Implications of the Vaginal Microbiome in Preterm Premature Rupture of Membranes (PPROM)

Lead Research Organisation: Imperial College London
Department Name: Dept of Surgery and Cancer

Abstract

Preterm birth (PTB) is a major health issue for the global community representing the main cause of infant death and illness in both developed and developing countries. PTB occurs in approximately 10% of all pregnancies placing immense financial and emotional burden on society. Despite decades of research effort, rates remain constant or are rising. In approximately 30% of cases, PTB is preceded by preterm premature rupture of membranes (PPROM), often referred to as when a pregnant woman's water breaks early. When this occurs, there may be an interval of several weeks before preterm labour (PTL) establishes during which there is a risk of infection ascending from the vagina into the uterus. There exists strong evidence of a link between infection-induced inflammation and both PTB and injury to the brains of developing babies.

There are many causes of PPROM but infection plays a significant role. Normal human labour involves the "switching on" of many pro-inflammatory genes causing the cervix to ripen (enabling the baby to pass though the birth canal) and the initiation of uterine contractions during labour. If these genes are switched on too early during pregnancy, for example by a bacterial infection, PPROM may result and uterine contractions will begin prematurely. The result is devastating for the baby, which has yet to reach its full developmental potential in the womb and may acquire an infection during the process.

The vagina plays host to an array of 'normal' or 'good' bacteria (collectively called the microbiome) that have an important role in reproductive health maintenance throughout pregnancy. These bacteria act to inhibit the growth of 'bad' or pathogenic bacteria by producing high concentrations of acid and releasing small molecules called bacteriocins that exhibit antibiotic properties. If 'bad' pathogenic bacteria are able to take hold during pregnancy, they can ascend and infect the upper reproductive tract, causing PTB via the activation of inflammatory pathways associated with uterine contractions. During delivery the baby is exposed to bacteria present in the birth canal and thus they play an important role in the outcome for the baby.

Clearly there is a need for a better understanding of the role played by the vaginal bacteria throughout pregnancy. There is also an urgent need for a method to identify early in pregnancy women at risk of PPROM. In this study I will use state of the art approaches to address both these needs. By using a technique known as "next-generation DNA sequencing", I will accurately identify and characterise the different bacteria residing in the vagina during pregnancy and examine how these bacteria differ in women who experience PPROM. I will also examine if these bacteria are exposed to the baby during delivery and analyse how specific bacteria implicated in PPROM activate inflammatory pathways that lead to PTL. The same bacterial DNA sequencing techniques will then be used to examine the effects of current clinical treatment (using the antibiotic erythromycin) on the vaginal microbiome. I will use techniques that will allow all of the chemicals in a body fluid to be examined simultaneously to develop personalised metabolic "fingerprints" of the vaginal fluid and urine from women who experience normal term pregnancy and those that result in PPROM. This will provide important information about how bacteria influence the chemical environment of the gestational tissues and will be used to rapidly and cost-effectively identify metabolite biomarkers that indicate risk of PPROM and PTL. Collectively this proposal will both shed new light upon the causes of PPROM and PTL and allow for rapid translation back into the clinic to improve management of risk of PTL. It will allow better patient monitoring and lead to improved strategies for the prevention of PPROM and its treatment when it occurs.

