Establishment of vaginal microbiota composition in early pregnancy and its association with subsequent preterm prelabor rupture of the fetal membranes
2018; Elsevier BV; Volume: 207; Linguagem: Inglês
10.1016/j.trsl.2018.12.005
ISSN1931-5244
AutoresRichard G. Brown, M. Al‐Memar, Julian R. Marchesi, Yun Sok Lee, Ann Smith, Denise Chan, Holly Lewis, Lindsay Kindinger, Vasso Terzidou, T. Bourne, Phillip R. Bennett, David A. MacIntyre,
Tópico(s)Urinary Tract Infections Management
ResumoVaginal bacterial community composition influences pregnancy outcome. Preterm prelabor rupture of the fetal membranes (PPROM), which precedes 30% of all spontaneous preterm births, is associated with high vaginal bacterial diversity prior to rupture. The point at which vaginal bacterial diversity is established before PPROM is unknown. In this study, we use metataxonomics to longitudinally characterize the vaginal bacterial composition from as early as 6 weeks of gestation in women at high (n = 38) and low (n = 22) risk of preterm birth who subsequently experience PPROM and in women delivering at term without complications (n = 36). Reduced Lactobacillus spp. abundance and high diversity was observed prior to PPROM in 20% and 26% of women at low and high risk of preterm births respectively, but in only 3% of women who delivered at term. PPROM was associated with instability of bacterial community structure during pregnancy and a shift toward higher diversity predominately occurring during the second trimester. This was characterized by increased relative abundance of potentially pathogenic species including Prevotella, Peptoniphilus, Streptococcus, and Dialister. This study identifies reduced Lactobacillus spp. abundance and increasing vaginal bacterial diversity as an early risk factor for PPROM and highlights the need for interventional studies designed to assess the impact of modifying vaginal bacterial composition for the prevention of preterm birth. Vaginal bacterial community composition influences pregnancy outcome. Preterm prelabor rupture of the fetal membranes (PPROM), which precedes 30% of all spontaneous preterm births, is associated with high vaginal bacterial diversity prior to rupture. The point at which vaginal bacterial diversity is established before PPROM is unknown. In this study, we use metataxonomics to longitudinally characterize the vaginal bacterial composition from as early as 6 weeks of gestation in women at high (n = 38) and low (n = 22) risk of preterm birth who subsequently experience PPROM and in women delivering at term without complications (n = 36). Reduced Lactobacillus spp. abundance and high diversity was observed prior to PPROM in 20% and 26% of women at low and high risk of preterm births respectively, but in only 3% of women who delivered at term. PPROM was associated with instability of bacterial community structure during pregnancy and a shift toward higher diversity predominately occurring during the second trimester. This was characterized by increased relative abundance of potentially pathogenic species including Prevotella, Peptoniphilus, Streptococcus, and Dialister. This study identifies reduced Lactobacillus spp. abundance and increasing vaginal bacterial diversity as an early risk factor for PPROM and highlights the need for interventional studies designed to assess the impact of modifying vaginal bacterial composition for the prevention of preterm birth. At a Glance CommentaryRichard G. Brown, et al.BackgroundPreterm birth (PTB) is the primary cause of death in children under 5yrs. Around 30% of cases are preceded by preterm prelabor rupture of fetal membranes (PPROM). A high diversity, Lactobacillus spp. deplete vaginal microbiome is a risk factor for PPROM, however it is unknown when in pregnancy this is established. By longitudinally characterizing vaginal composition from 6 weeks gestation, we show that PPROM is associated with bacterial community instability and shifts toward higher diversity, predominately during the second