Preterm labour and dysfunctional term labour lead to the most pre-eminent problems in obstetric practice today. We currently lack effective strategies for preventing spontaneous preterm labour, and prematurity remains the leading cause of perinatal morbidity and mortality. In contrast, some women do not labour effectively at term, or pregnancy becomes prolonged, both of which increase the risk of caesarean section, rates of which are on the rise in this country. Human labour is an inflammatory process, characterised by an influx of inflammatory cells into the myometrium and cervix at the onset of labour, and an increase in production of proinflammatory cytokines and expression of cell adhesion molecules. A substantial body of evidence has now accumulated in support of this theory, but relatively little attention has yet focused on anti-inflammatory pathways within this process. The resolution of inflammation is now recognised to be an active process, driven by a variety of endogenous pro-resolution factors. These include the lipid mediator lipoxin A4 (LXA4), derived from arachidonic acid and signalling through a G-protein coupled receptor, a member of the formyl peptide receptor family, FPR2/ALX. Another is Annexin A1 (ANXA1), a glucocorticoid-regulated peptide that also signals via FPR2/ALX. The aim of this thesis was to explore the role of these anti-inflammatory and pro-resolution mediators in myometrial and placental tissues and examine what role they may have within the inflammatory process of labour. Expression of the receptor FPR2/ALX and enzymes involved in the synthesis of LXA4 was determined by quantitative RT-PCR in labouring and non-labouring tissues of the reproductive tract and FPR2/ALX was localised in these tissues using immunohistochemistry. It was found that expression of the receptor and the enzyme 5-lipoxygenase (ALOX5) were upregulated in labouring tissues and that the receptor localised primarily to immune cells. A microarray and functional genomics were used to examine inflammation in myometrium and interesting parallels were drawn between artificially-induced inflammation and the process of labour, identifying pathways of interest for further study. Myometrial tissue was cultured in hypoxia, a pro-inflammatory environment, and it was demonstrated by ELISA and quantitative RT-PCR that LXA4 production was unaffected in these conditions, but that there may be changes in expression of ALOX5 and FPR2/ALX. Quantitative RT-PCR and immunohistochemistry were used to explore the expression of ANXA1 in labouring and non-labouring tissues and found localisation to immune cells and vascular endothelial cells but no difference in expression in labour. Tissue culture and quantitative RT-PCR demonstrated glucocorticoidregulation of FPR2/ALX but not ANXA1 in myometrium. This work has shown that LXA4 and ANXA1 are present in the reproductive tract during pregnancy and parturition and that a mechanism may exist, via upregulation of their common receptor, for a role within the inflammatory process of labour. Further work will aim to clarify the nature of this role and its functional effects, with the ultimate aim of better understanding the process of inflammation during parturition leading to the development of strategies to prevent or treat preterm and dysfunctional term labour.

Within my thesis I have shown that women with congenital heart disease are amenable to preconception counselling, but that many women are not referred to see a specialist cardiologist or obstetrician prior to pregnancy. Women with more severe forms of heart disease such as a Fontan repair have worse obstetric outcomes with high rates of miscarriage, preterm delivery and postpartum haemorrhage (PPH), hence the importance of comprehensive counselling prior to pregnancy. When women are counselled prior to pregnancy regarding the potential risks that they face, I have shown that the quoted risk of an adverse event assessed by an experienced clinician has a better correlation with actual outcome than other validated scoring systems such as the modified WHO score. Women with congenital heart disease are at much greater than average risk of postpartum haemorrhage (PPH). My analysis has shown that after correcting for known risk factors for PPH, women with a Fontan repair are at the highest risk of increased blood loss, and that low molecular weight heparin at both a prophylactic and therapeutic dose is associated with higher rates of PPH even when stopped at an appropriate time prior to delivery. I have focussed on the management of labour in women with heart disease, in particular of the second and third stages. Having shown that a restricted second stage for women with heart disease recommended by the multidisciplinary team (MDT) is often not adhered to in our actual clinical practice, my pilot study showed that there was only a modest measured haemodynamic effect of pushing (bearing down), with a small but significant increase in maternal heart rate in the active second stage of labour. I have found from a literature review and from our own historical dataset that the practice of a restricted oxytocin regimen for the management of the third stage (designed to minimise cardiovascular side-effects) is associated with greater rates of PPH, mainly due to uterine atony. In a prospective cohort study, women with heart disease were allocated to either a low dose infusion or a combination of the low dose infusion with an initial bolus of 2IU of oxytocin over ten minutes; those receiving the additional oxytocin had a significantly lower volume of blood loss and rate of PPH. Importantly, the women receiving the higher dose of oxytocin had no measured deleterious cardiovascular side effects.

