The pregnancy microbiome and preterm birth

Preterm birth is a global health concern and continues to contribute to substantial neonatal morbidity and mortality despite advances in obstetric and neonatal care. The underlying aetiology is multi-factorial and remains incompletely understood. In this review, the complex interplay between the vaginal microbiome in pregnancy and its association with preterm birth is discussed in depth. Advances in the study of bacteriology and an improved understanding of the human microbiome have seen an improved awareness of the vaginal microbiota in both health and in disease

The local and systemic immune response to intrauterine LPS in the prepartum mouse

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 μg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta

Specific Lipopolysaccharide Serotypes Induce Differential Maternal and Neonatal Inflammatory Responses in a Murine Model of Preterm Labor.

Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models

Modeling hormonal and inflammatory contributions to preterm and term labor using uterine temporal transcriptomics

BACKGROUND: Preterm birth is now recognized as the primary cause of infant mortality worldwide. Interplay between hormonal and inflammatory signaling in the uterus modulates the onset of contractions; however, the relative contribution of each remains unclear. In this study we aimed to characterize temporal transcriptome changes in the uterus preceding term labor and preterm labor (PTL) induced by progesterone withdrawal or inflammation in the mouse and compare these findings with human data. METHODS: Myometrium was collected at multiple time points during gestation and labor from three murine models of parturition: (1) term gestation; (2) PTL induced by RU486; and (3) PTL induced by lipopolysaccharide (LPS). RNA was extracted and cDNA libraries were prepared and sequenced using the Illumina HiSeq 2000 system. Resulting RNA-Seq data were analyzed using multivariate modeling approaches as well as pathway and causal network analyses and compared against human myometrial transcriptome data. RESULTS: We identified a core set of temporal myometrial gene changes associated with term labor and PTL in the mouse induced by either inflammation or progesterone withdrawal. Progesterone withdrawal initiated labor without inflammatory gene activation, yet LPS activation of uterine inflammation was sufficient to override the repressive effects of progesterone and induce a laboring phenotype. Comparison of human and mouse uterine transcriptomic datasets revealed that human labor more closely resembles inflammation-induced PTL in the mouse. CONCLUSIONS: Labor in the mouse can be achieved through inflammatory gene activation yet these changes are not a requisite for labor itself. Human labor more closely resembles LPS-induced PTL in the mouse, supporting an essential role for inflammatory mediators in human "functional progesterone withdrawal." This improved understanding of inflammatory and progesterone influence on the uterine transcriptome has important implications for the development of PTL prevention strategies

The interaction between vaginal microbiota, cervical length and vaginal progesterone treatment for preterm birth risk

Background: Preterm birth is the primary cause of infant death worldwide. A short cervix in the second trimester of pregnancy is a risk factor for preterm birth. In specific patient cohorts, vaginal progesterone reduces this risk. Using 16S rRNA gene sequencing we undertook a prospective study in women at risk of preterm birth (n=161) to assess 1) the relationship between vaginal microbiota and cervical length in the second trimester and preterm birth-risk, and 2) the impact of vaginal progesterone on vaginal bacterial communities in women with a short cervix. Results: Lactobacillus iners dominance at 16 weeks gestation was significantly associated with both a short cervix <25mm (n=15, P<0.05), and preterm birth <34+0 weeks (n=18, 38P<0.01; 69% PPV). In contrast, L. crispatus dominance was highly predictive of term birth (n=127, 98% PPV). Cervical shortening and preterm birth were not associated with vaginal dysbiosis. A longitudinal characterization of vaginal microbiota (<18, 22, 28 and 34 weeks) was then undertaken in women receiving vaginal progesterone (400mg/OD, n=25) versus controls (n=42). Progesterone did not alter vaginal bacterial community structure nor reduce L. iners-associated preterm birth (<34 weeks). Conclusions: L. iners dominance of the vaginal microbiota at 16 weeks gestation is a risk factor for preterm birth, whereas L. crispatus dominance is protective against preterm birth. Vaginal progesterone does not appear to impact the pregnancy vaginal microbiota. Patients and clinicians who may be concerned about ‘infection risk’ associated with use of a vaginal pessary during high-risk pregnancy can be reassured

The association between vaginal bacterial composition and miscarriage: a nested case-control study

