Preterm birth is a worldwide health concern accounting for 11.1% of all births. It is linked to adverse outcomes including cerebral palsy and chronic lung disease. Parturition is associated with marked inflammation in the gestational tissues with the myometrium being the key effector, responsible for coordinated contractions that lead to delivery of the fetus. Despite multiple theories, the mechanism underlying the onset of human labour remains unclear. As there is currently no effective method in stopping labour once the process has begun, research is focused on prevention. Currently, the only drug agent with proven efficacy is progesterone (P4), which has been shown to reduce the incidence of preterm labour (PTL) by up to 40%. P4 is known to have anti-inflammatory action, however its mode of action in the context of reducing the rate of PTL remains unclear. The overall aim of this project was to further understand the mechanism of P4 action in order to facilitate the development of superior drugs to combat PTL. Using a pregnant murine model, my studies have shown that vehicle control labour is associated with inflammation. Further, P4 supplementation was sufficient to delay labour by at least two days in association with a delay in inflammation and a loss of the rise in contraction-associated proteins including connexin-43. A second pregnant murine model was used to block P4 action with the mixed progesterone receptor (PR) and glucocorticoid receptor antagonist mifepristone (RU486), with animals delivering preterm at 17.4 ± 0.35 hours post administration. A pre-delivery (9 hours post RU486) significant up-regulation of the contraction-associated proteins connexin-43 and oxytocin receptor was observed in association with a peak in p65 and p38 phosphorylation. Inflammatory cytokines, such as IL-6, peaked at the time of labour at the RNA level and demonstrated a bimodal peak at the protein level (9 hours and labour). Much of the in vitro work on myometrial function is hampered by a lack of physiological relevance due low PR levels and the requirement for treatment with high P4 doses. In order to address this, I have developed and validated via microarray a myometrial explant model to study P4 function. Subsequently, I have shown that, in this model, P4 signals via PR and that this is associated with a reduction in p65 and c-Jun phosphorylation in an IL-1β-driven model of inflammation. I have also shown that in explants obtained from labouring patients, P4 does not lose the ability to repress inflammation. Finally, several authors have proposed a variety of mechanisms to account of the functional withdrawal of P4 in human parturition. In order to investigate some of these further, I have used the group's myometrial tissue bank. I have shown that myometrial P4 levels do not change with labour onset and confirmed other groups' reports that term labour is associated with a shift to PR-A dominance. I went on to show that, compared with idiopathic preterm labour, preterm labour secondary to chorioamnionitis is associated with a significant reduction in nuclear receptor corepressor (NCoR) and steroid receptor coactivator 1 (SRC1), as well as a significant increase in heat-shock protein 90 (HSP90) and FK506-binding protein 51 (FKBP51). Additionally, I demonstrated that preterm labour in twins is associated with a significant up-regulation of NCoR. In summary, the findings of this thesis broaden our understanding of P4 function and will help drive the field of parturition and PTL forward.

