Hot flushes are the most commonly reported symptom in postmenopausal women, occurring in approximately 73% of women and causing significant morbidity in 25%, affecting social life and even the ability to work. With improved healthcare and increased life expectancy (death rates decreased by 19% in the last 10 years), women spend a considerable proportion of their lives (30 years on average) in the menopause. At present 36% of the women in the UK are over 50 years of age. If left untreated, hot flushes resolve within one year, or less, in the majority of postmenopausal women. A third will report symptoms that last up to 5 years after natural menopause, and in 20% hot flushes persist for up to 15 years. This equates to as many as 1.5 million women in the UK. Despite this the mechanism of flushing is still poorly understood. A hot flush resembles a heat dissipation response, in that both are characterised by sweating and peripheral vasodilation. It follows that the underlying mechanism may involve some dysfunction in thermoregulation, which in humans is controlled by the medial preoptic area of the hypothalamus (MPOA), and effectors include cutaneous vessels for vasodilation and vasoconstriction. Therefore a dysfunction in thermoregulation may lie within the control centre (MPOA), its messengers (adrenergic neurones controlling vasoconstriction and cholinergic neurones controlling vasodilation) or the effectors (cutaneous vessels). Studies by Freedman et al, using an ultrasensitive temperature probe, suggest that hot flushes are triggered by small elevations in core body temperature (Tc) acting within a narrowed thermoneutral zone, mainly due to a lowering of the sweating threshold, in symptomatic postmenopausal women. However, the trigger remains unknown. Oestrogen is likely involved as these changes occur at times of relative oestrogen withdrawal; however, there is little correlation between hot flushes and circulating oestrogen levels. This suggests that other mechanisms are involved. Noradrenaline is thought to be the primary neurotransmitter responsible for lowering the thermoregulatory set point and triggering hot flushes. Animal studies have shown that intrahypothalamic injection of noradrenaline acts to narrow the thermoregulatory zone and hot flushes can be provoked in symptomatic postmenopausal women with the α2-adrenergic antagonist yohimbine, and ameliorated with clonidine, an α-adrenergic agonist. Furthermore, clonidine has been shown to widen the thermoregulatory zone in humans. Serotonin or 5-hydroxytriptamine (5-HT) is involved in many bodily functions including mood, anxiety, sleep, sexual behaviour and eating, and is thought to play a key role in thermoregulation. Oestrogen withdrawal is associated with decreased blood serotonin levels, which is returned to normal with oestrogen therapy. Furthermore, selective serotonin reuptake inhibitors (SSRI), designed to increase the available serotonin at the serotonergic synapse, have been shown in placebo-controlled trials to be effective in reducing the number and severity of hot flushes. It has also been shown that flushing women have a diminished vasoconstrictor response to cold and that they have increased blood flow to the forearm and hand during a flushing episode. Alterations in skin blood flow during a flushing attack have also been demonstrated in castrate men, and, as with women, improvements in symptoms are seen with hormone replacement. The aim of this thesis was to better understand the mechanism of flushing in postmenopausal women and hypogonadal men by assessing the role of cutaneous vessels. I measured cutaneous microvascular perfusion, using LASER Doppler imaging with iontophoresis, in postmenopausal women who flush and compared it with postmenopausal women with no flushing, and found that perfusion responses to vasoactive agents were increased in women with flushing. Paradoxically, these women with apparently ‘better’ endothelial function had evidence of serum cardiovascular risk factors. In a double-blind longitudinal cross over study, the role of the alpha-adrenergic system in the pathophysiology of flushing was investigated, by treating women with clonidine and placebo. There was an increase in perfusion responses with both clonidine and placebo. Clonidine was not shown to be superior to placebo in reducing the number and severity of flushes. The role of serotonin, both peripherally and centrally was studied by treating postmenopausal women, who experienced severe flushing, with venlafaxine (a serotonin and noradrenaline reuptake inhibitor that acts as a selective serotonin reuptake inhibitor at low doses). Flushing symptoms, as assessed by hot flush diaries and Greene climacteric scale (GCS) scores, were reduced, as were skin blood flow perfusion responses. Central serotonin transporters (SERT) were assessed in vivo using SPECT (single photon computed tomography) imaging and a radioligand, [123I] -beta-carbomethoxy-3-β-(4 iodophenyl)tropane ([123I] beta-CIT), with a high affinity for serotonin transporters. [123I] beta-CIT binding was significantly reduced, and this was associated with a significant reduction in BDI scores; in a group of non-depressed women. Adiposity is associated, both, with an increased risk of postmenopausal flushing and impaired endothelial function, but in this study, there were no differences demonstrated between obese and lean participants at baseline, despite significant differences in serum markers of endothelial dysfunction. Oestrogen receptors