Technical Summary

Preterm Premature Rupture of Membranes (PPROM) precedes preterm birth (PTB) in 30-40% of cases and is a cause of neonatal mortality and morbidity. A relationship exists between ascending vaginal infection, PPROM and preterm labour (PTL). The vaginal microbiota of pregnancy is dominated by Lactobacillus spp., which inhibit the growth of pathogens through lactic acid and bacteriocin secretion. Bacterial exposure to the neonate occurs during delivery therefore the composition of vaginal microbiota is an important factor in neonatal colonisation. The hypothesis of this study is that a dysbiosis of the vaginal microbiome is a determinate of PPROM and influences the risk of subsequent infection, PTL and neonatal colonisation. The objectives of the study are to:
i) Characterise the "normal" vaginal microbiome throughout pregnancy using 454 pyrosequencing methods and determine how advancing gestational age affects the composition of the vaginal microbiome.
ii) Assess how the vaginal microbiome differs in pregnancies complicated by PPROM and examine how vaginal microbiota may influence time to delivery (latency), risk of ascending infection, neonatal outcome and determine how current clinical treatment (erythromycin) impacts upon the vaginal microbiome.
iii) Understand how vaginal microbiota may be functionally implicit in PPROM by testing for their presence in gestational tissues following delivery and identifying which inflammatory pathways are activated in these tissues.
iv) Use MS and NMR-based metabolic profiling to explore the inter-relationship between the vaginal microbiome and vaginal metabolome and assess how host-microbe interaction is reflected in the urinary metabolic profile.
v) Identify urinary metabolites with prognostic and diagnostic potential for PPROM. By applying a systems biology approach to PPROM, this proposal will improve understanding of the vaginal microbiome in PPROM should lead to improved clinical management and treatment strategies.

Planned Impact

Preterm birth (PTB) is the major cause of neonatal mortality and morbidity and is preceded by preterm premature rupture of membranes (PPROM) in 30-40% of cases. While the mechanism is unclear a strong relationship exists between PPROM, ascending vaginal infection and preterm labour (PTL). PTL is increasingly recognised as a syndrome, with multiple aetiologies, although it is still clinically managed and studied as if were a single disease. Currently it is not possible to determine the aetiology of PPROM prospectively or retrospectively nor can it be accurately predicted. This means that treatment strategies for PPROM are often "one size fits all" approaches used in heterogeneous populations of women who may be at risk of PPROM and subsequent PTL for a variety of different reasons.
The purpose of this study is to apply a systems biology approach to understand the role of the vaginal microbiome in PPROM and PTL. The proposal will lead to the identification of metabolic biomarkers with prognostic and diagnostic potential that may be useful for patient stratification that would substantially improve current clinical strategies by enabling personalised therapeutic targeting.
In the immediate short-term (<5 years), beneficiaries of this research would obviously include clinicians and scientists with an interest in studying the aetiology of preterm birth. However in mid-term (5-10 years) this work would be beneficial to clinicians, hospital managers, primary care trust and health service policy-makers who may wish to introduce screening programs designed to more rationally and economically target surveillance and prevention of women at genuinely high risk of PPROM and PTL. Similarly, those women who experience PPROM could be stratified on the basis of underlying causative mechanism enabling more appropriate, personalised management of their treatment.
In this time frame, the results of this study will prove beneficial to the pharmaceutical industry by providing new biomarkers useful for the prediction of PPROM and PTL. Better understanding of the mechanisms of PPROM and PTL will also provide new targets for the rational development of treatments for these pathologies. The systems biology approach outlined in this proposal would also benefit clinicians and industry partners who wish to trial interventions in stratified/phenotyped subject cohorts.
The aforementioned beneficiaries would collectively impact and benefit the life and health of pregnant women, their children and their families. The ability to predict and prevent PPROM and subsequent PTL will have a major affect in reducing the economic, social, and psychological effects upon affected families and upon society in general. Prematurity represents an enormous financial burden to the NHS with initial hospitalisation costs increasing from £500 per term infant to £300,000 for an extremely preterm surviving infant. It is estimated that prolonging pregnancy by two weeks at gestational ages less than <33 weeks could result in cost savings of £50,000 per case. In the longer term, costs of education and health care for the preterm infant are dramatically increased- the annual economic burden of being born preterm is estimated to be £60,000. EU data shows that infants born preterm have lessened abilities to enter and contribute to the work force whereas the trauma associated with PTB for the mother can not only lead to immediate health problems (physical and mental) but also can impact on her willingness and ability to return to the workforce.