trimester.Translational SignificanceThese results enable improved PTB risk stratification and targeted intervention strategies, which are reliant upon accurate identification of etiology. Preterm birth (PTB) is the primary cause of death in children under 5yrs. Around 30% of cases are preceded by preterm prelabor rupture of fetal membranes (PPROM). A high diversity, Lactobacillus spp. deplete vaginal microbiome is a risk factor for PPROM, however it is unknown when in pregnancy this is established. By longitudinally characterizing vaginal composition from 6 weeks gestation, we show that PPROM is associated with bacterial community instability and shifts toward higher diversity, predominately during the second trimester. These results enable improved PTB risk stratification and targeted intervention strategies, which are reliant upon accurate identification of etiology. Preterm birth (PTB) is the greatest challenge facing obstetrics in the modern era. It is the world's leading cause of childhood mortality and is associated with 80% of all neonatal morbidity1WHO, World Health Organisation. Causes of child mortality. In: Global Health Observatory (GHO) Data. 2017. https://www.who.int/gho/child_health/mortality/causes/en/.Google Scholar resulting in major financial and emotional cost to families and society. Preterm prelabor rupture of the fetal membranes (PPROM) describes rupture of the fetal membranes prior to 37 weeks of gestation, before the onset of labor. PPROM is estimated to complicates 3% of pregnancies and is the largest contributor to spontaneous PTB, preceding 30% of cases,2Parry S. Strauss 3rd, J.F. Premature rupture of the fetal membranes.N Engl J Med. 1998; 338: 663-670Crossref PubMed Scopus (582) Google Scholar with 80% delivering within 9days3Peaceman A.M. Lai Y. Rouse D.J. Spong C.Y. Mercer B.M. Varner M.W. et al.Length of latency with preterm premature rupture of membranes before 32 weeks' gestation.Am J Perinatol. 2015; 32: 57-62PubMed Google Scholar and the overwhelming majority before 37 weeks. Despite much research effort, the causes of PPROM are incompletely understood and the incidence continues to rise on a global scale.4Liu L. Oza S. Hogan D. Perin J. Rudan I. Lawn J.E. et al.Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis.Lancet. 2015; 385: 430-440Abstract Full Text Full Text PDF PubMed Scopus (1995) Google Scholar One widely held hypothesis is that a proportion of PPROM cases are caused by colonization of the vagina by pathogenic bacteria that activate the local innate immune system2Parry S. Strauss 3rd, J.F. Premature rupture of the fetal membranes.N Engl J Med. 1998; 338: 663-670Crossref PubMed Scopus (582) Google Scholar, 5Chandiramani M. Bennett P.R. Brown R. Lee Y. MacIntyre D.A. Vaginal microbiome-pregnant host interactions determine a significant proportion of preterm labour.Fetal Matern Med Rev. 2014; 25: 73-78Crossref Scopus (9) Google Scholar precipitating an inflammatory cascade6Kanayama N. Terao T. Horiuchi K. 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An overview of mortality and sequelae of preterm birth from infancy to adulthood.Lancet. 2008; 371: 261-269Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar Consistent with this hypothesis, recent studies using culture independent techniques have shown that reduced Lactobacillus spp. abundance and increased bacterial diversity is associated with PPROM and PTB.13Brown R.G. Marchesi J.R. Lee Y.S. Smith A. Lehne B. Kindinger L.M. et al.Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin.BMC Med. 2018; 16: 9Crossref PubMed Scopus (142) Google Scholar, 14DiGiulio D.B. Callahan B.J. McMurdie P.J. Costello E.K. Lyell D.J. Robaczewska A. et al.Temporal and spatial variation of the human microbiota during pregnancy.Proc Natl Acad Sci USA. 2015; 112: 11060-11065Crossref PubMed Scopus (628) Google Scholar, 15Kindinger L.M. MacIntyre D.A. Lee Y.S. Marchesi J.R. Smith A. McDonald J.A. et al.Relationship between vaginal microbial dysbiosis, inflammation, and pregnancy outcomes in cervical cerclage.Sci Transl Med. 2016; 8: 350ra102Crossref PubMed Scopus (105) Google Scholar In contrast, healthy pregnancy is characterized by stable, low richness and low diversity community structures dominated by Lactobacillus spp.16Aagaard K. Riehle K. Ma J. Segata N. Mistretta T.A. Coarfa C. et al.A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy.PloS One. 2012; 7: e36466Crossref PubMed Scopus (465) Google Scholar, 17MacIntyre D.A. Chandiramani M. Lee Y.S. Kindinger L. Smith A. Angelopoulos N. et al.The vaginal microbiome during pregnancy and the postpartum period in a European population.Sci Rep. 2015; 5: 8988Crossref PubMed Scopus (151) Google Scholar, 18Romero R. Hassan S.S. Gajer P. Tarca A.L. Fadrosh D.W. Nikita L. et al.The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women.Microbiome. 2014; 2: 40Google Scholar These findings concur with earlier culture-based studies that reported absence of Lactobacillus spp. and polymicrobial colonization of the vagina as risk factors for PPROM19McGregor J.A. French J.I. Seo K. Premature rupture of membranes and bacterial vaginosis.Am J Obstet Gynecol. 1993; 169: 463-466Abstract Full Text PDF PubMed Scopus (64) Google Scholar and PTB.20Flynn C.A. Helwig A.L. Meurer L.N. Bacterial vaginosis in pregnancy and the risk of prematurity: a meta-analysis.J Fam Prac. 1999; 48: 885-892PubMed Google Scholar, 21Hillier S.L. Nugent R.P. Eschenbach D.A. Krohn M.A. Gibbs R.S. Martin D.H. et al.Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant. The Vaginal Infections and Prematurity Study Group.N Engl J Med. 1995; 333: 1737-1742Crossref PubMed Scopus (1199) Google Scholar In a recent study of 250 pregnant women, we showed that vaginal bacterial composition characterized by Lactobacillus spp. depletion and high diversity, was detectable prior to the rupture of fetal membranes in approximately a third of cases.13Brown R.G. Marchesi J.R. Lee Y.S. Smith A. Lehne B. Kindinger L.M. et al.Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin.BMC Med. 2018; 16: 9Crossref PubMed Scopus (142) Google Scholar Lactobacillus spp. depletion and high diversity was not observed in women who subsequently delivered at term without complications. However, the point during the pregnancy when vaginal bacterial composition shifts toward a high-diversity state in women who subsequently PPROM remains unknown. To address this gap in our knowledge, we prospectively sampled over 1500 women with and without risk factors for PTB to identify 60 women who subsequently experienced PPROM. Vaginal microbiota compositionwas examined in these women from 6 to 36 weeks of gestation and compared to samples from women who subsequently delivered at term, matched for maternal age, BMI, and ethnicity. Our data demonstrates that vaginal bacterial communities deplete in Lactobacillus species and high in diversity are a risk factor for subsequent PPROM and predominately emerge during the second trimester. We performed a prospective cohort study of women with and without risk factors for preterm birth between January 2013 and November 2016. The study was approved by the National Health Service, National Research Ethics Service Committees for London–Stanmore (REC 14/LO/0328), and London-Riverside (REC 14/LO0199) areas. All ethical guidelines for human research were followed and participants provided written informed consent. Women without pre-existing risk factors for PTB were recruited from the early pregnancy unit of Queen Charlotte's Hospital, London (n = 1003). Women with existing risk factors (history of preterm birth, midtrimester loss (MTL) or large loop excision of the transformation zone (LLETZ) were recruited from the preterm birth surveillance clinics at Queen Charlotte's, St Mary's, and Chelsea and Westminster hospitals, London (n = 535). Exclusion criteria for both cohorts included women under 18years of age, multiple pregnancy, and sexual intercourse within 72 hours of sampling and HIV or Hepatitis C positive