are also present on endothelial cells and as the most commonly used, and most effective, treatment for vasomotor symptoms, cutaneous microvascular perfusion was assessed following 8 weeks of HRT, and demonstrated an increase in both endothelium dependent and independent vasodilation. Hot flushes are also common in men on luteinising hormone releasing hormone (LHRH) agonists for prostate cancer therapy. Perfusion responses in these men were assessed prior to commencement of therapy (baseline), and after 8 and then 24 weeks of therapy. No differences were detected at baseline, between those who developed flushing as a result of treatment, and those who did not. At 8 weeks, those with flushing demonstrated increased skin blood flow compared to those without flushing. At 24 weeks, 2 gentlemen with flushing had kept diaries and these demonstrated an improvement in flushing, but no alteration in perfusion responses. ACh- and SNP-stimulated vasodilation was, however, reduced when compared to healthy controls. In this thesis, the data appear to support a role of skin blood flow in the mechanism of hot flushing in both postmenopausal women and hypogonadal men, which may be controlled or altered via neurotransmitters at a local level. The placebo response was significant, but alterations in skin blood flow do not appear to have mediated the adiposity effect.

Background: Perinatal mental illness, especially depression, is a leading cause of maternal morbidity and mortality in high-income countries. In the United Kingdom (UK), mental illness commonly presents to and is treated at primary care level; however there are no up-to-date estimates of the burden of different mental illnesses in women in and around pregnancy. The potential impact of mental illness with or without psychotropic medication on the risk of non-live pregnancy outcomes is unclear. In this context, the safety of psychotropic drugs, especially antidepressants, remains controversial. Aim and objectives: To estimate the clinical burden of depression, anxiety and serious mental illness (defined as bipolar disorder, schizophrenia and other related psychotic disorders) presenting to and/or being treated in UK primary care, and to investigate the effects on pregnancy outcomes while trying to differentiate the effects of psychotropic medication from mental illness itself. Methods: Women aged 15-45 years from 1990 to 2009 were identified from The Health Improvement Network, a UK primary care database. Coding of mental illness diagnoses and psychotropic drug prescriptions were examined by separately assessing the proportions of women with recordings of diagnoses, symptoms, and drug prescriptions over the study period. Three separate studies were then carried out. A cross-sectional study was firstly conducted to estimate the prevalence and diagnostic overlap of mental illnesses before, during and after pregnancy and the variation by maternal age, socioeconomic status and other maternal factors. The second study examined the risks of non-live pregnancy outcomes (defined as perinatal death, miscarriage, and termination) in women with no history of depression and anxiety, a diagnosis of such illness prior to pregnancy, illness during pregnancy or illness during pregnancy with use of medication (stratified by medication type). Multinomial logistic regression models were used to compare risks of non-live outcomes across these groups, adjusting for important socio-demographic and lifestyle characteristics. The third study examined the risks of major and system-specific congenital anomalies in children born to women with depression or anxiety that was untreated or treated with psychotropic medication. Logistic regression with a generalised estimating equation was used to compare risks of major congenital anomalies in children exposed and unexposed to psychotropic medication during the first trimester of pregnancy, adjusting for important socio-demographic, lifestyle and chronic comorbidity in the mother. Results: There were 344,042 women who had one or more singleton pregnancies identified between age 15 and 45 from 1990 to 2009. Recording of mental illness and prescriptions of psychotropic drugs increased considerably over the study period. There was high prevalence and overlap of different maternal mental illnesses, especially depression and anxiety, during and after pregnancy, and the prevalence was generally highest in younger, socioeconomically deprived women who had smoked before childbirth, were outside the normal range of BMI and had other chronic medical conditions, such as diabetes. Socioeconomic deprivation was associated with increased risk of all mental illnesses, although the impact of deprivation was more marked in older women. Those aged 35-45 in the most deprived group had 2.63 times the odds of antenatal depression (95% confidence interval [CI] 2.22-3.13) compared with the least deprived; in women aged 15-25 the increased odds associated with deprivation was more modest (odds ratio [OR]=1.35, 95%CI 1.07-1.70). Similar patterns were found for anxiety and serious mental illness. Women with antenatal exposure to antidepressant or anti-anxiety drugs showed the greatest increased risks for non-live pregnancy outcomes, relative to those with no history of depression or anxiety, although women with prior (but currently un-medicated) illness also showed modest increased risks. Compared with un-medicated antenatal morbidity, there was weak evidence of an excess risk in women taking tricyclic