The vaginal microbiome and preterm birth

Jennifer M. Fettweis; Myrna G. Serrano; J. Paul Brooks; David J. Edwards; Philippe H. Girerd; Hardik I. Parikh; Bernice Huang; Tom J. Arodz; Laahirie Edupuganti; Abigail L. Glascock; Jie Xu; Nicole R. Jimenez; Stephany C. Vivadelli; Stephen S. Fong; Nihar U. Sheth; Sophonie Jean; Vladimir Lee; Yahya A. Bokhari; Ana M. Lara; Shreni D. Mistry; Robert A. Duckworth; Steven P. Bradley; Vishal N. Koparde; X. Valentine Orenda; Sarah H. Milton; Sarah K. Rozycki; Andrey V. Matveyev; Michelle L. Wright; Snehalata V. Huzurbazar; Eugenie M. Jackson; Ekaterina Smirnova; Jonas Korlach; Yu-Chih Tsai; Molly R. Dickinson; Jamie L. Brooks; Jennifer I. Drake; Donald O. Chaffin; Amber L. Sexton; Michael G. Gravett; Craig E. Rubens; N. Romesh Wijesooriya; Karen D. Hendricks-Muñoz; Kimberly K. Jefferson; Jerome F. Strauss; Gregory A. Buck

2019

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes ‘omics’ data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

● The incidence of preterm birth exceeds 10% worldwide

● The resource features two comprehensive datasets of integrated microbiome and host functional properties measured longitudinally in pregnancy and the perinatal period (Fig. 1): (1) the Microbiome Study: Pregnancy Initiative (MOMS-PI) Preterm Birth (PTB) study dataset generated from a case–control study of 45 women predominantly of African ancestry, who delivered spontaneously preterm, and 90 case-matched women who delivered at term; and (2) the MOMS-PI Term Birth (TB) study dataset generated from an ethnically diverse retrospective cohort study of 90 women, who delivered at term or early term42

● Previous studies16,21 have shown that carriage of L. crispatus, which is negatively associated with Preterm birth (PTB), is more prevalent in women of European ancestry, and BV-associated bacterium 1 (BVAB1), which is positively associated with PTBs, is more common in women of African ancestry

● Our findings are consistent with a proposed framework in which there is a spectrum of vaginal microbiome states linked to risk for PTB, and that these states vary across populations

● In a complementary analysis of an ethnically diverse cohort of women who delivered at term or early term in the MOMS-PI TB study, we show differences in the longitudinal dynamics of the microbiome in women of African ancestry compared with women of European ancestry42

● Samples in which the largest proportion was less than 30% were not assigned a community state types (CSTs)/vagitype

● We developed a proof-of-concept model that suggests the presence of BVAB1, Prevotella cluster 2, S. amnii and TM7-H1 early in pregnancy may be useful for prediction of risk for PTB, in high-risk populations

Introduction There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States.

A vaginal microbiome with high species diversity, as observed with bacterial vaginosis, has been associated with increased risk for acquisition and transmission of sexually transmitted infections, PTB and pelvic inflammatory disease.

Several studies that focused on populations predominantly of European descent have associated Lactobacillus crispatus with a lower risk of PTB, and the finding was replicated in a cohort of predominantly African descent.

Population-specific studies may be required to assess the broad impacts of the vaginal microbiome on risk of PTB and to identify contributing taxa that may be carried by only a small subset of women

Methods Participants for this study were enrolled from women visiting maternity clinics in Virginia and Washington State.

Participants were enrolled at multiple sites in Washington State by the partner registry, the GAPPS which was at the time under the umbrella of Seattle Children’s IRB (FWA00002443), IRB Application number 12879.

Study protocols were harmonized across sites, and data and samples from participants enrolled in Washington State were distributed to the VCU site.

All study participants enrolled in Virginia and most participants enrolled at Washington State sites were enrolled in the RAMS Registry at VCU.

RAMS Registry protocols were approved at VCU

HM15528); GAPPS-associated sites ceded review to the VCU IRB through reliance agreements.

Additional samples were collected from the participants enrolled in Washington State, which are archived at the GAPPS.

Written informed consent was obtained from all participants and parental permission and assent were obtained for participating minors aged at least 15 years

Results The Multi-Omic Microbiome Study: Pregnancy Initiative The longitudinal iHMP study, the Multi-Omic Microbiome Study: Pregnancy Initiative (MOMS-PI) includes a total of 1,572 pregnancies, with 992 pregnancies from clinics associated with the Research Alliance for Microbiome Science (RAMS) Registry, based at Virginia Commonwealth University (VCU) in Virginia, and 580 pregnancies from sites associated with the Global Alliance to Prevent Prematurity and Stillbirth (GAPPS) in Washington State.

In the overall MOMS-PI study, the authors collected a total of 206,437 samples from pregnant women and their neonates, which have been archived in the RAMS Registry (Fig. 1c).

Both cohorts were selected from the phase 1 RAMS Registry cohort (n = 627).

Omics data types include 16S rRNA amplicon sequencing, metagenomic sequencing (MGS), metatranscriptomic sequencing (MTS), host cytokine assays and lipidomics. c, A total of 206,437 samples were collected at more than 7,000 visits from 1,572 pregnancies in the MOMS-PI study, and are archived in the RAMS Registry

Conclusion From a subset of 597 of the 1,572 pregnancies longitudinally sampled for the MOMS-PI study, the authors have generated omics data from more than 12,000 samples in one of the largest and most comprehensive multi-omic studies published to date.

The authors' analyses of longitudinal omics data from vaginal samples from 45 women who delivered preterm and 90 controls showed a signature of PTB in a cohort of women of predominantly African ancestry, including several taxa that have previously been implicated in adverse outcomes of pregnancy, including premature delivery, in addition to taxa that have not been previously linked to adverse pregnancy outcomes.

The authors' longitudinal modeling showed taxa associated with PTB tended to decrease in abundance in the vaginal environment throughout pregnancy, in women of African ancestry

This finding is consistent with previous observations that pregnancy is associated with reduced carriage of bacterial vaginosis-associated organisms.