status. Women were recruited upon presentation to the early pregnancy unit, (typically between 6 and 10weeks gestation),or at their initial appointment in the preterm birth surveillance clinic (12–14 weeks gestation). All women had cervicovaginal fluid sampled from the posterior vaginal fornix using a BBL™ CultureSwab MaxV Liquid Amies swab (Becton, Dickinson and Company, Oxford, UK). Swabs were placed immediately on ice before being stored at −80°C within 5minutes of collection. Repeat samples were taken where possible within the gestational time windows of; 12–17+6, 18–23+6, 24–29+6, 30–36+6 weeks+days of completed gestation, however, not all women could be sampled at each of the prespecified timepoints. Detailed clinical and outcome data were collected for all participants. PPROM was defined as rupture of the fetal membranes, diagnosed by pooling of amniotic fluid on speculum examination, prior to 37 weeks gestation more than 24hours prior to spontaneous preterm delivery or clinically indicated delivery or induction of labor. Where speculum examination was equivocal, evidence of oligohydramnios or anhydramnios on ultrasonography, patient history and the decision of the attending clinician to administer steroids, erythromycin and induce labor at 34 weeks was accepted as a diagnosis of PPROM. Uncomplicated term deliveries without antepartum bleeding, antibiotic use, fetal growth restriction, pre-eclampsia, gestational diabetes, or other significant medical comorbidities were selected to match PPROM cases for age, ethnicity, and BMI. DNA extraction from vaginal swabs and assessment of DNA integrity by PCR amplification was performed as previously described.22MacIntyre D.A. Chandiramani M. Lee Y.S. Kindinger L. Smith A. Angelopoulos N. et al.The vaginal microbiome during pregnancy and the postpartum period in a European population.Sci Rep. 2015; 5: 8988Crossref PubMed Scopus (334) Google Scholar, 23Kindinger L.M. Bennett P.R. Lee Y.S. Marchesi J.R. Smith A. Cacciatore S. et al.The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk.Microbiome. 2017; 5: 6Crossref PubMed Scopus (201) Google Scholar The V1–V2 hyper variable regions of 16S rRNA genes were amplified for sequencing using forward and reverse fusion primers with the forward primer consisting of an Illumina i5 adapter (5′-AATGATACGGCGACCACCGAGATCTACAC-3′), an 8-base pair (bp) bar code, a primer pad (forward, 5′-TATGGTAATT-3′), and the 28F primer (5′-GAGTTTGATCNTGGCTCAG-3′).24Sundquist A. Bigdeli S. Jalili R. Druzin M.L. Waller S. Pullen K.M. et al.Bacterial flora-typing with targeted, chip-based Pyrosequencing.BMC Microbiol. 2007; 7: 108Crossref PubMed Scopus (188) Google Scholar The reverse fusion primer consisted of an Illumina i7 adapter (5′-CAAGCAGAAGACGGCATACGAGAT-3′), an 8-bp bar code, a primer pad (reverse, 5′-AGTCAGTCAG-3′), and the 388R primer (5′-TGCTGCCTCCCGTAGGAGT-3′). Sequencing was performed at RTL Genomics (Lubbock, TX) using an Illumina MiSeq platform (Illumina Inc.) and the resulting sequence data analyzed using the MiSeq SOP Pipeline of the Mothur package.25Kozich J.J. Westcott S.L. Baxter N.T. Highlander S.K. Schloss P.D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.Appl Environ Microbiol. 2013; 79: 5112-5120Crossref PubMed Scopus (4129) Google Scholar Sequence alignment was performed using the Silva bacterial database (www.arb-silva.de/), classification was performed using the Ribosomal Database Project database reference sequence files26Wang Q. Garrity G.M. Tiedje J.M. Cole J.R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Appl Environ Microbiol. 2007; 73: 5261-5267Crossref PubMed Scopus (13299) Google Scholar and the Ribosomal Database Project MultiClassifier script was used for determination of operational taxonomic unit taxonomies (phylum to genus). Species-level taxonomies were determined using USEARCH.27Edgar R.C. Search and clustering orders of magnitude faster than BLAST.Bioinformatics. 