The microbiome in prematurity: Key messages from emerging science

Although preterm birth (PTB) (<37 weeks at delivery) is a leading cause of perinatal morbidity and mortality worldwide, reliable tools to clinically predict both its occurrence and severity or the co-occurrence of infant morbidity are lacking. This clinical dilemma is further exacerbated by the absence of highly efficacious primary and secondary prevention measures, as well as reliable interventions.^1,2 Perhaps the greatest obstacle to their discovery is the likely multifactorial and varied etiology of PTB, which is undoubtedly a reflection of the “lumping” of what is actually a syndrome with varied etiologies into a single disorder. Indeed, PTB can be readily separated into spontaneous and indicated PTB, and, thus, treatment or prevention for one might be counterproductive for the other.

Take for example any day on a labor and delivery ward around the United States: 2 women deliver at 32 and 2/7 weeks’ gestation. The first was induced for 2 days secondary to her diagnosis of severe preeclampsia with concern for worsening symptoms. The second woman presented 3 hours ago in active labor with advanced cervical dilatation to 7 cm and just ruptured her membranes. Working to identify the etiology of early severe preeclampsia in the first patient could be of benefit in future pregnancies (eg, treating underlying systemic lupus erythematosus, providing low-dose aspirin). Conversely, in our second patient, attempts at preventing recurrent spontaneous PTB will be of benefit (eg, via administration of 17-alpha hyrdoxyprogesterone caproate). However, both outcomes would be classified as PTB even though their etiologies are likely quite distinct. Given the heterogeneous nature of PTB, we will focus this review on the spontaneous PTB syndrome and the potential of underlying inflammatory and infectious causes of its occurrence.

Etiologies of spontaneous preterm birth

Observational studies have identified a number of environmental and host factors that carry a greater risk of spontaneous PTB, including a prior history, maternal smoking, and maternal race/ethnicity.³ Intrauterine infection and associated inflammation has also been hypothesized as a potential contributor to spontaneous PTB, but a specific infectious etiology has yet to be identified. Moreover, empirical administration of antimicrobials for presumptive infectious processes have been uniformly shown to be nonefficacious in preventing spontaneous PTB, and may instead increase risk.⁴ In addition, bacterial vaginosis (BV) increases risk of PTB, but while antibiotic treatment of symptomatic BV is efficacious, PTB rates are unaffected.^5,6 Even more concerning, several studies have found that empiric antibiotic administration to asymptomatic women increased the rate of PTB.^7,8 These outcomes highlight the notion that PTB may be a result of aberrant shifts in maternal microbiota rather than by a single infectious microorganism per se. However, despite several decades documenting the co-association of altered vaginal microbiota and inflammation with spontaneous PTB, neither a clear pathologic agent nor targeted therapy has shown unmitigated success in combatting presumptive infectious or inflammatory PTB.

Lack of clear pathogenic microbes driving spontaneous PTB has spurred interest in the potential role of commensal microbes. Research on the trillions of commensal microorganisms that reside on and within our bodies, collectively known as the human microbiota, has begun to reorient how we think microorganisms may influence pregnancy outcomes. Recent advances in sequencing technologies have enabled in-depth interrogation of these microbial communities and their function without the limitations of culture-based methods.⁹The normal vaginal microbiota of healthy non-pregnant and pregnant women has since been comprehensively characterized, providing a reference for the typical microbiota associated with obstetrical health.^9–12 Furthermore, the low biomass microbial community of placentae, their membranes, and amniotic fluid in healthy pregnancies has been similarly identified and characterized by several investigators, which has contributed to the emerging body of evidence that is challenging the notion of a sterile intrauterine environment.^13–18These and other studies have subsequently begun to characterize the placental microbiota in cases of PTB, chorioamnionitis and other adverse pregnancy outcomes.^18–20 In this review, we will briefly review the current literature exploring associations between the vaginal and placental microbiota and PTB, highlight the remaining gaps in our knowledge, and speculate on how this information may impact future clinical practice.