antidepressants (TCAs), and stronger evidence for other medications. The absolute risks of major and system-specific congenital anomalies were small in the general population (269 per 10,000 children for major congenital anomalies). Compared with un-medicated antenatal depression or anxiety (278 per 10,000 children for major congenital anomalies), the use of antidepressants during early pregnancy was associated with excess risks, especially for selective serotonin reuptake inhibitors (SSRIs) (290 per 10,000 children for major congenital anomalies). Compared with children born to women with no depression or anxiety, there was an increased risk of heart anomalies in children with antenatal exposure to SSRIs (adjusted OR=1.25, 95% 95%CI 1.02-1.53), particularly in those exposed to paroxetine (adjusted OR=1.89, 95%CI 1.24-2.88). Children exposed to sertraline and escitalopram also had similar increased risks, although fewer women were exposed to these drugs. No increased risks of major congenital anomalies were found in children exposed to TCAs or benzodiazepines; however, the risks of right ventricular outflow tract anomalies were notably higher for all drug classes. Conclusion: Strong socioeconomic inequalities in perinatal mental illnesses occur and persist with increasing maternal age. Women with depression or anxiety have higher risks of miscarriage, perinatal death and therapeutic terminations than women without these diagnoses and the risks are even higher if prescribed psychotropic medication during early pregnancy than if not. There is also an increased risk of congenital heart anomalies in children exposed to paroxetine and other SSRIs during the first trimester compared with those who are unexposed, although the absolute risk is small. There could be other associated factors also related to depression, anxiety or use of medications, which yet unlikely fully explain the observed excess risks. Whilst medicated depression or anxiety could be a marker of more severe illness than un-medicated ones, my findings indicate there may be some specific drug effects Targeting detection and effective interventions to women at risk of mental illness during pregnancy may reduce inequity and avoid substantial psychiatric morbidity, and subsequently reduce the need for further psychotropic treatment. GPs and other health care professionals should take a cautious approach when managing mental illness in pregnant women. The findings in this thesis provide vital information for this purpose, namely helping communicate the magnitude of risk of major congenital anomalies to women with the use of different psychotropic drugs in the context of the baseline risk in the general population.

Communication, anxiety and satisfaction during pregnancy was examined. Ley (1977) presents a cognitive model to explain satisfaction and its links with understanding. Janis (1958, 1971) notes a curvilinear relationship between anxiety and post-operative coping and postulates that information, anxiety and cognitive preparation are the variables accounting for this. Kumar and Robson note that obstetric anxiety is related to concerns for maternal and infant well being rather than irrational anxiety. This study examined the experience of women, stressors, communication satisfaction, knowledge and information and looked at the extent to which these three theories could interrelate to provide a fuller explanation of the psychological experience of women. Five studies were undertaken. Initially a pilot study revealed many negative statements about communication when transcripts were analysed. Communication factors and anxiety laden instances were correlated. The next study was set up to examine knowledge levels as Ley predicts that these, together with misunderstandings could contribute to dissatisfaction. Desire for knowledge was high. Knowledge varied according to social class but not parity. Doctors felt parity would be a factor. Women had difficulty approaching their doctor for information yet still desired doctors as their primary information source. Doctors delegated much information imparting to classes. Study three examined anxiety, communications and satisfaction in labour with pain management (a noted stressor in study 1). Patients receiving Pethidine were dissatisfied. Their pain experience did not differ markedly,but their psychological preparation did. In study 4 anxiety and outcome was monitored, together with information gathering strategies. Linear, rather than curvilinear relationships were found (unlike those predicted by Janis). The course of anxiety was a useful measure and the impact of anxiety on caregivers in the cycle of communications and recovery were explored. The final study looked at the impact of intervention on anxiety and satisfaction in ante-natal care. Women were randomly allocated to groups receiving no intervention, information and information plus feedback. The latter group had significantly lower post-consultation anxiety and higher satisfaction than the other two. The role of knowledge and accuracy in relation to satisfaction was explored. Kumar and Robsons propositions about anxiety were supported in these studies. Ley's cognitive model contributed much to the understanding but limitations in this model are explored, especially in relation to process and interaction factors and the routes to understanding. Janis' curvilinear relationship was not upheld, but his theoretical explanations involving the use of information and worry needs further testing.