Publications

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Description Institute for Translational Medicine and Therapeutics (ITMAT)
Amount £140,377 (GBP)
Funding ID P58434 
Organisation National Institute for Health Research 
Department NIHR Imperial Biomedical Research Centre
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 07/2015 
End 03/2016
 
Title Cervical stitch for preterm birth prevention dataset 
Description This database provides 16S rRNA bacterial sequencing data from longitudinal samples collected from women receiving either monofilament or braided cervical sutures for preterm birth prevention. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Preterm birth, the leading cause of death in children under five, is an inflammatory driven process frequently triggered by ascending vaginal infection. Some 2 million cervical cerclages are performed annually to prevent preterm birth. The procedure is thought to provide structural support and maintain the endocervical mucus plug as a barrier to ascending infection. Multifilament braided suture material is predominantly used for cerclage without an evidence base. In a study of 678 women receiving cervical cerclage in 5 UK university hospitals, we showed that braided cerclage was associated with increased rates of intrauterine death (16% v 5%, P < 0.0001) and preterm birth (28% v 17%, P < 0.001) compared to monofilament alternative. A prospective, longitudinal study of women at risk of preterm birth due to short cervical length (<25mm) randomised to braided (n=25) or monofilament cerclage (n=24) revealed that braided suture induces a persistent shift towards vaginal microbiome dysbiosis characterized by reduced Lactobacillus spp. and enriched numbers of pathobionts. Increased vaginal dysbiosis was associated with inflammatory cytokine and interstitial collagenase excretion into cervicovaginal fluid (CVF) and premature cervical remodeling. In comparison, monofilament suture has minimal impact upon the vaginal microbiome and maternal host-interactions. These data provide in vivo evidence that a dynamic shift of the human vaginal microbiome toward dysbiosis is a determinant of preterm birth. 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB11895
 
Title The interaction between the vaginal microbiome, cervical length and vaginal progesterone treatment for preterm birth risk 
Description 16S rRNA bacterial sequencing data collected longitudinally from women with short (<25mm) or normal (>25mm) cervical length who subsequently experience preterm or term delivery. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact A short cervix in the second trimester of pregnancy is a risk factor for preterm birth (PTB). Vaginal progesterone reduces this risk in specific patient cohorts. In a group of women at high risk of PTB (n=161) we examined the relationship between second trimester vaginal microbiome using 16S rRNA gene sequencing and subsequent preterm (<34 weeks, n=34) or term (>37 weeks, n=127) delivery. Additionally we assessed the effect of vaginal progesterone therapy on the vaginal microbiota. Lactobacillus iners dominance at 16 weeks was shown to be significantly associated with both a short cervix <25mm (P<0.05), and PTB <34+0 weeks (P<0.01; 69% PPV). In contrast, L. crispatus dominance was highly predictive of term birth (98% PPV). Cervical shortening or PTB were not associated with vaginal dysbiosis. A longitudinal characterization of vaginal microbial structure at <18, 22, 28 and 34 weeks in women receiving vaginal progesterone (400mg/OD, n=42) or controls (n=25) showed that progesterone does not alter the vaginal microbiome and is not associated with a reduction of L. iners-associated early PTB (<34 weeks). In summary, Lactobacillus iners dominance of the vaginal microbiome at 16 weeks gestation is a risk factor for preterm birth, whereas L. crispatus dominance is protective against preterm birth. Vaginal progesterone does not reduce the risk of PTB through effects upon the vaginal microbiome. 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB12577
 
Title Uncomplicated UK vaginal microbiome 
Description Abnormal vaginal microbiomes have been implicated in PTL risk. Following vaginal delivery the gut microbiome of the neonate is strongly influenced by the maternal vaginal microbiome. Studies using next-generation DNA sequencing approaches to explore the vaginal microbiome in pregnancy have been historically limited to Northern American populations. We have undertaken the first study of the vaginal microbiome in a mixed ethnic pregnant UK population (n=42) who all experienced uncomplicated, term pregnancies, using the MiSeq platform. While the results of this study are currently under review, we have uploaded the raw sequence data and accompanying metadata for public access at the European Nucleotide Archive's (ENA) Sequence Read Archive (SRA) (Accession number: PRJEB7703). 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Our results show that the vaginal microbiome of our UK population differs markedly from Northern American populations and shows for the first time that there is a dynamic shift in the structure of the vaginal microbiome in the weeks following delivery. Our dataset will enable others in the field to analyse our data using new or alternative approaches and as a basis for similar studies investigating implications of the vaginal microbiome in pregnancy outcomes. 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB7703