2010; 26: 2460-2461Crossref PubMed Scopus (14014) Google Scholar To avoid sequencing bias, data were resampled and normalized to the lowest read count. Assessment of statistical differences between taxa was performed at genera and species level in the Statistical Analysis of Metagenomic Profiles software package .28Parks D.H. Beiko R.G. Identifying biologically relevant differences between metagenomic communities.Bioinformatics. 2010; 26: 715-721Crossref PubMed Scopus (720) Google Scholar Samples were further classified into 3 groups according to Centroid linkage hierarchical clustering analysis of bacterial genera using a clustering density threshold of 0.75 with the 25most abundant genera displayed to facilitate visual presentation. Three separate clusters were identified and characterized on the basis of Lactobacillus spp. abundance into; dominant (75%–100% abundance), intermediate (50%–74% abundance), and deplete (0%–43% abundance). No samples were found to have relative Lactobacillus spp. abundance values between 43% and 49%. Comparison of mean bacterial richness (species observed), diversity (inverse Simpson index) and relative abundances of bacterial genera was performed using nonparametric testing (Mann-Whitney U) and Fisher's exact test to compare frequency of vaginal microbiota compositions between the following groups; (1) women sampled at the last timepoint prior to PPROM vs gestational age matched samples from women delivering at term; (2) women sampled at 5 gestational age windows (6–11+6, 12–17+6, 18–23+6, 24–29+6, 30–36+6 weeks) who subsequently have PPROM vs those who subsequently deliver at term with no patient contributing more than 1 sample within any time window; (3) cases resulting in PPROM and delivery at less than 28, 34, or 37 weeks of gestation; (4) women with and without risk factors for PTB who experienced subsequent PPROM; (5) women with and without cervical cerclage who experienced PPROM; and (6) women who did or did not receive progesterone treatment prior to PPROM. The relative risk of subsequent PPROM was calculated based using the relative abundance of lactobacilli (dominant, intermediate, depleted), the dominant Lactobacillus species and the presence of a nonlactobacilli dominated microbiome in women destined for term delivery or PPROM, at each of the 5 gestational age windows. The LEfSe method29Segata N. Izard J. Waldron L. Gevers D. Miropolsky L. Garrett W.S. et al.Metagenomic biomarker discovery and explanation.Genome Biol. 2011; 12: R60Crossref PubMed Scopus (7650) Google Scholar was used to identify differentially abundant taxonomic features between the PPROM and term delivery groups. An α value of 0.05 was used for factorial Kruskal-Wallis test between classes, and a minimum threshold of 2.0 was used for logarithmic latent discriminatory analysis score for discriminative features. The significance of differences between percentage abundance of bacterial genera identified as discriminatory between the 2 outcome groups by LEfSe was calculated by Mann-Whitney. To assess the dynamics of vaginal bacterial communities across pregnancy, longitudinal samples for pregnancies culminating in term delivery and PPROM were analyzed. The dominant microbiota and Lactobacillus spp. abundance for each sample prior to term delivery (62 samples, 16 term deliveries) and PPROM (167 samples, 45 PPROM cases) were highlighted and transitions between dominant microbiota and different abundances of Lactobacillus spp. examined. Between March 2013 and November 2016, 6324 women were seen Early Pregnancy Assessment Unit of Queen Charlotte's Hospital with 2667 meeting the inclusion criteria and 1048 being recruited, resulting in a participation rate of 38%. There were 5 withdrawals, 32 losses to follow-up, 20 terminations of pregnancy, 99 first trimester miscarriages and 14 second trimester miscarriages resulting in 847 ongoing singleton pregnancies. The large majority (800) delivered at term (94%), 47 delivered preterm (5.5%) of which 22 were spontaneous preterm deliveries (2.6%) and 23 experienced PPROM (2.7%). Of the 23 cases that experienced PPROM, 1 participant did not have samples collected. Simultaneously between January 2013 and November 2016, 502 women with risk factors for PTB including previous PTB, previous midtrimester spontaneous miscarriage, previous rescue or ultrasound indicated cervical cerclage and previous LLETZ procedure, were recruited from the preterm birth surveillance clinics at Queen Charlotte's, St Mary's, and Chelsea and Westminster hospitals. These women were recruited from 810 eligible participants resulting in a recruitment rate of 60%. Of these, 2 withdrew from the study, 3 were lost to follow-up and 7 experienced miscarriage resulting in 490 pregnancies that entered the third trimester. The majority (361) delivered at term, (75%) and of the 129 preterm births (26%), 38 (7.8%) cases experienced PPROM. Thus a total of 60 PPROM cases combined from the 2 cohorts were selected for sequencing (Fig 1) and were compared to 40 matched controls at a minimum ratio of 2:1 (cases:controls). Control women were matched for maternal age, gestational age at sampling, BMI, and ethnicity and included those without antepartum bleeding, antibiotic use, fetal growth restriction, pre-eclampsia, gestational diabetes or other significant medical comorbidities. Exclusion of 4 control samples due to amplification failure or low sequence reads resulted in a final sample size of 36 (TableI).Table IClinical and demographic characteristics of the study cohortUncomplicated term deliveryPre-PPROMP valueTotal number3660Total number of samples82172Age (y)33.3 (31.9–34.6)33.8 (31.2–34.3)0.70BMI24.8 (22.9–26.6)24.5 (23.5–25.6)0.82Smoking status1 (3%)4 (7%)0.65EthnicityCaucasian18 (50 %)31 (52%)0.78Black9 (25%)13 (21%)Asian9(25%)16 (27%)GA at PPROM (wk)30+5 (29+1–32+2)GA at sample (wk)24+6 (24+2–27+3)25+5 (24–28+2)0.78GA at Delivery(wk)39+3 (38+4–40+4)32+3 (31+1–33+5)<0.0001Birth weight (g)3342 (3094–3589)2033 (1837–2230)<0.0001Abbreviations: BMI, body mass index; GA, gestational age; MR, membrane rupture; PPROM, preterm prelabour rupture of the fetal membranes.Data presented as median (interquartile range) or number (%).P values: t test/ Mann-Whitney U (depending upon distribution), Fisher's exact for proportional data. Open table in a new tab Abbreviations: BMI, body mass index; GA, gestational age; MR, membrane rupture; PPROM, preterm prelabour rupture of the fetal membranes. Data presented as median (interquartile range) or number (%). P values: t test/ Mann-Whitney U (depending upon distribution), Fisher's exact for proportional data. A total of 254 swab samples were analyzed providing 4,593,060 high quality reads with an average read count of 18,083 per sample. After removal of singletons and rare operational taxonomic units (OTUs) (defined as 95% of all sequence reads), permitting samples to be classified into 3 vaginal microbiota groups characterized by the relative abundance of Lactobacillus spp. and termed Lactobacillus spp. dominant (75%–100%, abundance), intermediate (50%–74%, abundance) and depleted (0%–43%, abundance) (Sup Fig 2). The final sample prior to PPROM (n = 60) and gestational age matched samples (n = 36) from women delivering at term were separated on the basis of ethnicity to examine vaginal bacterial composition in women of Caucasian, Asian, and Black ethnicities. Each of the predominant Lactobacillus spp. (L. crispatus, L. iners, L. jensenii, and L. gasseri) were identified in women of Asian and Caucasian ethnicity, but L. gasseri was not present in women of black ethnicity as previously reported in low risk pregnancies from the same background population.17MacIntyre D.A. Chandiramani M. Lee Y.S. Kindinger L. Smith A. Angelopoulos N. et al.The vaginal microbiome during pregnancy and the postpartum period in a European population.Sci Rep. 2015; 5: 8988Crossref PubMed Scopus (151) Google Scholar A Lactobacillus spp. deplete microbiome was not observed in Asian or Caucasian women delivering at term, but was present in 1/8 black women. Richness