Background: The Growmore Study was an observational cohort study of adolescents born preterm, in Newcastle-upon-Tyne. The cohort were born between 1993 and 1998: mean gestation at birth was 31 weeks (range 24+5d to 36+2d) and birthweight 1392g (690-2200g). Individuals were originally recruited into one of two randomised, controlled trials (‘growth’, n=113; and ‘protein’, n=134). As separate groups they were followed up at intervals, undergoing assessment of growth and development. From 247 children originally recruited, 220 completed assessments to 24 months of age. At age 10 underwent cognitive assessment. Between 9 and 13 years old, the two cohorts were amalgamated to a single cohort and underwent further auxological and metabolic testing (n=153/247) including DEXA scan (n=109) and bloods (n=139). The current study revisited the cohort, aged between 12 and 18 years old. Aims: The study aimed to explore relationships between: growth in early life; body fat deposition; mitochondrial oxidative capacity; and quantitatively assessed diet and activity in ex-preterms. Methods: 60 of the 235 traceable members of the original cohort were recruited into this study. They underwent multimodal assessment, including: auxological measures; body composition measurement by air-displacement plethysmography and skinfold thickness; magnetic resonance spectroscopy (MRS), using a 3-Tesla scanner, custom-built coils and a tailored scanning routine to quantify skeletal muscle mitochondrial oxidative capacity, lipid content of the liver, and fat-containing tissue at the L2/3 vertebral level; a standard OGTT (bloods taken at 0 and 120minutes); and serum insulin, glucose, lipid profile, liver function and Vitamin D measurement. Dietary intake was assessed using a computer-based recall diary and physical activity by wearing of accelerometers. Data was analysed by using a variety of statistical methods including comparative, correlation and regression analysis. Results: The 60 adolescents recruited for this study had a mean gestation at birth of 31 weeks (range 26+1d to 34+4d) with birthweight of 1370g (range 840-1870g). Their mean age at study was 15.5 years and M:F ratio was 1:1.4. Analysis showed they were not significantly different from their peers at the previous cohort assessment and both of the two original RCTs were almost equally represented (‘growth’: ‘protein’ = 1:1.07). The current study showed that amongst this cohort subgroup, vitamin D status iii correlated with time of year (p=0.046) and current weight SDS (p= 0.039). Skeletal muscle oxidative function was significantly related to vitamin D status (p=0.021) and gestational age at birth (p=0.005); combined r2:0.31; p=0.002). Earlier gestational age (GA) at birth and lower serum vitamin D was associated with reduced oxidative capacity. Physical activity was not associated with oxidative capacity. Visceral adipose tissue (VAT), circulating triglyceride (TG) and waist circumference were strongly associated with hepatic lipid content (all p<0.001); dietary intake was not. VAT and TG were highly significant when the model was adjusted for Tanner Stage (r2: 0.4; p=0.0002). GA and birthweight were not related to hepatic lipid deposition. Insulin sensitivity by two different measures was predicted by triglyceride levels (p<0.001), light activity (P<0.05) and vitamin D levels (p<0.05). Conclusion: Environment and early life both have an influence on adolescent physiology. The strength of association between vitamin D and muscle oxidative capacity has been observed in other conditions, but the contribution of gestation at birth in those born preterm is a novel finding. This may reflect either a variance in muscle fibre type or mitochondrial density directly related to developmental arrest or delay as a result of preterm birth. Vitamin D status also influences insulin sensitivity, as seen in other populations: Vitamin D status is an obvious target for dietary advice. Absence of an association between gestation and adiposity, and correlation between VAT and hepatic lipid deposition suggests that there are opportunities for children born preterm to improve their health in adolescence, and by implication, their future adult health.

Pre-eclampsia is a disease unique to pregnancy. Prevalence in the UK is between 5- 8% of pregnancies yet diagnosis remains challenging. The PELICAN study was a multi-centre, observational cohort study. The primary aim was to evaluate the diagnostic accuracy of plasma placental growth factor (PlGF) in the second half of pregnancy, in predicting the need for delivery for pre-eclampsia within 14 days of testing. 649 women presenting with suspected pre-eclampsia were recruited between January 2011 and February 2012, across seven consultant-led units within England and Ireland. Blood samples were taken at enrolment; PlGF measurements were performed but results blinded until the study was complete and diagnoses and pregnancy outcome known. A further 47 biomarkers were measured (using 57 assays) to evaluate whether the diagnostic potential of PlGF could be improved further. Using a pre-specified cut off of <5th centile, a low (>12pg/ml < 5th centile) or very low (<12pg/ml) PlGF concentration was shown to have high sensitivity (0.95 CI (0.89- 0.99) in women < 35 weeks’ gestation) to determine need for delivery within 14 days. When compared with other biologically plausible biomarkers, the area under the ROC curve for low or very low PlGF (0.87, standard error 0.03), was greater than all other commonly utilised tests either singly or in combination (range 0.58–0.76; p<0.001 for all comparisons). Data from 100 women were then used to perform a budget impact analysis. A hypothetical decision analytical model using data extracted from case note review and reference cost tariffs, suggested a mean cost saving associated with the PlGF test (in the PlGF plus management arm) of £35,087 (95% CI -£33,181 to -£36,992) per 1,000 women, equating to a saving of £582 (95% CI -£552 to -£613) per woman tested. PlGF testing could be used to risk-stratify women with suspected pre-eclampsia with the aim of improving pregnancy outcome.