The authors developed a proof-of-concept model that suggests the presence of BVAB1, Prevotella cluster 2, S. amnii and TM7-H1 early in pregnancy may be useful for prediction of risk for PTB, in high-risk populations.

The authors' MTS and MGS analyses supported the microbial signatures identified using 16S rRNA profiles

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes ‘omics’ data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

Approximately 15 million preterm births at less than 37 weeks of gestation occur annually worldwide1. Preterm birth (PTB) remains the second most common cause of neonatal death across the globe, and the most common cause of infant mortality in middle- and high-income economies2. The consequences of PTB persist from early childhood into adolescence and adulthood3,4. In the United States, striking population differences with respect to PTB exist, with women of African ancestry having a substantially larger burden of risk. The estimated annual cost of PTB in the United States alone is over US$26.2 billion5. Despite these statistics, there remains a paucity of effective strategies for predicting and preventing PTB.

Although maternal and fetal genetics, and gene–environment interactions, clearly play roles in determining the length of gestation, environmental factors, including the microbiome, are the most important contributors to PTB, particularly among women of African ancestry6. Microbe-induced inflammation resulting from urinary tract infection, sexually transmitted infections, including trichomoniasis, or bacterial vaginosis is thought to be a cause of PTB7,8. Ascension of microbes7,9 from the lower reproductive tract to the placenta, fetal membranes and uterine cavity, and hematogenous spread of periodontal pathogens from the mouth, have also been invoked to explain the up to 40–50% of preterm births that are associated with microbial etiologies10,11.

A homogeneous Lactobacillus-dominated microbiome has long been considered the hallmark of health in the female reproductive tract. In contrast, a vaginal microbiome with high species diversity, as observed with bacterial vaginosis, has been associated with increased risk for acquisition and transmission of sexually transmitted infections, PTB and pelvic inflammatory disease12,13,14,15. However, many asymptomatic healthy women have diverse vaginal microbiota. More refined approaches are needed to assess risk, promote health, and prevent and treat disease16,17,18,19,20,21.

Recent reports of the microbiome in pregnant women22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 have suggested that the composition of the vaginal microbiome has a significant population-specific impact on PTB risk. Several studies that focused on populations predominantly of European descent22,23,24,25 have associated Lactobacillus crispatus with a lower risk of PTB, and the finding was replicated in a cohort of predominantly African descent25. As first reported by Ravel et al.16, and subsequently confirmed in other studies21,40, the vaginal microbiome profiles of women of African and European ancestry differ significantly. Although distinct taxa have been associated with PTB in women of African ancestry in some studies25,26, others have not found significant associations27,30. Women of African descent are less likely to exhibit vaginal lactobacilli, frequently have vaginal L. crispatus predominance and are more likely to exhibit increased vaginal microbial diversity16,21. Consequently, population-specific studies may be required to assess the broad impacts of the vaginal microbiome on risk of PTB and to identify contributing taxa that may be carried by only a small subset of women.

Participants for this study were enrolled from women visiting maternity clinics in Virginia and Washington State. All study procedures involving human subjects were reviewed and approved by the institutional review board at VCU (IRB no. HM15527). Participants were enrolled at multiple sites in Washington State by our partner registry, the GAPPS (see www.gapps.org) which was at the time under the umbrella of Seattle Children’s IRB (FWA00002443), IRB Application number 12879. Study protocols were harmonized across sites, and data and samples from participants enrolled in Washington State were distributed to the VCU site. Samples were collected from ten maternal body sites (that is, vaginal, cervical, buccal and rectal mucosa, blood, urine, chest, dominant palm, antecubital fossa and nares), five types of birth products (that is cord blood, amniotic fluid, placental membranes, placental tissue and umbilical cord) and seven infant body sites (that is, buccal and rectal mucosa, meconium/stool, chest, right palm, nares and respiratory secretions if intubated). All study participants enrolled in Virginia and most participants enrolled at Washington State sites were also enrolled in the RAMS Registry at VCU. RAMS Registry protocols were approved at VCU (IRB no. HM15528); GAPPS-associated sites ceded review to the VCU IRB through reliance agreements. Additional samples were collected from the participants enrolled in Washington State, which are archived at the GAPPS. The present study was performed with compliance with all relevant ethical regulations. Written informed consent was obtained from all participants and parental permission and assent were obtained for participating minors aged at least 15 years.

The Multi-Omic Microbiome Study: Pregnancy Initiative The longitudinal iHMP study, the Multi-Omic Microbiome Study: Pregnancy Initiative (MOMS-PI) includes a total of 1,572 pregnancies, with 992 pregnancies from clinics associated with the Research Alliance for Microbiome Science (RAMS) Registry, based at Virginia Commonwealth University (VCU) in Virginia, and 580 pregnancies from sites associated with the Global Alliance to Prevent Prematurity and Stillbirth (GAPPS) in Washington State. The resource features two comprehensive datasets of integrated microbiome and host functional properties measured longitudinally in pregnancy and the perinatal period (Fig. 1): (1) the MOMS-PI Preterm Birth (PTB) study dataset generated from a case–control study of 45 women predominantly of African ancestry, who delivered spontaneously preterm, and 90 case-matched women who delivered at term; and (2) the MOMS-PI Term Birth (TB) study dataset generated from an ethnically diverse retrospective cohort study of 90 women, who delivered at term or early term42. From a selection of 12,039 samples from 597 pregnancies, we generated: (1) 16S ribosomal RNA (rRNA) taxonomic profiles from 6,452 samples from pregnant women and 2,753 samples from neonates; (2) metagenome profiles from 930 samples from pregnant women and 146 samples from neonates; (3) metatranscriptome profiles from 297 samples from pregnant women; (4) cytokine profiles from 1,223 samples from pregnant women and 173 samples from neonates; and (5) lipid profiles from 63 samples from pregnant women. In the overall MOMS-PI study, we collected a total of 206,437 samples from pregnant women and their neonates, which have been archived in the RAMS Registry (see ramsregistry.vcu.edu) (Fig. 1c). Comprehensive health history and outcome data were also collected longitudinally. a, An overview of the study designs for the MOMS-PI PTB study (45 spontaneous preterm (sPTB) cases and 90 term controls) and the MOMS-PI TB study (90 women who delivered at term or early term and their neonates). Both cohorts were selected from the phase 1 RAMS Registry cohort (n = 627). b, Omics data were generated from samples from the MOMS-PI PTB and MOMS-PI TB studies and 384 additional pregnancies from the overall MOMS-PI cohort. Samples from the 12 women who were selected for both the MOMS-PI PTB study and the MOMS-PI TB study are depicted under both studies. Omics data types include 16S rRNA amplicon sequencing, metagenomic sequencing (MGS), metatranscriptomic sequencing (MTS), host cytokine assays and lipidomics. c, A total of 206,437 samples were collected at more than 7,000 visits from 1,572 pregnancies in the MOMS-PI study, and are archived in the RAMS Registry.