Betel (areca) nut is the fourth most widely used psychoactive substance globally, accounting for 10-20% of the world’s population. Its most basic form is betel ‘quid’ which consists of betel leaf, betel nut (the main psychoactive ingredient) and slaked lime. Evidence that betel quid and betel nut alone are associated with oral cancer has been established. Background: While there is a substantial body of evidence on the impact of health-risk behaviours including smoking and drinking alcohol on adverse pregnancy outcomes, studies on the impact of betel quid chewing on pregnancy outcomes are sparse and heterogeneous. Although several studies report the negative impact of betel quid chewing on pregnancy outcomes, the evidence is inconclusive. One of the challenges in understanding the impact of betel quid is to distinguish the impact of betel quid chewing from the impact of smoking. Bhutan, where low prevalence of smoking and high prevalence of betel-quid chewing are reported, provides a natural experimental environment for taking a close look at the impact of betel quid chewing alone. As a part of the global agenda to address preterm births (PTB) as a public health priority and in order to provide evidence to inform efforts to reduce neonatal morbidity and mortality in Bhutan, this study explores the impact of betel quid chewing on birth outcomes and its importance in relation to other risk factors. Methods: This study used a multi-centre case-control design. A case was defined as a mother of a singleton live born infant whose gestational age is less than 37 completed weeks and/or an infant whose birth weight is less than 2500 g. A control was defined as a mother of singleton live born term babies whose birth weight was more than 2500g and gestational age was greater than 37 weeks. Information was collected using a semi-structured questionnaire from February 2015 to the beginning of March 2016 at the three referral hospitals in Bhutan. Study participants were recruited by a trained interviewer during their post-delivery stay before discharge from each hospital. A statistical approach and a causal directed acyclic graph (DAG) approach were used for building logistic regression models. Results: Of the 669 study participants, 55% of the case mothers and 52% of the control mothers chewed betel quid during pregnancy. About 22% of cases and 22% of controls used commercial betel products during pregnancy. In total, 60% of the case mothers and 57% of the control mothers chewed either betel quid or packaged betel products during pregnancy. Neither the statistical approach nor DAG approach provided clear evidence of an association between betel quid use and low birth weight (LBW) or PTB. The adjusted odds ratio (aOR) of term LBW was 1.07 (95% CI: 8 0.54-2.13, p=0.845) in the statistical approach while the aOR of term LBW was 1.30 (95% CI: 0.74-2.27, p=0.439) in the DAG approach. Using the DAG approach, the aOR of PTB in association with betel quid chewing during pregnancy was 1.20 (95% CI: 0.72-2.00, p=0.614). When the total number of betel nuts consumed during the last three months of pregnancy was used as an exposure variable, the aOR for mothers who consumed more than one nut per day was 1.39 for term LBW (95%:0.52-3.68, p=0.514) and the aOR of PTB was 0.66 (95% CI: 0.27-1.66, p=0.383) compared to non-chewers. For a secondary outcome, the data suggest betel quid chewing is associated with increased odds of anaemia (aOR 2.09, 95% CI 1.27-3.43, p=0.004). Using the DAG approach, tobacco and alcohol use during pregnancy, low gestational weight gain, and urinary tract infection showed a clear association with term LBW and PTB. Conclusion: In the present study, the results provide no clear evidence of an association between term LBW or PTB and betel quid chewing during pregnancy. For a secondary outcome, the data suggest betel quid chewing is associated with increased odds of anaemia. The present study provides rich baseline data for mothers and established a cohort of cases and controls, which could be followed up to understand the short- and long-term effects of LBW and PTB and may help design effective interventions.

