressed. Email: relmanestanford.edu. oral indigenous bacterial species have been recovered in 15-50%0 ticlecontainssupportinginformationonlineatwww.pnasorg/lookup/suppw/doi:10. of cases, and their relative abundances have correlated directly as.1502875112/-/DCSupplementaL. 11060-11065|PNAs| September1,2015|vol.112|no.35 ww.pnas. org/cgi/doi/10. 1073/pnas. 1502875112Temporal and spatial variation of the human microbiota during pregnancy Daniel B. DiGiulioa,b,c,1, Benjamin J. Callahana,d,1, Paul J. McMurdiea,d, Elizabeth K. Costelloa,e, Deirdre J. Lyella,f, Anna Robaczewskaa,b,c, Christine L. Suna,e, Daniela S. A. Goltsmana,e, Ronald J. Wonga,g, Gary Shawa,g, David K. Stevensona,g, Susan P. Holmesa,d, and David A. Relmana,b,c,e,2 a March of Dimes Prematurity Research Center, Stanford University School of Medicine, Stanford, CA 94305; b Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305; c Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304; d Department of Statistics, Stanford University, Stanford, CA 94305; e Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; f Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305; and g Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305 Edited by Jeffrey I. Gordon, Washington University School of Medicine in St. Louis, St. Louis, MO, and approved July 17, 2015 (received for review February 11, 2015) Despite the critical role of the human microbiota in health, our understanding of microbiota compositional dynamics during and after pregnancy is incomplete. We conducted a case-control study of 49 pregnant women, 15 of whom delivered preterm. From 40 of these women, we analyzed bacterial taxonomic composition of 3,767 specimens collected prospectively and weekly during gestation and monthly after delivery from the vagina, distal gut, saliva, and tooth/ gum. Linear mixed-effects modeling, medoid-based clustering, and Markov chain modeling were used to analyze community temporal trends, community structure, and vaginal community state transi￾tions. Microbiota community taxonomic composition and diversity remained remarkably stable at all four body sites during pregnancy (P > 0.05 for trends over time). Prevalence of a Lactobacillus-poor vaginal community state type (CST 4) was inversely correlated with gestational age at delivery (P = 0.0039). Risk for preterm birth was more pronounced for subjects with CST 4 accompanied by elevated Gardnerella or Ureaplasma abundances. This finding was validated with a set of 246 vaginal specimens from nine women (four of whom delivered preterm). Most women experienced a postdelivery distur￾bance in the vaginal community characterized by a decrease in Lac￾tobacillus species and an increase in diverse anaerobes such as Peptoniphilus, Prevotella, and Anaerococcus species. This disturbance was unrelated to gestational age at delivery and persisted for up to 1 y. These findings have important implications for predicting pre￾mature labor, a major global health problem, and for understanding the potential impact of a persistent, altered postpartum microbiota on maternal health, including outcomes of pregnancies following short interpregnancy intervals. 16S rRNA gene | pregnancy | preterm birth | microbiome | premature labor The human body harbors diverse, complex, and abundant microbiota whose composition is determined largely by body site but also by host genetics, environmental exposures, and time (1, 2). The microbiota plays critical roles in health and in disease, including nutrient acquisition, immune programming, and pro￾tection from pathogens (3). Normal pregnancy represents a unique, transient, and dynamic state of altered anatomy, physiology, and immune function. Preterm birth, i.e., before 37 wk of gestation, occurs in 11% of pregnancies and is the leading cause of neonatal death (4). In both term and preterm pregnancies, the interplay between the microbiota and the host remains poorly understood. Approximately 25% of preterm births are associated with oc￾cult microbial invasion of the amniotic cavity (5). Evidence suggests that the most common source of invading microbes is the host microbiota. In studies of amniotic fluid from women with preterm labor and either intact or ruptured membranes, 16S ribosomal RNA (rRNA) sequences of known vaginal, gut, and oral indigenous bacterial species have been recovered in 15–50% of cases, and their relative abundances have correlated directly with markers of inflammation and inversely with time to delivery (6–9). Preterm birth also is associated with bacterial vaginosis, a community-wide alteration of the vaginal microbiota (10, 11) that increases the risk of preterm birth approximately twofold (12, 13). Several studies have examined the vaginal microbiota during pregnancy using cultivation-independent techniques (14–19). Collectively, these studies found the vaginal communities of pregnant women to be dominated by Lactobacillus species and characterized by lower richness and diversity than in nonpregnant women but with higher stability. Of the two studies that evaluated pregnancy outcomes, one found preterm birth to be linked with higher intracommunity (alpha) diversity in the vagina (16), but the other found no significant association between preterm birth and any specific community type or microbial taxon (17). Other (nonvaginal) body sites have been even less well studied in the setting of pregnancy. The subgingival crevice has been investigated only with cultivation (20, 21) or with taxon-specific molecular approaches (22). Two studies of the fecal microbiota reported differences in bacterial community structure between the first and third trimesters (23, 24); in each study, however, samples were collected at only two time points. These limited Significance The human indigenous microbial communities (microbiota) play critical roles in health and may be especially important for mother and fetus during pregnancy. Using a case-control co￾hort of 40 women, we characterized weekly variation in the vaginal, gut, and oral microbiota during and after pregnancy. Microbiota membership remained relatively stable at each body site during pregnancy. An altered vaginal microbial community was associated with preterm birth; this finding was corrobo￾rated by an analysis of samples from an additional cohort of nine women. We also discovered an abrupt change in the vaginal microbiota at delivery that persisted in some cases for at least 1 y. Our findings suggest that pregnancy outcomes might be predicted by features of the microbiota early in gestation. Author contributions: D.B.D., D.J.L., G.S., D.K.S., S.P.H., and D.A.R. designed research; D.B.D., A.R., and R.J.W. performed research; B.J.C., P.J.M., and S.P.H. contributed new reagents/ analytic tools; D.B.D., B.J.C., P.J.M., E.K.C., C.L.S., D.S.A.G., S.P.H., and D.A.R. analyzed data; and D.B.D., B.J.C., P.J.M., E.K.C., C.L.S., D.S.A.G., S.P.H., and D.A.R. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: Raw sequence data have been deposited at the Sequence Read Archive (SRP no. 288562). 1 D.B.D. and B.J.C. contributed equally to this work. 2 To whom correspondence should be addressed. Email: relman@stanford.edu. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1502875112/-/DCSupplemental. 11060–11065 | PNAS | September 1, 2015 | vol. 112 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1502875112