From a subset of 597 of the 1,572 pregnancies longitudinally sampled for the MOMS-PI study, we have generated omics data from more than 12,000 samples in one of the largest and most comprehensive multi-omic studies published to date. In addition, our analyses of longitudinal omics data from vaginal samples from 45 women who delivered preterm and 90 controls showed a signature of PTB in a cohort of women of predominantly African ancestry, including several taxa that have previously been implicated in adverse outcomes of pregnancy, including premature delivery22,26,57,58,59, in addition to taxa that have not been previously linked to adverse pregnancy outcomes. Women of African ancestry have a greatly increased risk of PTB compared with women of European ancestry60. Previous studies16,21 have shown that carriage of L. crispatus, which is negatively associated with PTB (see Fig. 2 and refs. 22,23,24,25), is more prevalent in women of European ancestry, and BVAB1, which is positively associated with PTBs, is more common in women of African ancestry. Thus, our findings are consistent with a proposed framework in which there is a spectrum of vaginal microbiome states linked to risk for PTB, and that these states vary across populations. Our longitudinal modeling showed taxa associated with PTB tended to decrease in abundance in the vaginal environment throughout pregnancy, particularly in women of African ancestry. This finding is consistent with previous observations that pregnancy is associated with reduced carriage of bacterial vaginosis-associated organisms21,27,57. Considering that an adverse pregnancy outcome may be caused by ascension of pathogenic microbes, this trend suggests that the microbiome composition early in pregnancy may be most useful in the prediction of adverse outcomes. In a complementary analysis of an ethnically diverse cohort of women who delivered at term or early term in the MOMS-PI TB study, we show differences in the longitudinal dynamics of the microbiome in women of African ancestry compared with women of European ancestry42. In the present study, we developed a proof-of-concept model that suggests the presence of BVAB1, Prevotella cluster 2, S. amnii and TM7-H1 early in pregnancy may be useful for prediction of risk for PTB, particularly in high-risk populations. It is possible that BVAB1, Prevotella cluster 2, S. amnii and TM7-H1, and other taxa may have roles in the causation of PTB. As BVAB1 and TM7-H1 had not been cultivated or genetically characterized, we assembled their genomes from MGS data to search for clues to their pathogenic potential. We previously characterized the genome of S. amnii, identified potential cytotoxin genes and showed that cultured bacteria kill eukaryotic cells in vitro61. Although culture is not available for BVAB1 or TM7-H1, genomic factors identified in their genomes can now be genetically amplified and recombined into heterologous reporter systems and tested for pathogenic activity. Our MTS and MGS analyses supported the microbial signatures identified using 16S rRNA profiles.

This study was funded by NIH grant nos

UH3AI083263 and U54HD080784 to G.A.B., K.K.J. and J.F.S. and was supported with funds from the Common Fund, the National Center for Complementary and Integrative Health, and the Office of Research on Women’s Health

Other grants that provided partial support include a GAPPS BMGF PPB grant to G.A.B. and J.M.F., and NIH grants R21HD092965 to J.M.F. and E

T.A. was supported by NSF grant 1453658

N.R.J. was supported by grant no

There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes ‘omics’ data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry

In the present study, we report a community resource that includes ‘omics’ data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa

Women with any of the following conditions were excluded from sampling at a given visit:. We initially selected 47 preterm cases of singleton, non-medically indicated PTBs from women who delivered between 23 weeks of gestation and 36 weeks and 6 days of gestation, and were selected from the 627 pregnancies in the Virginia arm of the study for whom gestational age at delivery was available at the time of the study design (phase 1 RAMS Registry cohort). From the phase 1 RAMS Registry cohort, 82 delivered before 37 weeks of gestation

Two of the 47 PTBs did not have 16S rRNA that passed quality control, so these PTB samples and their controls were excluded from the MOMS-PI PTB study. From an early subset of women who delivered in the MOMS-PI, we selected 90 pregnancies, including 41 women of European descent and 49 of African descent, who experienced term (≥39 weeks of gestation) or early term birth (between 37 weeks of gestation and 38 weeks and 6 days of gestation). An early pilot study was selected from a subset of 69 women in the MOMS-PI cohort from whom vaginal samples had been collected before 14 weeks of gestation

From an early subset of women who delivered in the MOMS-PI, we selected 90 pregnancies, including 41 women of European descent and 49 of African descent, who experienced term (≥39 weeks of gestation) or early term birth (between 37 weeks of gestation and 38 weeks and 6 days of gestation). An early pilot study was selected from a subset of 69 women in the MOMS-PI cohort from whom vaginal samples had been collected before 14 weeks of gestation. Targeted vaginal lipidomic profiles, vaginal cytokine profiles and vaginal 16S rRNA taxonomic profiles were generated

The samples were randomized at the PCR stage and again at the sequencing stage. Samples were multiplexed (384 samples per run) and sequenced on our Illumina MiSeq sequencer using 600 cycles creating 2 × 300 bp paired-end reads to generate a depth of coverage of at least 50,000 reads per sample. The raw sequence data were demultiplexed into sample paired-end fastq files based on unique barcode sequences using a customized Python script

DNA libraries were prepared using KAPA Biosystems HyperPlus Library Kit and sequenced on our Illumina HiSeq 4000 (2 × 150 b PE). We sequenced all available vaginal samples for the PTB and TB cohorts, multiplexed 24 samples per lane and obtained ~1–2 × 107 150-nucleotide reads per sample. For MGS, we subjected 555 vaginal samples to sequencing and 542/555 (97.7%) had a minimum of 100,000 post-quality control (QC)-filtered read pairs