<p> Women and men&rsquo;s preferences for delivery services in rural Ethiopia Nancy Beam Aims: This study aims to determine the combination of facility-based delivery care attributes preferred by women and men; if gender differences exist in attribute preferences; and key demographic factors associated with attribute preferences. </p><p> <b>Background:</b> Despite programs to promote facility-based delivery, which has been shown to decrease maternal and neonatal mortality, 80% of women in rural Ethiopia deliver at home without a skilled birth attendant. </p><p> A review of the Ethiopian literature on factors associated with delivery location revealed several weaknesses in research methods that need to be addressed. First, research participants were almost exclusively women, although male partners often make decisions about delivery location. Second, most quantitative study designs are similar in content to the Ethiopian Demographic Health Survey, limiting the generation of new knowledge. Third, cultural practices identified in qualitative studies as barriers to facility-based delivery have not been included in quantitative studies. This study addressed these weaknesses by using discrete choice experiment methodology to elicit preferences for delivery service attributes, including support persons in the delivery room, staff training and attitude, cost, distance and transportation availability. </p><p> <b>Methods:</b> A cross-sectional, discrete choice experiment was conducted in 109 randomly selected households in rural Ethiopia in September-October 2015. Women, who were pregnant or who had a child &lt; 2 years old, and their male partners were interviewed. After completing a demographic questionnaire, male and female respondents were asked separately to choose between facility-based scenarios that reflected various attributes for delivering their next baby. Data were analyzed using a multilevel mixed-effects logistic regression model. </p><p> <b>Results:</b> Both women and men preferred health facilities where medications and supplies were available, a support person was allowed in the delivery room, cost was low, and doctors performed the delivery. Women also valued free ambulance service, while men favored nearby facilities with friendly providers. Men are disproportionately involved in making household decisions, including decisions about whether their wives seek health care. Yet, men are often unaware of their partners&rsquo; prenatal care attendance. </p><p> <b>Implications:</b> The Ethiopian government and health facilities could increase facility births in rural areas by responding to families&rsquo; delivery service preferences.</p>

It is important therefore that maternal smoking is accurately recorded on screening request forms and in this study, the accuracy of self reported smoking status was assessed by analysis of cotinine in serum. Results showed that the percentage of self-reported smokers (24.1%) at booking was significantly lower than the cotinine-validated estimate of 30.1%. Also, smoking was associated with low birth weight, delivery prior to 39 weeks, increased AFP level (3.1%) and reduced hCG level (28.7%) in the second trimester. An increasing AFP level (but not hCG level) was associated with lower birth weight and delivery prior to 39 weeks in both smokers and non smokers but the effect was most marked in smokers. The difference in birth weight between the highest and the lowest AFP category for non-smokers was 448.3g and for smokers was 619.2g, suggesting that smoking exacerbates the effect of an elevated AFP on birth weight. Overall the difference in birth weight between the lowest AFP category in non smokers and the highest AFP category in smokers was 931.6g. Summary In summary, this study has shown that a cross-trimester contingent screening protocol with repeat measures has the potential to meet the UK NSC aspirational standard of 90% detection of Down’s syndrome pregnancies with a screen positive rate of less than 2%. Around 90% of women would complete screening in the first trimester without the need for a second stage sequential test. Correcting for factors such as maternal smoking habits, ethnicity and ART would further improve screening performance. Also it has been shown that where ultrasound resources are scarce, within-trimester and across-trimester protocols can reduce the need for NT measurement in all women and still deliver excellent screening performance although this falls short of the higher performance standard. The potential of these new screening protocols now need to be tested in prospective multicentre trials to confirm their performance in prospective practice.