Mutations in maternally inherited mitochondrial DNA (mtDNA) can cause a range of complex diseases for which there are currently no curative treatments. Using IVF based techniques involving nuclear genome transplantation, it may be possible enable women who carry mtDNA mutations to have a genetically related child without the risk of transmitting disease. The central aim of this project is to perform preclinical studies testing the safety and efficiency of pronuclear transfer (PNT). Surprisingly, the PNT technique developed using abnormally fertilised zygotes was detrimental to survival of normally fertilised zygotes. We tested the possibility that this might be due to the relatively accelerated development of normally fertilised zygotes allowing insufficient time for recovery following transplantation of the pronuclei. Switching the timing of PNT to shortly after pronuclei appearance (ePNT) rather than shortly before disappearance resulted in increased survival. Further modification of the enucleation and embryo culture media resulted in improved blastocyst quality. As part of the optimisation process, I tested the effect and reversibility of drugs that are used to inhibit the cytoskeleton of oocytes and zygotes in preparation for manipulations. Comparison of two compounds, which directly inhibit actin polymerisation, revealed marked differences in the reversibility. However, latrunculin B, which is rapidly reversed, has a detrimental effect on blastocyst development compared with latrunculin A, which is more potent and less readily reversible. Finally, I analysed single-cell RNA-sequencing data to determine whether gene expression in human blastocysts is altered by ePNT. This work was done in collaboration with Dr Kathy Niakan at the Francis Crick Institute. The findings indicate no detectable differences in global or lineage-associated gene expression between control and good quality ePNT blastocysts. Analysis of mitochondrial gene expression revealed high variability in the level of expression both within and between blastocysts. However, this variability was observed in ePNT and control blastocysts, and there was no detectable difference between them. In conclusion, this study has tested PNT in normally fertilised human zygotes for the first time; results indicate no detectable harmful effects of the ePNT procedure. We therefore conclude that it is likely to give rise to normal pregnancies.

Introduction Preterm birth is a common obstetric syndrome with multiple potential causes. The consequences of preterm delivery are significant for the baby, their family and wider society. Our understanding of the biological mechanisms underlying preterm birth is incomplete and there is no available strategy to predict preterm labour in asymptomatic primiparous women. MicroRNAs (miRNAs) are small, non-coding RNA molecules, which regulate gene expression at the post-transcriptional level. MiRNAs play a modulatory role in pathways leading to labour onset, and are shed from their tissue of origin in to the peripheral circulation, where they are stable and easily measurable. Methods Myometrial biopsies were collected from pregnant women undergoing either prelabour or intrapartum oxytocin augmented caesarean section. Tissue samples were subjected to miRNA profiling using microarray. Differentially expressed miRNAs were identified and then validated using RT-PCR and expression was then compared with samples from women in spontaneous labour. Human uterine myocytes were cultured and subjected to electroporation transfection experiments which examined the functional effects of earlier identified labour/oxytocin associated miRNAs. Plasma samples and cervical length measurements were prospectively collected from women in the second trimester of pregnancy. Plasma miRNA profiling was undertaken using the nCounterTM assay to describe expression across gestation in uncomplicated pregnancies and then compared with samples from women who went on to display cervical shortening or deliver preterm. MiRNAs predictive of later cervical shortening were identified, and validated with RT-PCR. ROC curves were constructed. Results 494 miRNAs were detected in human myometrium and six were differentially expressed in samples from women receiving oxytocin. Four of the validated miRNAs (hsa-miR-146a-5p, hsa-miR-146b-3p, hsa-miR-196b-3p and hsa-miR-876-5p) were expressed in primary human myocytes and oxytocin treatment of these cells replicated the directional changes observed in