We sequenced all available vaginal samples for the PTB and TB cohorts, multiplexed 24 samples per lane and obtained ~1–2 × 107 150-nucleotide reads per sample. For MGS, we subjected 555 vaginal samples to sequencing and 542/555 (97.7%) had a minimum of 100,000 post-quality control (QC)-filtered read pairs. For MTS, we selected samples in the second trimester and the last collected sample for sequencing

If samples were not available in the second trimester, samples before and after the targeted second trimester window were selected. RNA was extracted from a total of 337 samples, of which 243 (72.10%) had at least 10 ng μl–1 of RNA and were submitted for sequencing. Of these 242/243 (99.6%) met our minimum of 100,000 post-QC-filtered read pairs

RNA was extracted from a total of 337 samples, of which 243 (72.10%) had at least 10 ng μl–1 of RNA and were submitted for sequencing. Of these 242/243 (99.6%) met our minimum of 100,000 post-QC-filtered read pairs. The rRNA-depleted messenger RNA was prepared for sequencing by constructing complementary DNA libraries using the KAPA Biosystems KAPA RNA HyperPrep Kit

The rRNA-depleted messenger RNA was prepared for sequencing by constructing complementary DNA libraries using the KAPA Biosystems KAPA RNA HyperPrep Kit. Indexed complementary DNA libraries were pooled in equimolar amounts and sequenced on the Illumina HiSeq 4000 instrument, running four multiplexed samples per lane, with an average yield of ~100 Gb per lane, sufficient to provide >100× coverage of the expression profiles of the most abundant 15–20 taxa in a sample. Raw sequence data were demultiplexed into sample-specific fastq files using bcl2fastq conversion software from Illumina

Where necessary, gaps were closed by sequencing of PCR amplicons using primers directed to contig ends. Coverage of the final genome averaged over 40× and completion was confirmed by the presence of all 40 highly conserved marker genes commonly used to assess genome assemblies69. Genome sequences were annotated with in-house pipelines using Prokka and ASGARD66

Chimeras were excluded, only primary alignments were mapped and duplicate reads were excluded. Paired MGS and MTS data were available for 41 women who delivered preterm and 81 term controls. To confirm that the microbial signatures of PTB identified using 16S rRNA data were not attributable to protocol choices or protocol biases, we used MGS and MTS data to support these findings

We analyzed one time point per participant, with a mean gestational age of sampling at 25 weeks for the preterm cohort and 26 weeks for the full-term cohort, and used a global scaling approach to normalize to all genes in the 56 taxa in our database. DESeq2 (ref. 71) was used to compare term and preterm cohorts using an organism-independent, global-scaling approach; genes with fewer than 1,000 total mapped genes across samples were excluded from analysis. Note that, with global scaling, it is not possible to differentiate differences caused by differential abundance and those due to differential expression in MTS data

Univariate analysis to identify taxa very different in abundance in PTB and TB cohorts. We analyzed vaginal 16S rRNA profiles from 135 participants, 45 who delivered preterm and 90 who delivered term. The microbiome profile of the earliest sample from each of these women was used in this analysis

We constructed a linear predictive model of PTB as follows: from the full cohort, we selected subjects who had at least one vaginal 16S rRNA sample early in the pregnancy, in days 42–167 (inclusive) of gestation. A total of 31 PTB and 59 TB subjects had at least one sample in this time window; if multiple samples were present in that window, we used the earliest sample. We first filtered out low-abundant species in this dataset: 25 passed the selection criteria

We performed a systematic literature review to identify previous studies that used taxonomic markers to assess the vaginal microbiome and PTB22,23,24,25,26,27,30,31,32,33,34,35,36,37,57. Three studies included cohorts of pregnant women who were predominantly of African descent, were at high risk for PTB and had publicly available 16S rRNA reads25,27,30 (bioProjects: PRJNA242473, PRJNA294119, PRJNA393472-(University of Alabama (UAB) cohort). De-identified clinical data for bioProject PRJNA294119 was kindly made available by Molly Stout

Controls were matched 2:1 to cases from the same original study, based on the matching criteria used in the present study, as far as possible. This included ancestry/ethnicity for all three studies and age for two of the studies. Age was not available the Stout et al study27 (that is, PRJNA294119)

Age was not available the Stout et al study27 (that is, PRJNA294119). Annual household income was not used for matching because it was not available for any of the three studies. In the high-risk cohort from the Callahan et al.25 study (that is, PRJNA393472-UAB cohort), there was not a sufficient number of controls who met the criteria, and therefore cases for the available controls to preserve the matching ratio

Selection of rematched cases/controls was performed blinded to other data. We applied a standardized bioinformatics pipeline to the 16S rRNA reads from the three original studies to harmonize with the present study as far as possible. Reads were downloaded from the SRA (bioProjects: PRJNA242473, PRJNA294119, PRJNA393472-UAB cohort)

For all studies, only samples with a minimum of 1,000 quality-filtered reads were used in downstream analyses. A total of 85 of the 149 samples in the Stout et al study met this QC threshold and 348 of 349 samples in the Romero et al.16 study. All 1,280 samples from the Callahan et al UAB cohort25 had a minimum of 1,000 quality-filtered reads

A total of 85 of the 149 samples in the Stout et al study met this QC threshold and 348 of 349 samples in the Romero et al.16 study. All 1,280 samples from the Callahan et al UAB cohort25 had a minimum of 1,000 quality-filtered reads. Clinical data were available for two additional samples in the Callahan et al UAB cohort for which sequence data were not available at the SRA

In the present study, we report a community resource that includes samples collected longitudinally during 1,572 pregnancies of women from diverse ancestries, and omics data generated from samples collected from 597 pregnancies in a collaborative effort under the umbrella of the National Institutes of Health’s integrative Human Microbiome Project (iHMP)41. Furthermore, we provided an analysis of the longitudinal, comprehensive, multi-omic profiling of vaginal samples from 45 women who experienced spontaneous PTB and 90 case-matched controls, in a cohort of women of predominantly African ancestry. In an initial analysis of this dataset, which represents one of the largest and most comprehensive studies of the vaginal microbiome to date, we identified vaginal microbial signatures in women who went on to experience PTB