OBJECTIVE: To characterise vaginal bacterial composition in early pregnancy and investigate its relationship with first and second trimester miscarriages. DESIGN: Nested case-control study. SETTING: Queen Charlotte's and Chelsea Hospital, Imperial College Healthcare NHS Trust, London. POPULATION: 161 pregnancies; 64 resulting in first trimester miscarriage, 14 in second trimester miscarriage and 83 term pregnancies. METHODS: Prospective profiling and comparison of vaginal bacteria composition using 16S rRNA gene-based metataxonomics from 5 weeks gestation in pregnancies ending in miscarriage or uncomplicated term deliveries matched for age, gestation and body-mass index. MAIN OUTCOME MEASURES: Relative vaginal bacteria abundance, diversity and richness. Pregnancy outcomes defined as first or second trimester miscarriage, or uncomplicated term delivery. RESULTS: First trimester miscarriage associated with reduced prevalence of Lactobacillus spp.-dominated vaginal microbiota classified using hierarchical clustering analysis (65.6% vs. 87·7%; P=0·005), higher alpha diversity (mean Inverse Simpson Index 2.5 (95% confidence interval 1.8-3.0) vs. 1.5 (1.3-1.7), P=0·003) and higher richness 25.1 (18.5-31.7) vs. 16.7 (13.4-20), P=0·017), compared to viable pregnancies. This was independent of vaginal bleeding and observable before first trimester miscarriage diagnosis (P=0·015). Incomplete/complete miscarriage associated with higher proportions of Lactobacillus spp.-deplete communities compared to missed miscarriage. Early pregnancy vaginal bacterial stability was similar between miscarriage and term pregnancies. CONCLUSIONS: These findings associate the bacterial component of vaginal microbiota with first trimester miscarriage and indicate suboptimal community composition is established in early pregnancy. While further studies are required to elucidate the mechanism, vaginal bacterial composition may represent a modifiable risk factor for first trimester miscarriage

The vaginal microbiome during pregnancy and the postpartum period in a European population

The composition and structure of the pregnancy vaginal microbiome may influence susceptibility to adverse pregnancy outcomes. Studies on the pregnant vaginal microbiome have largely been limited to Northern American populations. Using MiSeq sequencing of 16S rRNA gene amplicons, we characterised the vaginal microbiota of a mixed British cohort of women (n = 42) who experienced uncomplicated term delivery and who were sampled longitudinally throughout pregnancy (8–12, 20–22, 28–30 and 34–36 weeks gestation) and 6 weeks postpartum. We show that vaginal microbiome composition dramatically changes postpartum to become less Lactobacillus spp. dominant with increased alpha-diversity irrespective of the community structure during pregnancy and independent of ethnicity. While the pregnancy vaginal microbiome was characteristically dominated by Lactobacillus spp. and low alpha-diversity, unlike Northern American populations, a significant number of pregnant women this British population had a L. jensenii-dominated microbiome characterised by low alpha-diversity. L. jensenii was predominantly observed in women of Asian and Caucasian ethnicity whereas L. gasseri was absent in samples from Black women. This study reveals new insights into biogeographical and ethnic effects upon the pregnancy and postpartum vaginal microbiome and has important implications for future studies exploring relationships between the vaginal microbiome, host health and pregnancy outcomes

Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs

Herein we demonstrate the first example of oligonucleotide-templated reaction (OTR) performed on paper, using lateral flow to capture and concentrate specific nucleic acid biomarkers on a test line. Quantitative analysis, using a low-cost benchtop fluorescence reader showed very high specificity down to the single nucleotide level and proved sensitive enough for amplification-free, on-chip, detection of endogenous concentrations of miR-150-5p, a recently identified predictive blood biomarker for preterm birth

Lateral Flow Test (LFT) detects cell-free microRNAs predictive of preterm birth directly from human plasma

Despite extensive research toward the development of point-of-care nucleic acid tests (POC NATs) for the detection of microRNAs (miRs) from liquid biopsies, major hurdles remain including the strict requirement for extensive off-chip sample preprocessing. Herein, a nucleic acid lateral flow test (NALFT) is reported on that enables the direct detection of endogenous miRs from as little as 3 μL of plasma without the requirement for any enzyme-catalyzed target amplification or complex miR extraction steps. This is achieved through integration of a denaturing hydrogel composite material onto the LFT, allowing for near-instantaneous on-chip release of miRs from their carriers (extracellular vesicles or transport proteins) prior to detection. This next-generation LFT is sensitive enough to detect endogenous concentrations of miR-150-5p, a predictive biomarker for preterm birth (PTB) found deregulated in maternal blood from as early as 12th week of pregnancy. Herein, a key step is represented toward a first bedside test for risk-stratification during pregnancy by predicting true outcome at a very early stage. More generally, the universal and versatile nature of this novel sample preprocessing platform can further improve the robustness of existing NALFTs and facilitate their application at the POC