Background: Infants of mothers who smoked (S) or substance misused (SM) during pregnancy have an increased risk of sudden infant death syndrome (SIDS). Hypotheses: Infants of SM and S mothers will have poorer ventilatory responsiveness to hypercarbia and hypoxia and reduced chemoreceptor sensitivity compared to controls. Impairment of ventilatory responsiveness and chemoreceptor sensitivity will be greater in the infants of mothers who both substance abuse and smoke compared to those whose mothers only smoke. Methods: Three groups were recruited: 1. Infants of mothers with a history of substance misuse during pregnancy (SM infants) 2. Infants of mothers with a history of smoking during pregnancy (S infants). 3. Infants of mothers who neither smoked nor misused substances during pregnancy (Controls). Ventilatory responses to hypercarbia and hypoxia were assessed in the newborn period and at 6-12 weeks of age. Results: In the newborn period both the SM and S infants had a lower ventilatory response to 2% and 4% CO2 than the controls. The ventilatory response to CO2 was lower in the SM infants compared to the S infants. In response to hypoxic challenge in the newborn period, SM infants had a greater magnitude of decline in minute volume than the S infants and the controls. In addition, the rate of decline in minute volume was greater in the SM infants and the S infants compared to the controls. At 6-12 weeks of age S and SM infants had a dampened ventilatory response to hypercarbia and a greater magnitude of decline in minute volume in response to hypoxia compared to controls. Dampening of the ventilatory response to hypercarbia was greater at the peak age of SIDS compared to the perinatal period.

Background: Preeclampsia (PE) affects 2-3% of all pregnancies and is a major cause of maternal and perinatal morbidity and mortality. It is thought to occur due to abnormal placentation characterised by poor trophoblastic invasion resulting in oxidative stress and release of factors that promote endothelial dysfunction and inflammation. The current approach of screening for PE is to identify risk factors from maternal demographic characteristics and medical history. In the United Kingdom, the National Institute for Health and Clinical Excellence (NICE) has issued guidelines recommending that women should be considered to be at high risk of developing PE if they have any 1 high-risk factor or any 2 moderate-risk factors. With this approach, defined by NICE, at a screen positive rate of 11%, the detection rate (DR) for total PE is 35%. Such a screening approach has two main limitations. Firstly, it does not provide individualised, patient specific results and secondly, it does not allow the integration of biomarkers for improving the performance of the screening test. However, the integration of such biomarkers is essential in achieving an effective screening strategy for PE. Objectives: The aims of the papers included in this thesis are firstly, to identify and quantify the effects of variables from maternal characteristics and medical history on specific biochemical markers, secondly to present a model for standardising biochemical marker measurements in all three trimesters of pregnancy into multiples of the normal median (MoM) values, thirdly to summarize the distribution of MoM values in pregnancies with normal outcomes and those that subsequently develop PE and fourthly, to examine the potential improvement in performance of screening for PE at 30-34 weeks’ gestation by maternal factors alone with the addition of biophysical and biochemical markers. Methods: The data for this thesis were derived from prospective screening of women with singleton pregnancies attending for three routine hospital visits at 12, 22 and 32 or 36 weeks’ gestation. We have recorded a series of maternal characteristics and history, measured the maternal weight and height as well as the uterine artery pulsatility index (UTPI), mean arterial pressure (MAP), serum concentration of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFLIT-1). The pregnancy outcomes were obtained from the hospital maternity records or the general medical practitioners of the women. Bayes theorem was used to combine the a priori risk from maternal factors with various combinations of biomarker MoM values. The potential value of biophysical and biochemical markers in improving the performance in screening for PE were evaluated. Results: Firstly, in pregnancies that developed PE, serum PlGF is decreased, while sFLIT-1, MAP and UTPI were increased, secondly, the separation in MoM values from normal is greater with earlier than later gestational age at which delivery for PE is necessary and thirdly, the slope of the regression lines of PlGF MoM with gestational age at delivery in pregnancies that develop PE increases with gestational age at screening. Combined screening at 30-34 weeks’ gestation by maternal factors, MAP, UTPI, PlGF, and sFLIT-1 predicted 98% (95% confidence interval, 88- 100%) of preterm PE and 49% (95% confidence interval, 42-57%) of term PE, at a false positive rate of 5%. Conclusions: This thesis has demonstrated that biophysical and biochemical markers increase significantly the performance of screening for PE and as a result the timing and content of clinical visits can be defined by the patient-specific risk of developing the disease. The vast majority of women would be screened low risk and these can follow the routine antenatal care, whereas those few who are high risk could be directed to a more specialized pathway, where early therapeutic interventions prophylactically may lead to the prevention of the disease and close follow-up will reduce the adverse consequences of PE.