The longitudinal iHMP study, the Multi-Omic Microbiome Study: Pregnancy Initiative (MOMS-PI) includes a total of 1,572 pregnancies, with 992 pregnancies from clinics associated with the Research Alliance for Microbiome Science (RAMS) Registry, based at Virginia Commonwealth University (VCU) in Virginia, and 580 pregnancies from sites associated with the Global Alliance to Prevent Prematurity and Stillbirth (GAPPS) in Washington State. The resource features two comprehensive datasets of integrated microbiome and host functional properties measured longitudinally in pregnancy and the perinatal period (Fig. 1): (1) the MOMS-PI Preterm Birth (PTB) study dataset generated from a case–control study of 45 women predominantly of African ancestry, who delivered spontaneously preterm, and 90 case-matched women who delivered at term; and (2) the MOMS-PI Term Birth (TB) study dataset generated from an ethnically diverse retrospective cohort study of 90 women, who delivered at term or early term42. From a selection of 12,039 samples from 597 pregnancies, we generated: (1) 16S ribosomal RNA (rRNA) taxonomic profiles from 6,452 samples from pregnant women and 2,753 samples from neonates; (2) metagenome profiles from 930 samples from pregnant women and 146 samples from neonates; (3) metatranscriptome profiles from 297 samples from pregnant women; (4) cytokine profiles from 1,223 samples from pregnant women and 173 samples from neonates; and (5) lipid profiles from 63 samples from pregnant women

The resource features two comprehensive datasets of integrated microbiome and host functional properties measured longitudinally in pregnancy and the perinatal period (Fig. 1): (1) the MOMS-PI Preterm Birth (PTB) study dataset generated from a case–control study of 45 women predominantly of African ancestry, who delivered spontaneously preterm, and 90 case-matched women who delivered at term; and (2) the MOMS-PI Term Birth (TB) study dataset generated from an ethnically diverse retrospective cohort study of 90 women, who delivered at term or early term42. From a selection of 12,039 samples from 597 pregnancies, we generated: (1) 16S ribosomal RNA (rRNA) taxonomic profiles from 6,452 samples from pregnant women and 2,753 samples from neonates; (2) metagenome profiles from 930 samples from pregnant women and 146 samples from neonates; (3) metatranscriptome profiles from 297 samples from pregnant women; (4) cytokine profiles from 1,223 samples from pregnant women and 173 samples from neonates; and (5) lipid profiles from 63 samples from pregnant women. In the overall MOMS-PI study, we collected a total of 206,437 samples from pregnant women and their neonates, which have been archived in the RAMS Registry (see ramsregistry.vcu.edu) (Fig. 1c)

From a selection of 12,039 samples from 597 pregnancies, we generated: (1) 16S ribosomal RNA (rRNA) taxonomic profiles from 6,452 samples from pregnant women and 2,753 samples from neonates; (2) metagenome profiles from 930 samples from pregnant women and 146 samples from neonates; (3) metatranscriptome profiles from 297 samples from pregnant women; (4) cytokine profiles from 1,223 samples from pregnant women and 173 samples from neonates; and (5) lipid profiles from 63 samples from pregnant women. In the overall MOMS-PI study, we collected a total of 206,437 samples from pregnant women and their neonates, which have been archived in the RAMS Registry (see ramsregistry.vcu.edu) (Fig. 1c). Comprehensive health history and outcome data were also collected longitudinally

Comprehensive health history and outcome data were also collected longitudinally. a, An overview of the study designs for the MOMS-PI PTB study (45 spontaneous preterm (sPTB) cases and 90 term controls) and the MOMS-PI TB study (90 women who delivered at term or early term and their neonates). Both cohorts were selected from the phase 1 RAMS Registry cohort (n = 627). b, Omics data were generated from samples from the MOMS-PI PTB and MOMS-PI TB studies and 384 additional pregnancies from the overall MOMS-PI cohort

a, An overview of the study designs for the MOMS-PI PTB study (45 spontaneous preterm (sPTB) cases and 90 term controls) and the MOMS-PI TB study (90 women who delivered at term or early term and their neonates). Both cohorts were selected from the phase 1 RAMS Registry cohort (n = 627). b, Omics data were generated from samples from the MOMS-PI PTB and MOMS-PI TB studies and 384 additional pregnancies from the overall MOMS-PI cohort. Samples from the 12 women who were selected for both the MOMS-PI PTB study and the MOMS-PI TB study are depicted under both studies

Both cohorts were selected from the phase 1 RAMS Registry cohort (n = 627). b, Omics data were generated from samples from the MOMS-PI PTB and MOMS-PI TB studies and 384 additional pregnancies from the overall MOMS-PI cohort. Samples from the 12 women who were selected for both the MOMS-PI PTB study and the MOMS-PI TB study are depicted under both studies. Omics data types include 16S rRNA amplicon sequencing, metagenomic sequencing (MGS), metatranscriptomic sequencing (MTS), host cytokine assays and lipidomics. c, A total of 206,437 samples were collected at more than 7,000 visits from 1,572 pregnancies in the MOMS-PI study, and are archived in the RAMS Registry

Samples from the 12 women who were selected for both the MOMS-PI PTB study and the MOMS-PI TB study are depicted under both studies. Omics data types include 16S rRNA amplicon sequencing, metagenomic sequencing (MGS), metatranscriptomic sequencing (MTS), host cytokine assays and lipidomics. c, A total of 206,437 samples were collected at more than 7,000 visits from 1,572 pregnancies in the MOMS-PI study, and are archived in the RAMS Registry. Vaginal microbiome profiles show PTB-associated trends

Prevotella cluster 2 comprises several closely related taxa of that genus47, including Prevotella timonensis and Prevotella buccalis. Through an analysis of samples collected from the 31 PTB and 59 TB subjects who had samples collected early (6–24 weeks of gestational age) in pregnancy, we identified two additional taxa that were significantly increased in PTB samples: Megasphaera type 1 and TM7-H1 (that is, BVAB-TM7) (see Extended Data Fig. 3). Both of these taxa have been previously associated with adverse conditions of vaginal health12