Vaginal microbiota and preterm birth

Given its proximity to the intrauterine environment, vaginal microbiota are hypothesized to play a role in maintaining a healthy pregnancy. Recently, the typical microbiota associated with healthy women before and during pregnancy have been catalogued using deep sequencing methods in an attempt to provide a “normal” reference before ascribing specific microbiota to disease risk or prevention trials. It has long been known that the vaginal microbiome of non-pregnant woman tends to be dominated by Lactobacillus species, which produce lactic acid and other bacteriocidins that likely provide protection against pathogenic microorganisms.^21,22 However, deep sequencing of the vaginal microbiota of reproductive-aged women has uncovered further complexities within and between microbial communities not previously described. A seminal study of approximately 400 non-pregnant reproductive-aged women of various ethnicities and ages found that individuals could be discriminated by the dominant species of Lactobacillus found within their vaginal microbiota (eg, L crispatus, L jensenii or lack thereof ), which grouped them into 1 of 5 “community state types” (CSTs).¹¹ While the CST representation was found to vary significantly among different ethnicities, indicating that host genetics, diet, or environment may influence the type and abundance of bacteria present within the vagina,¹¹ caution must be used when using this terminology in clinical practice because CST identification may vary with analytical methods and may underestimate the true variation of vaginal microbiota that likely exists within the population.²³ Moreover, the vaginal microbiome can be highly variable over time, thus it would be difficult to assign these women to a single CST at any given point.²⁴ Nevertheless, these studies have provided an initial framework with which to interrogate the impact of microbiota in pregnancy.

Comparative studies of pregnant and non-pregnant women have found that the vaginal microbiome undergoes specific rearrangements that accompany pregnancy. In pregnancy, the vaginal microbiome tends to exhibit increased stability over time, harbors fewer unique bacteria, and experiences fewer shifts in community composition.^10,12,25 These observations are likely a reflection of the increased abundance of Lactobacilli in the vagina, which tends to eventually dominate the vaginal flora as the pregnancy progresses.¹² The role that Lactobacilli play in maintaining a healthy pregnancy remains poorly understood, but they may synergistically metabolize increased glycogen stores within the vaginal epithelium to acidify the vaginal environment to foster its own growth while inhibiting growth of other species.^26,27 Despite these important observations, our understanding of the vaginal microbiome in healthy pregnancies remains incomplete. Individuals of different racial or ethnic backgrounds have varied risk profiles for adverse pregnancy outcomes and in a similar manner, the vaginal microbiota appear to vary substantially according to race and ethnicity.^11,28 In an attempt to disentangle the genetic and cultural differences attributed to ethnicity, we previously demonstrated that heritable mitochondrial DNA variation, which is often used as a genetic surrogate for race or ethnicity, is associated with differences in the vaginal microbiome, providing a potential genetic explanation to the aforementioned observations.²⁸ It is therefore imperative that large studies encompassing pregnant individuals of a diverse demographic range be conducted to better understand how host or environmental factors may impact the dynamics of the vaginal flora throughout pregnancy and in the pathophysiology of PTB.

Although the relationship between vaginal microbiota and PTB has been probed, studies to date are limited and have yet to identify a consensus signature in the vaginal microbiome predictive or causative of PTB. Work by Romero et al. reported no difference in the composition and abundance of microbiota in the vagina between preterm and term deliveries.²⁹ Hyman et al. similarly did not identify any microbiota associated with PTB.³⁰ In the latter study, the overall diversity of the vaginal microbiome was significantly reduced in Caucasian women who delivered preterm, but the impact of such a reduction is unknown.³⁰ In contrast, a subsequent study by DiGiulio et al. reported that women with reduced Lactobacilli and increased Gardnerella or Ureaplasma were at increased risk of PTB, but frequent sampling to detect these subtle variations was necessary.²⁵ Differences in the ethnic demographics between these study cohorts may account for the disparate conclusions reached by each, with 90% of subjects in the Romero et al., study identifying as African American, while a majority of subjects in the DiGiulio et al. study identified as Caucasian. Thus, the discrepancies between the overall conclusions of these studies may reflect the difference in impact on host genetics and other factors on vaginal microbiome composition in the context of PTB, although it is clear that with such little evidence to date, additional studies are sorely needed to evaluate vaginal microbiota in cases of PTB.

