This study represents the largest next generation sequencing-based analysis of vaginal microbiota in pregnancies at risk of preterm birth to date. We demonstrate a significant association between L. iners dominance of the vaginal microbiome at 16 weeks of gestation with subsequent preterm birth and conversely show that L. crispatus dominance correlates with reduced risk or preterm birth. Moreover, we show that the insertion of a progesterone pessary for prevention of preterm birth has no adverse impact on vaginal microbial communities.

A healthy vaginal microbiome in non-gravid and gravid subjects is often described as being synonymous with low bacterial diversity and Lactobacillus species dominance [63–66]. Examination of vaginal microbiota at the time of delivery using culture and/or microscopy-based techniques has shown that Lactobacillus species dominance is negatively associated with delivery before 37 weeks of gestation (odds ratio 0.2) whereas bacterial dysbiosis is positively associated with preterm delivery (odds ratio 2.3) [66]. Using similar methodology, Donders and colleagues recently reported that a lactobacilli-dominated vaginal microbiome in the first trimester was associated with a 75% lower risk of delivery before 35 weeks of gestation (0.26; 95% confidence interval (CI) 0.12–0.56] compared to women harboring a vaginal microbiome void of Lactobacillus species (OR 2.4; 95% CI 1.2–4.8) [67]. Using culture-independent characterization of vaginal bacterial communities in a high-risk pregnant population, we show that the perceived benefit of lactobacilli dominance in pregnancy is species specific; L. crispatus is advantageous and associated with term delivery whereas L. iners is associated with increased risk of preterm delivery. Furthermore, L. iners is associated more specifically with a risk of early (<34 weeks) rather than late (34–37 weeks) preterm birth. High relative abundance of L. crispatus is highly specific for term birth, with a false positive rate (1 specificity) of just 3% in our population of women at high risk because of a previous preterm birth. In this population, second trimester dominance of L. iners carries a 67% detection rate (i.e., sensitivity) for preterm birth before 34 weeks; a screening sensitivity comparable to cervical length, the current and primary screening tool used for preterm birth surveillance [32, 68]. Consistent with our findings, Petricevic and colleagues recently reported an over-representation of L. iners dominance in vaginal swab samples collected from 13 preterm births derived from a low-risk cohort of 111 pregnancies, and none of whom delivered before 33 weeks of gestation [25]. However, this study was limited by the use of denaturing gradient gel electrophoresis (DGGE) for the characterization of only major Lactobacillus species and could not identify other pathobionts in the samples.

While our study reveals a clear relationship between relative abundance of vaginal Lactobacillus species and risk of subsequent preterm birth, our data indicate that Lactobacillus spp. depletion or vaginal dysbiosis in the second trimester does not appear to contribute to preterm birth risk. The role of early gestational vaginal dysbiosis in the pathology of preterm birth is controversial. In agreement with our findings, a recent longitudinal analysis of the vaginal microbiome by Romero and colleagues in 18 women experiencing preterm birth (<34 weeks gestation) reported no association between preterm birth and vaginal microbial dysbiosis when compared to controls experiencing term delivery (n = 72) [24]. However, in their study, 95% (17/18) of preterm birth samples and 86% of control samples were collected from African American women who exhibit a higher pregnant and non-pregnant background prevalence of vaginal dysbiosis (CST IV) [13, 24]. In contrast to these findings, Digiulio and co-workers [19] reported, in a small yet densely sampled cohort of women experiencing preterm birth (n = 15), which vaginal bacterial diversity does correlate with risk of preterm delivery [19]. The clinical relevance of these findings however are difficult to establish considering the small sample size and the heterogeneous nature of the cohort; only five women delivered preterm spontaneously and almost half delivered within 1 week of term dates (>36 weeks+3/7 days).

Our presented data provide some suggestion that vaginal microbiota in Black women may not play as an important contributory role to preterm birth pathogenesis as Caucasians and Asians. We did not however have sufficient power to demonstrate the significance of this, but this may be worth examining in future studies.

In our study, we also observed a high rate of CST-shifting in women with an L. iners-dominated microbiome in the second trimester compared to women with an L. crispatus-dominated microbiome. L. iners has been reported as an intermediary between lactobacilli dominance and CST IV-associated states and is the predominant microbiome in peri-menopausal women as they transition through to postmenopausal dominance of anaerobic bacteria [16]. Interactions between L. iners and the maternal host likely provides a vaginal mucosal environment permissible to colonization by BV-associated pathogens, a setting in which it tolerates co-existence well [69, 70]. Unlike other Lactobacillus species, L. iners also induces secretion of pro-inflammatory cytokines when human vaginal epithelial cells are observed in vitro, whereas L. crispatus does not [71, 72].