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Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects and neurodevelopmental delay, with worst outcomes in congenital infection. HCMV exacerbates neurological disease and progression to AIDS in HIV co-infected infants. In Zambia HCMV has adverse effects on growth and development even in maternally HIV-exposed uninfected infants, affecting a third. Transmission to infants can be congenital or postnatal primarily through breast milk, similar to HIV. However, the route affecting infant development is not established, but critical to understand for deployment of appropriate interventions and hospital diagnostics. In this thesis, congenital HCMV infection was investigated in newborns using PCR and nucleotide sequencing of saliva and umbilical cord DNA, and showed 1% prevalence, which was too low to account for the widespread adverse growth effects. Therefore, maternal HCMV secretion via breast milk and the effects of maternal HIV were investigated. HCMV viral loads were measured by qPCR in 461 breast milk samples from both HIV-infected and uninfected mothers at postpartum day 3 and weeks 2, 4, 9, 12, and 16. Results showed higher HCMV viral loads and longer secretion in HIV-positive compared to negative mothers, peaking at week 4 (p=.026) and remaining elevated at week 16 (p < .001). HCMV strains were investigated using the hypervariable glycoprotein gO, with all eight genotypes detected, but no relationship to viral load. Next, Illumina NGS was used to further analyse strains and mixed infections in order to assess the burden of infection. A script was developed to identify linked gO and gN 'Molecular tags' via analyses of FASTQ reads. In 21 samples, mixed infections were identified, showing 1-7 genotypes and a dominant genotype constituting 45-100%. ZMB240, a full-length HCMV genome - the first reference strain from a healthy donor representing a transmission population, and also first from Africa - was derived directly from breast milk DNA and found similar to elsewhere, including the reference Merlin. Overall, the thesis provided evidence that breast milk is the principal route for early HCMV infection in breastfed Zambian infants, and that maternal HIV increases HCMV load, burden of infection, and duration of secretion in breast milk, thereby escalating and prolonging risk of early infant HCMV infection.

While it is clear that the control of implantation is multifactorial, one emerging component that appears to be important in the success or failure of embryo implantation is the endocannabinoid system. The primary ligand of the endocannabinoid system (ECS) is anandamide (AEA) which is synthesised by a N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degraded by fatty acid amide hydrolase (FAAH). A careful balance in the activities of NAPE-PLD and FAAH is required to ensure the appropriate levels of AEA are available during implantation. The purpose of this thesis was to explore further the role of the ECS, specifically its role in uterine receptivity using both in-vivo and in-vitro models. In-vivo models were used to study the expression of the ECS by measuring plasma concentrations of AEA, along with OEA and PEA (two other ligands of the ECS). A statistically significant change in plasma PEA concentrations was noted when comparing urine pregnancy test results. No significant differences in either AEA or OEA plasma concentrations were demonstrated. In-vitro models were used to investigate the interactions between galectin 3, integrin β3 and the ECS. The results show that while galectin 3 did not have any effect on the ECS, up-regulation of the expression of integrin β3 in receptive endometrial cells both increased the expression of FAAH and decreased the expression on NAPE-PLD in a dose-dependent manner. However, no effect was demonstrated in non-receptive endometrial cells, suggesting that integrin β3 expression, in collaboration with the ECS, plays an important role in endometrial receptivity. Overall, this work gives us further insight into the immensely complex processes involved in ensuring the endometrium is receptive to an embryo. Most importantly, it has suggested a link between the expression of integrin β3 and the enzymes of the ECS that is absent in the non-receptive endometrium.