Placental microbiota and preterm birth

The prevailing paradigm indicates that the intrauterine environment and its associated tissues are sterile, and that bacterial infection of the placenta results in adverse outcomes, including preterm labor. However, bacteria within the placenta are not restricted to subjects with PTB, as a number of studies have documented DNA and culture evidence of bacteria within the amniotic fluid, cord blood and placenta of healthy, term pregnancies.^{13,15,16,31–33} More recently, we used deep sequencing methods to describe the microbial community within the placenta of healthy, term pregnancies.¹⁸ Across 320 pregnancies, we identified a large diversity of microbiota present in the placental parenchyma with a notable predominance of Escherichia coli.¹⁸ Numerous groups have subsequently repeated this work and have further shown that the placental microbiota share similarity to those within the amniotic fluid and neonatal meconium.^32,33 Interestingly, because the placental microbiome bore the greatest similarities to that of the oral cavity, we’ve speculated that many of the microorganisms found within the placenta originate from the oral gingiva. This could potentially explain the known association of periodontitis with PTB, although studies in well-controlled models are required to delineate this potential link further.

But what is the normal impact and function of these microbiota and how is this changed in disease? In our initial characterization of the normal placental microbiome, we found that the placental microbiome was significantly different in cases of PTB, in mothers with a remote history of antenatal infection, and more recently, in relation to histological chorioamnionitis.^18,20Burkholderia was increased in the placentae of PTB subjects, while Streptococcus and Acinetobacter were enriched in the placentae of subjects with a remote history of antenatal infection.¹⁸ We’ve further demonstrated that the “preterm” placental microbiome can be further distinguished in mothers with excess gestational weight gain, independent of maternal obesity.¹⁹ Interestingly, bacterial gene pathways related to butanoate metabolism were also decreased in mothers with excess gestational weight gain, which may have substantial implications on placental biology as butanoate has been shown to modulate inflammation in the gastrointestinal tract.^19,34 Although additional studies are needed to ensure the robustness of these findings, these initial observations have nevertheless provided tangible hypotheses to drive future interrogations of the complex interplay between host and microbiota in the etiology of PTB.

Microbiota of other body sites

Studying microbiota in other areas of the body, including the maternal gut and oral cavity, may further inform our understanding of PTB. Periodontal disease is an associated risk factor for PTB, and a number of common oral pathogens, including Fusobacterium nucleatum, are frequently found in diagnostic cultures from patients with preterm labor, premature rupture of membranes and, stillbirth.^35,36 In agreement with these clinical observations, the placental microbiome has been similarly shown to harbor many oral commensal bacteria, including Streptococcus species.¹⁸ But how do microbiota typical of the oral cavity come to inhabit the intrauterine space? One hypothesis indicates a potential hematogenous route. Translocation of gingival-associated microbes into the blood stream can occur as a result of periodontal disease or after dental procedures.³⁷ In keeping with that thesis, studies in mouse models have demonstrated that oral commensals can hematogenously spread to the placenta, potentially facilitated by specific bacterial cell surface proteins that can compromise endothelial adhesion junctions.^38,39 Thus the possibility of oral to placental transmission exists, though further studies are necessary to investigate this proposed mechanism in greater detail. If such a link does exist, then the gastrointestinal tract, which by far harbors the greatest biomass of bacteria, becomes another important organ system that may impact the placental microbiome. Certain disease states, including obesity, can compromise the intestinal barrier and increase permeability to microbiota.⁴⁰ If and how such a state may impact the placental microbiome and pregnancy outcomes remains unknown, but together these observations indicate that the gut and oral microbiomes may be an important component of the complex pathophysiology that underlies PTB.

Summary

Advancements in sequencing technologies have provided more sophisticated tools to interrogate the role of maternal microbiota in obstetrical health and disease. This has greatly expanded our understanding of the microbiota present in the vagina and uncovered new areas of research into the low-biomass microbiome of the intrauterine space. Although studies of the vaginal microbiome have yet to consistently discriminate a microbial signature associated with PTB, studies of the placental microbiome are beginning to provide insight into the complex interplay between microbiota and host in the context of spontaneous PTB. Further investigation of this unique microbiome (including culturing of live microbes) and those of other tissues not typically associated with PTB (eg, gut or oral cavity), may be necessary to fully understand how microbiota influence pregnancy outcomes. Microbiome science is emerging as a powerful approach to unraveling the complexities underlying PTB, but more questions have arisen than investigators have been able to answer. Adequately powered, large cohort studies inclusive of a diverse demographic population are required to fill the remaining gaps in our knowledge, but nevertheless, studies on our microbial counterparts, which have been aptly termed our second genome, offers tremendous promise to understand and eliminate PTB.

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