Overall, our observations are consistent with previous reports of dynamic changes in the vaginal microbiome in pregnancy18,48,49 and results from the MOMS-PI TB study, which show that the dynamics of vaginal microbiome differ by ancestry, with women of African ancestry exhibiting a more pronounced decrease in microbial diversity throughout the course of a term pregnancy42. A total of 496 longitudinal vaginal samples from participants in the MOMS-PI PTB study (see Fig. 1b) were subjected to metagenomic sequencing (MGS) and a subset of 243 samples was subjected to metatranscriptomic sequencing (MTS) (see Extended Data Fig. 4). At the pathway level, the functional and metabolic potentials of the microbial communities were largely conserved, with the exception of L. crispatus-dominated samples, which exhibited a much higher proportional metabolic potential and transcriptional activity of the UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) biosynthesis pathway (see Extended Data Fig. 5)

Although proportional differences were observed across detection methods, there was concordance in detection of taxa using 16S rRNA profiles, MGS and MTS (see Extended Data Fig. 6). Paired MGS and MTS data were available for 41 women who delivered preterm and for 81 term controls. Thus, we analyzed a single time point per participant, with a mean gestational age of sampling at 25 weeks for the preterm cohort and 26 weeks for the full-term cohort; we also used a global scaling approach to normalize to all genes in the 56 taxa in our database

L. jensenii, L. gasseri, L. iners and G. vaginalis had relatively few genes that showed very different transcript levels between the term and preterm cohorts (Fig. 4). Analysis was performed by mapping reads from PTB (n = 41) and TB (n = 81) samples to a custom database of genomes representing 56 taxa. Comparative analysis was performed with DESeq2 using a global scaling approach

Furthermore, BVAB1 was negatively correlated with IP-10/CXCL10 in these samples. a,b, Cytokine abundance in vaginal samples from women who experienced TB (n = 90) (a) or PTB (n = 41) (b) were subjected to an integrative sCCA using log-transformed cytokine levels and log-transformed taxonomic profiling data (see Methods). Blue circles represent bacterial taxa and red diamonds represent cytokines

For example, the present study confirms an association between Sneathia sanguinegens and PTB, which was reported as significant (P < 0.05) before adjustment for multiple testing by Romero et al.30. Although there were only five spontaneous PTB cases reanalyzed in the Stout et al cohort27, and only ten in the reanalyzed Callahan et al.25 cohort, we observed high concordance in the directionality of differences in abundance levels of preterm and term groups between these cohorts and the present study. We were also able to confirm that BVAB1, Megasphaera phylotype 1 and Sneathia species were elevated in a preterm cohort, which Nelson et al.26 previously reported as related to an increased risk for PTBs among women reporting a prior preterm delivery

We also confirmed that L. iners and G. vaginalis, which were identified as vaginal microbial signatures associated with PTB in low-risk cohorts25, did not generalize to cohorts of African ancestry. From a subset of 597 of the 1,572 pregnancies longitudinally sampled for the MOMS-PI study, we have generated omics data from more than 12,000 samples in one of the largest and most comprehensive multi-omic studies published to date. In addition, our analyses of longitudinal omics data from vaginal samples from 45 women who delivered preterm and 90 controls showed a signature of PTB in a cohort of women of predominantly African ancestry, including several taxa that have previously been implicated in adverse outcomes of pregnancy, including premature delivery22,26,57,58,59, in addition to taxa that have not been previously linked to adverse pregnancy outcomes

From a subset of 597 of the 1,572 pregnancies longitudinally sampled for the MOMS-PI study, we have generated omics data from more than 12,000 samples in one of the largest and most comprehensive multi-omic studies published to date. In addition, our analyses of longitudinal omics data from vaginal samples from 45 women who delivered preterm and 90 controls showed a signature of PTB in a cohort of women of predominantly African ancestry, including several taxa that have previously been implicated in adverse outcomes of pregnancy, including premature delivery22,26,57,58,59, in addition to taxa that have not been previously linked to adverse pregnancy outcomes. Women of African ancestry have a greatly increased risk of PTB compared with women of European ancestry60

MOMS-PI PTB study case–control design. We initially selected 47 preterm cases of singleton, non-medically indicated PTBs from women who delivered between 23 weeks of gestation and 36 weeks and 6 days of gestation, and were selected from the 627 pregnancies in the Virginia arm of the study for whom gestational age at delivery was available at the time of the study design (phase 1 RAMS Registry cohort). From the phase 1 RAMS Registry cohort, 82 delivered before 37 weeks of gestation

MOMS-PI TB study design. From an early subset of women who delivered in the MOMS-PI, we selected 90 pregnancies, including 41 women of European descent and 49 of African descent, who experienced term (≥39 weeks of gestation) or early term birth (between 37 weeks of gestation and 38 weeks and 6 days of gestation). Early pregnancy study design

Early pregnancy study design. An early pilot study was selected from a subset of 69 women in the MOMS-PI cohort from whom vaginal samples had been collected before 14 weeks of gestation. Targeted vaginal lipidomic profiles, vaginal cytokine profiles and vaginal 16S rRNA taxonomic profiles were generated

The rRNA-depleted messenger RNA was prepared for sequencing by constructing complementary DNA libraries using the KAPA Biosystems KAPA RNA HyperPrep Kit. Indexed complementary DNA libraries were pooled in equimolar amounts and sequenced on the Illumina HiSeq 4000 instrument, running four multiplexed samples per lane, with an average yield of ~100 Gb per lane, sufficient to provide >100× coverage of the expression profiles of the most abundant 15–20 taxa in a sample. Whole shotgun metagenomic/metatranscriptomic data pre-processing

Replication cohort processing of 16S rRNA data. We applied a standardized bioinformatics pipeline to the 16S rRNA reads from the three original studies to harmonize with the present study as far as possible. Reads were downloaded from the SRA (bioProjects: PRJNA242473, PRJNA294119, PRJNA393472-UAB cohort)

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For vaginal samples, the dominant bacterial taxon is one clinically meaningful measure by which to stratify samples16,46. Women who went on to deliver at term were more likely to exhibit L. crispatus predominance in the vaginal microbiome (P = 0.014, Fig. 2a,b, and see Supplementary Table 2a and Extended Data Fig. 1), paralleling earlier observations17,22,23,24,25. A Markov chain analysis to assess vagitype changes throughout pregnancy did not reveal statistically significant differences in transition rates between case and control groups

