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

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.

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.

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.