vivo. Additionally, hsa-miR-146b-3p was increased in both oxytocin receiving and spontaneous labouring samples. Myocytes treated with IL1β demonstrated increased expression of hsa-miR-146a-5p and hsa-miR-146b-3p. Knockdown of the p65 NFκB subunit resulted in reduced expression of hsa-miR-146a-5p whilst overexpression of the p65 NFκB subunit resulted in increased expression of both hsa-miR-146a-5p and hsa-miR-146b-3p. Myocytes transfected with hsa-miR-146a-5p and hsa-miR-146b-3p mimics exhibited a tendancy in suppression at the mRNA level of IL1β mediated NFκB dependent genes: CCR1, MMP10, IL8R, CCL4 and OXTR. Overexpression of hsa-miR-146a-5p resulted in a tendency of reduction of the IL1β induced inflammatory cytokines. Fifty-six miRNAs were expressed in maternal plasma samples and the profile was stable across gestation in uncomplicated pregnancies. Levels of nine miRNAs were significantly higher in plasma from women who went on to exhibit cervical shortening. Of the nine, hsa-miR-150-5p had both the highest AUC (0.86) and the greatest detection rate at a given false positive rate (FPR). The minimum FPR required for hsa-miR-150-5p to achieve 100% sensitivity is 39%. Hsa-miR-150-5p appears to be the strongest candidate biomarker to predict cervical shortening and therefore cervical weakness associated preterm labour. Conclusions This thesis describes how oxytocin alters the expression of a set of myometrial miRNAs and identifies a further role for oxytocin as a signalling molecule involved in the regulation of gene expression during parturition. This thesis also demonstrates that hsa-miR-146a-5p and hsa-miR-146b-3p are regulated by NFκB in human myocytes and that these miRNAs may, in turn, regulate NFκB dependent genes which are key to the inflammatory process of parturition. Furthermore, this work identifies a panel of nine plasma miRNAs which predict cervical shortening from 12 weeks gestation, prior to ultrasonographic evidence of cervical change. The second trimester is a key period in pregnancy when pathological changes can be identified, but time remains to deliver outcome-modifying interventions.

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Experimental animal models and observational studies suggest that maternal obesity in pregnancy influences the development of obesity in early infancy. As the trajectory of later adiposity is thought to be determined in early life; an understanding of potential contributing factors and associative mechanisms are essential to inform targeted interventions. The analyses reported in this thesis have sought to address these associations in mothers and their offspring from the UK Pregnancies Better Eating and Physical Activity Trial (UPBEAT), a randomised controlled trial of a behavioural intervention (diet and physical activity) in obese pregnant women. As the trial, did not show a reduction in the primary outcomes; incidence of gestational diabetes and delivery of a large for gestational age neonate, the dataset was treated as a cohort. Modifiable maternal risk factors including early pregnancy measures of maternal adiposity, were linearly associated with neonatal adiposity. An independent association with maternal birthweight and neonatal adiposity was also identified. Mode of early infant feeding (breast, formula and mixed feeding) and measures of general appetite influenced measures of infant growth and body composition at 6 months in offspring born to obese women from the UPBEAT cohort; providing evidence of a potential target for intervention within a sensitive window of opportunity. To address mechanistic pathways underpinning the associations between in-utero exposures and offspring adiposity, assessment of the cord blood metabolic profile was undertaken including measurement of candidate biomarkers and metabolome. A novel relationship between cord lysophosphatidylcholine with neonatal and infant anthropometry was identified, which was associated with maternal fasting glucose in late second trimester. Maternal fasting glucose in late second trimester was also found to partially mediate the effect of maternal parity and early pregnancy adiposity with neonatal adiposity. The UPBEAT intervention was associated with a reduction in a measure of offspring central adiposity, which was mediated through changes in antenatal diet and gestational weight gain instigated by the intervention. This provides evidence, within a randomised control trial setting that a behavioural intervention may potentially reduce offspring adiposity.