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The resulting model incorporates four taxa: S. amnii, BVAB1, Prevotella cluster 2 and TM7-H1, which are all positively correlated with PTB (Fig. 2d, and see Extended Data Fig. 3b,c). The discriminative model is significant (P = 0.0024) and has an expected sensitivity of 77.4%, specificity of 76.3%, and an area under the receiver operating characteristics (AUROC) curve of 0.723 for samples not used during training. This model, based on microbiome composition data, had 5–7% greater sensitivity and specificity than a model constructed using only clinical variables with a slight reduction in the AUROC curve (that is, 0.723 versus 0.764)

A network analysis of these four taxa (Fig. 2c) shows them to be positively correlated with taxa associated with vaginal dysbiosis. A stratification of the longitudinal GAMM analysis by ancestry (Fig. 3b, and see Supplementary Fig. 1b) showed that, over the duration of pregnancy, women of African ancestry who delivered preterm experienced significant decreases in the prevalence of A. vaginae (P = 0.0011), BVAB1 (P = 0.0003), G. vaginalis (P = 0.0002), P. amnii (P= 0.0013), S. amnii (P = 0.0219) and TM7-H1 (P = 0.0014). Women of African ancestry (AA) who delivered at term exhibited fewer changes in the modeled taxa throughout pregnancy, although decreases in A. vaginae (P = 0.0001) and G. vaginalis (P = 0.0003) and an increase in L. iners (P = 0.0404) were observed

A stratification of the longitudinal GAMM analysis by ancestry (Fig. 3b, and see Supplementary Fig. 1b) showed that, over the duration of pregnancy, women of African ancestry who delivered preterm experienced significant decreases in the prevalence of A. vaginae (P = 0.0011), BVAB1 (P = 0.0003), G. vaginalis (P = 0.0002), P. amnii (P= 0.0013), S. amnii (P = 0.0219) and TM7-H1 (P = 0.0014). Women of African ancestry (AA) who delivered at term exhibited fewer changes in the modeled taxa throughout pregnancy, although decreases in A. vaginae (P = 0.0001) and G. vaginalis (P = 0.0003) and an increase in L. iners (P = 0.0404) were observed. Women of European ancestry (EA) generally exhibited stable microbiome profiles during pregnancy, although an increase in prevalence of G. vaginalis (P = 0.0401) was noted for women who delivered preterm

Women of African ancestry (AA) who delivered at term exhibited fewer changes in the modeled taxa throughout pregnancy, although decreases in A. vaginae (P = 0.0001) and G. vaginalis (P = 0.0003) and an increase in L. iners (P = 0.0404) were observed. Women of European ancestry (EA) generally exhibited stable microbiome profiles during pregnancy, although an increase in prevalence of G. vaginalis (P = 0.0401) was noted for women who delivered preterm. G. vaginalis has been previously reported as a microbial signature for PTB in cohorts of women of predominantly European ancestry25

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Interestingly, 12.55% of the G. vaginalis genes analyzed using MGS data were significantly higher in the term cohort, whereas only one gene (that is, 0.02% of genes analyzed) that was identified as a hypothetical protein was higher (Padj < 0.05) in the preterm cohort. We found the overall relative transcriptional rate of G. vaginalis was higher in preterm samples compared with term samples, using a calculated ratio of the proportion of reads mapped to genes in G. vaginalis reference genomes by MTS to reads mapped by MGS (Wilcoxon’s, P < 0.05; see Extended Data Fig. 7b,c). Previous studies have suggested that PTB risk differs with carriage of different G. vaginalis clades25

Host cytokine expression in PTB. Of the nine cytokine levels examined in the present study (interleukin (IL)-1β, IL-6, IL-8, eotaxin, tumor necrosis factor (TNF)-α, IL-17A, macrophage inflammatory protein (MIP)-1β, interferon-γ-induced protein (IP)-10/chemokine ligand (CXCL)10, RANTES (regulated on activation, normal T cell expressed and secreted)), four (eotaxin, IL-1β, IL-6 and MIP-1β) were greatly increased in PTB relative to TB samples (false discovery rate (FDR)-adjusted P < 0.05 for each), consistent with previous reports of elevated IL-1, IL-6, MIP-1, IP10/CXCL10 and other proinflammatory cytokines associated with PTB in blood, amniotic fluid or cervical–vaginal lavage samples54. For further examination of the role of cytokines in the progression of pregnancy to PTB, we performed an integrative sparse canonical correlation analysis (sCCA) to assess the association of specific bacterial taxa with the abundance levels of nine key cytokines

Although not statistically significant, likely due to sample size, cohort characteristics, and differences in experimental design and details as outlined above, we found partial support for candidate taxa identified in the present study (see Extended Data Fig. 10a–d). For example, the present study confirms an association between Sneathia sanguinegens and PTB, which was reported as significant (P < 0.05) before adjustment for multiple testing by Romero et al.30. Although there were only five spontaneous PTB cases reanalyzed in the Stout et al cohort27, and only ten in the reanalyzed Callahan et al.25 cohort, we observed high concordance in the directionality of differences in abundance levels of preterm and term groups between these cohorts and the present study

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Blencowe, H. et al. National, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic analysis and implications. Lancet379, 2162–2172 (2012).

Liu, L. et al. Global, regional, and national causes of under-5 mortality in 2000–15: an updated systematic analysis with implications for the Sustainable Development Goals. Lancet388, 3027–3035 (2016).

Front matter

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Subjects

Abstract

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes ‘omics’ data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.

Approximately 15 million preterm births at less than 37 weeks of gestation occur annually worldwide1. Preterm birth (PTB) remains the second most common cause of neonatal death across the globe, and the most common cause of infant mortality in middle- and high-income economies2. The consequences of PTB persist from early childhood into adolescence and adulthood3,4. In the United States, striking population differences with respect to PTB exist, with women of African ancestry having a substantially larger burden of risk. The estimated annual cost of PTB in the United States alone is over US$26.2 billion5. Despite these statistics, there remains a paucity of effective strategies for predicting and preventing PTB.