Other evidence for a role of bacterial dysbiosis in the pathology of preterm birth includes the long recognized association between bacterial vaginosis (BV) and increased risk of preterm birth; however, evidence suggests that screening and treating BV in pregnancy reduces preterm delivery in certain cohorts [73], but not in others [74]. We propose an alternate concept, which is that it is the presence of L. iners that promotes risk of early preterm birth, but because an L. iners-dominated vaginal microbiome has less stability, there is a tendency for transition to BV-associated CST-IV [69, 75]. Indeed, L. iners is the prominent vaginal species following antibiotic treatment for BV [76]. Older studies aimed at detecting BV, which could not differentiate Lactobacillus species and concluded that it was BV rather than species of Lactobacillus that conferred the risk.

Recent investigations into the protective role of Lactobacillus species in the context of reproductive health have revealed major species-specific differences in the capacity to prevent pathobiont colonization and viral infections [12, 72, 77, 78] that are driven largely by maternal host-bacterial metabolite interactions at the vaginal mucosal interface. For example, although lactic acid-producing bacteria including Lactobacillus spp. produce both the d- and l-lactic acid isomers [79], the chirality of the isomer has major functional implications. In women exhibiting a vaginal microbiome dominated by L. iners, an increased ratio of l to d-lactic acid has previously been shown to promote expression of vaginal extracellular matrix metalloproteinase inducer (EMMPRIN) and the activation of matrix metalloproteinase-8 (MMP8), which may subsequently modulate cervical integrity [80]. Conversely, no such relationship has been observed in women with vaginal microbial communities dominated by L. crispatus, which instead preferentially excretes high levels of d-lactic acid and greater overall levels of lactic acid than L. iners [80]. Apart from modulating local tissue inflammation, recent studies have also implicated lactate isomers in vaginal mucosal trapping mechanisms. High concentrations of d-lactic acid are associated with L. crispatus dominance and enhanced trapping of HIV-1 virions in cervico-vaginal mucosa whereas low concentrations of d-lactic acid associated with L. iners dominance permits comparatively rapid diffusion of virions through cervico-vaginal mucosa [77]. Collectively, these data suggest mechanisms by which L. iners dominance of vaginal microbial communities during pregnancy may lead to the modulation of local tissue inflammation and remodeling pathways and to disruption of chemical and mechanical mucosal barriers protective against ascending infection and increase the risk of preterm birth. Such mechanisms may account for the observed association between L. iners dominance and a short cervical length (<25 mm) seen in our study at 16 weeks of gestation, which itself is highly specific for preterm birth [81].

Considering the potential pro-inflammatory roles played by L. iners in the vagina during pregnancy, we postulated that any associated poor pregnancy outcomes might be attenuated by the anti-inflammatory actions of progesterone [45, 82]. However, in this study, no effect of vaginal progesterone therapy upon the frequency of vaginal community state structure was observed across pregnancy indicating that the mode of action of progesterone in the prevention of preterm birth is not through modulation of the vaginal microbiome. The data also show, however, that there is no detrimental effect upon the vaginal microbiome of either progesterone itself or of the daily vaginal insertion of a pessary.

A particular strength of our study is that we characterized the vaginal microbiome in a comparatively large patient cohort at high risk of preterm birth. This strength was demonstrated by a high spontaneous preterm birth rate (n = 34/161). The mean gestation at birth of 32+6 weeks within our preterm birth cohort, and a distribution of gestational ages ranging from 24 to 36 weeks, enabled the characterization of microbial profiles associated with both early (<34 weeks) and late (34 > 37 weeks) preterm birth, providing a broader observational base for microbial-host interactions in pregnancy. The primary limitation was the small number of women receiving progesterone (n = 25) and the lack of an equivalent control group with a short CL <25 mm not receiving any intervention or receiving a placebo. A short CL significantly increases subsequent preterm birth risk [33]; therefore, once detected, clinicians are ethically obliged to provide a preventative intervention such as progesterone. Consequently, a “placebo” intervention for a short cervix could not be included for study in this clinical study. As such, the control women are not true controls as their cervical lengths were all greater than 25 mm at entry. A further potential confounding factor was the impact of ethnicity of vaginal microbiota and gestation at birth, although we demonstrated this not to be significant in our cohort.