and diversity measures were comparable between ethnic groups in women delivering at term. In women with subsequent PPROM there was an increase in the number of women with a Lactobacillus spp. depleted vaginal microbiome across all ethnicities, with the greatest increase of Lactobacillus spp. deplete communities in black women (Sup Fig 3, Sup Table 1). The last sample obtained before membrane rupture from the 60 PPROM cases was compared to samples taken from the term delivery group (n = 36) matched for gestational age at sampling, maternal age, BMI, and ethnicity (TableI). The average gestation of the last sample before PPROM was 24+6 weeks compared to 25+5 weeks for those delivering at term (P = 0.78, Mann-Whitney). The great majority of women (35/36, 97%) who delivered at term had a vaginal microbiome with >75% abundance of Lactobacillus spp. and 83% (30/36) had Lactobacillus spp. abundance above 98%. Samples obtained prior to PPROM were comparatively enriched for intermediate or Lactobacillus spp. depleted communities (PPROM; 14/60, 23% vs Control; 1/36, 3%, P = 0.011, Mann-Whitney), decreased total Lactobacillus spp. abundance (PPROM; 79% vs Control; 96%, P = 0.016 Mann-Whitney) and increased richness (total number of species observed PPROM; 65vs Control; 10, P = 0.0086) (Fig 2). LEfSe analysis of differentially abundant taxonomic features between PPROM and term delivery groups identified bacteria from the genera Prevotella, Peptoniphilus, Dialister, Streptococcus, Catonella, Parvimonas, and Anaerococcus as being discriminatory for pre-PPROM while Lactobacillus spp. were positively associated with term delivery (Fig 3). Percentage abundance of bacteria belonging to the genera Prevotella (P = 0.0014), Peptoniphilus (P = 0.022) and Dialister (P = 0.027) were significantly higher in samples pre-PPROM whereas Lactobacillus spp. were significantly reduced compared to term controls (P = 0.016; Fig 3). These corresponded at species level to Prevotella bivia, Prevotella timonensis, and Dialister micraerophilus. Lactobacillus vaginalis was associated with term delivery, but was only present in low abundance, comprising a maximum of 8.5% of sequence reads, and was always found in combination with other Lactobacillus species. Longitudinal analyses showed that uncomplicated term delivery was associated with stable alpha diversity (Inverse Simpson index; P = 0.33, Kruskal Wallis, Dunn's post hoc), L crispatus dominance and reduced richness between 12–17+6 and 24–29+6 weeks (P = 0.03, Kruskal Wallis, Dunn's post hoc) compared to women who subsequently had PPROM (Fig 4). In contrast, subsequent PPROM was associated with a significant increase in richness between 6–11+6 and 24–29+6 weeks (P = 0.02, Kruskal Wallis, Dunn's post hoc) (Fig 4, Sup Table 2). A total 45 cases of subsequent PPROM had multiple antenatal samples and were compared to 16 individuals with term delivery. The majority (13/16, 81%) of individuals delivering at term displayed a vaginal microbiome that was dominated by a single Lactobacillus spp. and remained stable throughout the pregnancy with no detected transition events recorded (Fig5). Only 2 women demonstrated transition from the microbiome present in the first trimester, transitioning from L. iners to L. jensenii and L. crispatus respectively prior to term delivery. There was a single case of term delivery where Lactobacillus spp. abundance was reduced and in which L. iners co-colonized with Sneathia, Megasphaera, and Prevotella spp. In contrast only 24/46 (56%) women with subsequent PPROM maintained a vaginal microbiome dominated by a single species of Lactobacillus, 7/46 (15%) transitioned between different Lactobacillus spp. maintaining an overall dominance of Lactobacillus spp. Within the PPROM group, 22% (10/46) transitioned between a Lactobacillus spp. dominant and deplete community structure dominated by genera including Streptococcus, Prevotella, Atopobium, and Megasphaera. Overa
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