Although maternal and fetal genetics, and gene–environment interactions, clearly play roles in determining the length of gestation, environmental factors, including the microbiome, are the most important contributors to PTB, particularly among women of African ancestry6. Microbe-induced inflammation resulting from urinary tract infection, sexually transmitted infections, including trichomoniasis, or bacterial vaginosis is thought to be a cause of PTB7,8. Ascension of microbes7,9 from the lower reproductive tract to the placenta, fetal membranes and uterine cavity, and hematogenous spread of periodontal pathogens from the mouth, have also been invoked to explain the up to 40–50% of preterm births that are associated with microbial etiologies10,11.

A homogeneous Lactobacillus-dominated microbiome has long been considered the hallmark of health in the female reproductive tract. In contrast, a vaginal microbiome with high species diversity, as observed with bacterial vaginosis, has been associated with increased risk for acquisition and transmission of sexually transmitted infections, PTB and pelvic inflammatory disease12,13,14,15. However, many asymptomatic healthy women have diverse vaginal microbiota. More refined approaches are needed to assess risk, promote health, and prevent and treat disease16,17,18,19,20,21.

Recent reports of the microbiome in pregnant women22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 have suggested that the composition of the vaginal microbiome has a significant population-specific impact on PTB risk. Several studies that focused on populations predominantly of European descent22,23,24,25 have associated Lactobacillus crispatus with a lower risk of PTB, and the finding was replicated in a cohort of predominantly African descent25. As first reported by Ravel et al.16, and subsequently confirmed in other studies21,40, the vaginal microbiome profiles of women of African and European ancestry differ significantly. Although distinct taxa have been associated with PTB in women of African ancestry in some studies25,26, others have not found significant associations27,30. Women of African descent are less likely to exhibit vaginal lactobacilli, frequently have vaginal L. crispatus predominance and are more likely to exhibit increased vaginal microbial diversity16,21. Consequently, population-specific studies may be required to assess the broad impacts of the vaginal microbiome on risk of PTB and to identify contributing taxa that may be carried by only a small subset of women.

In the present study, we report a community resource that includes samples collected longitudinally during 1,572 pregnancies of women from diverse ancestries, and omics data generated from samples collected from 597 pregnancies in a collaborative effort under the umbrella of the National Institutes of Health’s integrative Human Microbiome Project (iHMP)41. Furthermore, we provided an analysis of the longitudinal, comprehensive, multi-omic profiling of vaginal samples from 45 women who experienced spontaneous PTB and 90 case-matched controls, in a cohort of women of predominantly African ancestry. In an initial analysis of this dataset, which represents one of the largest and most comprehensive studies of the vaginal microbiome to date, we identified vaginal microbial signatures in women who went on to experience PTB.

Results

The longitudinal iHMP study, the Multi-Omic Microbiome Study: Pregnancy Initiative (MOMS-PI) includes a total of 1,572 pregnancies, with 992 pregnancies from clinics associated with the Research Alliance for Microbiome Science (RAMS) Registry, based at Virginia Commonwealth University (VCU) in Virginia, and 580 pregnancies from sites associated with the Global Alliance to Prevent Prematurity and Stillbirth (GAPPS) in Washington State. The resource features two comprehensive datasets of integrated microbiome and host functional properties measured longitudinally in pregnancy and the perinatal period (Fig. 1): (1) the MOMS-PI Preterm Birth (PTB) study dataset generated from a case–control study of 45 women predominantly of African ancestry, who delivered spontaneously preterm, and 90 case-matched women who delivered at term; and (2) the MOMS-PI Term Birth (TB) study dataset generated from an ethnically diverse retrospective cohort study of 90 women, who delivered at term or early term42. From a selection of 12,039 samples from 597 pregnancies, we generated: (1) 16S ribosomal RNA (rRNA) taxonomic profiles from 6,452 samples from pregnant women and 2,753 samples from neonates; (2) metagenome profiles from 930 samples from pregnant women and 146 samples from neonates; (3) metatranscriptome profiles from 297 samples from pregnant women; (4) cytokine profiles from 1,223 samples from pregnant women and 173 samples from neonates; and (5) lipid profiles from 63 samples from pregnant women. In the overall MOMS-PI study, we collected a total of 206,437 samples from pregnant women and their neonates, which have been archived in the RAMS Registry (see ramsregistry.vcu.edu) (Fig. 1c). Comprehensive health history and outcome data were also collected longitudinally.

a, An overview of the study designs for the MOMS-PI PTB study (45 spontaneous preterm (sPTB) cases and 90 term controls) and the MOMS-PI TB study (90 women who delivered at term or early term and their neonates). Both cohorts were selected from the phase 1 RAMS Registry cohort (n = 627). b, Omics data were generated from samples from the MOMS-PI PTB and MOMS-PI TB studies and 384 additional pregnancies from the overall MOMS-PI cohort. Samples from the 12 women who were selected for both the MOMS-PI PTB study and the MOMS-PI TB study are depicted under both studies. Omics data types include 16S rRNA amplicon sequencing, metagenomic sequencing (MGS), metatranscriptomic sequencing (MTS), host cytokine assays and lipidomics. c, A total of 206,437 samples were collected at more than 7,000 visits from 1,572 pregnancies in the MOMS-PI study, and are archived in the RAMS Registry.

In the present study, we focus our analysis on a comprehensive multi-omic profiling of vaginal samples in the MOMS-PI PTB study. We analyzed 45 single gestation pregnancies that met the criteria for spontaneous PTB (23–36 weeks 6 days of gestational age) and 90 single gestation pregnancies that extended through term (≥39 weeks) to avoid issues possibly associated with early term births43,44,45. The TB controls in the MOMS-PI PTB study were case matched to the PTB group (2TB:1PTB) for age, race and annual household income. On average, the earliest samples were collected at 18 weeks of gestation, and the mean number of sampling visits per participant was 7. The respective mean and median gestational age at delivery was 34, 0/7 and 35, 6/7 for the PTB group and 40, 0/7 and 39, 6/7 for the TB group.

Abstract

Key points

Summary

Introduction

Methods

Results

Discussion

Funding

Participants and statistics

References

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