It has been established that placing the C57BL6/J mouse on a high fat diet induces obesity and impaired glucose homeostasis, and produces a model that is pathophysiologically relevant to the human condition of the metabolic syndrome. The cardiovascular changes in this model have been relatively poorly explored. I have focussed my work on understanding the effects of this diet on cardiac function using in vivo cardiac functional assessment in combination with in vitro analysis of intact single cardiomyocyte dynamics and Ca2+ handling, as well as demembranated left ventricle trabeculae for the study of myofilament function. High fat diet caused increased left ventricular end diastolic pressure after 30 weeks as assessed by in vivo pressure catheterisation. This change was associated with, increased passive tension in demembranated trabeculae at 30 w, increased collagen 3 expression on mRNA level and increased total tissue collagen from high fat diet after 40 weeks. Reversion to normal diet for 10 weeks had no discernible effect on whole organ function compared with animals continually fed with the high fat diet, despite body fat content and glucose homeostasis becoming normalised. Isolated cardiomyocytes exhibited increased relaxation rate after 40 weeks which was partly reversed by a return to normal diet. The increased cardiomyocyte relaxation rate occurred without changes to the intracellular Ca2+ transient, thus suggesting it may be caused by altered myofilament Ca2+ sensitivity. There was no established decreased myofilament Ca2+ sensitivity seen in demembranated trabeculae at week 40, even though trends were seen towards increased phosphorylation of serines 23/24 of cardiac troponin I, a modification known to induce Ca2+ desensitisation. It is concluded that high fat diet feeding in the C57BL6/J mouse is a mild but valid model for studying the effects on cardiac function of obesity and impaired glucose handling. It results in impaired diastolic function at the whole organ level after 30 weeks feeding. This effect is likely principally caused by extracellular deposition of collagen (which reduces compliance) rather than by changes at the cardiomyocyte level. This whole organ diastolic alteration appears to become established, not responding to dietary fat reduction. The increased relaxation rate of cardiomyocytes more likely originates from changes at the myofilament level as opposed to Ca2+ handling although this could not be statistically verified in this study. As high fat diet feeding was found to increase myocardial NADPH oxidase activity, myofilament function was also assessed in a mouse model ofNADPH oxidase 2 over-expression. NADPH oxidase 2 over-expression was found to cause myofilament sensitisation to Ca2+ and increased actomyosin cross bridge cycling rate at maximum Ca2+ activation. This was associated with an increased total phosphorylation of cardiac troponin I independent of phosphorylation of serines 23/24. This finding may represent a new signalling pathway linking NADPH oxidase 2 activity to contractile activation via the myofilament but appears opposite to myofilament changes induced by high fat diet feeding.

Pregnant women are routinely offered serum screening tests for Down's syndrome, open neural tube defects and Edward's syndrome. Informed decisions rely on health professionals communicating probabilities, and service users understanding this information. However, cognitive psychology research has established that people do not draw mathematically correct inferences from probabilistic screening information. This thesis applies this theoretical research to the context of prenatal genetic screening. The empirical work involved presenting screening scenarios to prenatal screening service users and providers in different forms (prenatal/neutral context, frequency/percentage presentation), arid assessing the effect of these manipulations on participants' ability to estimate risk probabilities. The understanding of medical students, postgraduate students, pregnant women, partners and companions,midwives, and obstetricians was investigated. The majority of responses to the scenario were incorrect. Most respondents gave extreme under- or over-estimates (close to 0 or 100). Obstetricians gave significantly more correct responses to frequency scenarios than percentages scenarios, replicating past studies. Other participants gave significantly fewer correct answers overall, and were unaffected by the change from percentage to frequency information. The reasoning of midwives and service users had not previously been investigated, and this thesis indicates that there are fundamental differences in the ways that different groups interpret probabilities, creating potential communication barriers between stakeholders.