Maternal metabolism undergoes dramatic changes in pregnancy in order to sustain and nourish the developing fetus. During healthy pregnancy the mother goes from an anabolic state in early pregnancy to a state of catabolism in late pregnancy with increased lipolysis together with a significant reduction in insulin sensitivity. Pre-eclampsia (PE) characterised by hypertension and proteinuria is a major cause of maternal and perinatal morbidity. There is acute ‘atherosis’ in PE placenta, and lipid accumulation within glomerular cells and liver. PE women have an early, excessive triglyceride and free fatty acid (FFA) rise and greater cardiovascular disease (CVD) risk in later life. The cause of these lipid abnormalities in PE is unknown but disordered adipocyte function including exaggerated lipolysis and aberrant release of adipokines (such as IL-6 and TNF alpha) is a major candidate pathway. Elevations in FFAs, and pro-inflammatory adipokines could underpin the oxidative stress, endothelial dysfunction, inflammation, and insulin resistance - characteristic features of PE. The aims of this thesis were to acquire a better understanding of lipid metabolism and function in normal pregnancy, to determine if adipocyte function was altered in PE and, if so, to establish mechanisms. In addition I planned to corroborate epidemiological evidence of increased future CVD risk and to establish which risk factors accounted for this increased risk. I collected subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) biopsies in non-labouring pregnant healthy (n=31) and PE (n=14) women who underwent caesarean section. Maternal blood was collected prior to delivery and phenotyping of the mother was performed including plasma assay for cholesterol, triglyceride, HDL-cholesterol, IL-6, TNF-α, leptin, adiponectin, high sensitivity CRP, glucose and insulin concentrations. Maternal BMI at booking, standardised blood pressure measurements and birth weight centile were also recorded. I determined ex vivo lipolytic activity (basal, isoprotenerol stimulated and insulin suppression of lipolysis) and adipokine production in response to lipopolysaccharide (LPS) stimulation from these biopsies. The gene expression of relevant target genes and macrophage densities in each adipose depot by immunocytochemistry (ICC) was also performed. In addition I performed carotid ultrasound assessment of women with a previous history of PE (n=31) and matched controls (n=29). Ethical approval was obtained from Glasgow Royal Infirmary LREC and all patients gave their informed consent. I found that in normal pregnancy, adipocyte lipolytic function is independent of maternal BMI. Adipocyte lipolytic function of SAT and VAT are also independent of each other. Adipose tissue is very metabolically flexible and the rate of whole body lipolysis is still insulin sensitive in late gestation. VAT is more closely related to markers of maternal insulin resistance (IR) and is more sensitive to catecholamine stimulation and less sensitive to insulin suppression of lipolysis than SAT, the basis of the “portal paradigm”. Increasing BMI is associated with an increase in VAT cell size, with increased lipolysis and an increase in pro-inflammatory adipokines, a potential mechanism through which increasing obesity could predispose to metabolic complications of pregnancy. In contrast SAT cell size is not closely related to BMI and this may reflect the adaptation of this depot to increasing fat mass through both hypertrophy and hyperplasia, a metabolically advantageous response. TNF alpha is an important correlate of basal lipolysis in SAT. In PE there is decreased insulin sensitivity of both SAT and VAT compared to controls as calculated by the fat cell insulin sensitivity index (or responsiveness to insulin once the tissue is stimulated by isoproterenol). This would potentially make a significant impact on total circulating FFA as almost 60% of circulating FFA are from these adipose depots. The rise in FFA in PE occurs early in pregnancy and contributes significantly to IR. Therefore the IR of adipose tissue could lead to a vicious cycle of increased lipolysis, increased FFA and further exacerbation of IR. In contrast to controls, SAT cell size is intimately related to BMI suggesting that adaptation to increasing fat mass is mainly through adipocyte hypertrophy which could lead to increased endoplasmic reticulum stress, increased IR and increased release of inflammatory adipokines. I have shown that SAT cell size does relate to adipokine release in PE, with increased release of leptin, CRP and PAI-1 and paradoxical increase in the anti-inflammatory IL-10. I had hypothesised that in addition to an inherent defect in adipocyte function there was an additional factor present in maternal serum of women with PE released from the placenta which excessively stimulated lipolysis. I failed to demonstrate any effect of maternal serum on adipocyte lipolysis in either controls or PE. I also found that after stimulation with LPS, there was increased release of TNF alpha and IL-6 in VAT in PE but not in controls, with higher gene expression of these adipokines. TNF alpha release also correlated negatively with the fat cell insulin sensitivity index (FCISI) of VAT implicating a paracrine effect in this tissue. I also demonstrated an increase in gene expression of cfms (activated macrophages) relative to control gene, and increased density of cfms+ macrophages/adipocytes in the VAT of PE women implicating activated adipose tissue macrophages as a potential source of the increased release of inflammatory adipokines. Lastly I attempted to corroborate epidemiological evidence for the increase future risk of CVD women with a history of PE by assessing two surrogate markers for atherosclerosis - carotid IMT and carotid plaque scores. Both were found to be increased, with plaque scores significantly so. Classic risk factors such as age, lipids, BP and smoking did not attenuate this effect and BMI only marginally attenuated it, therefore only partially explaining this increased risk. In summary the data presented in this thesis provides further evidence that PE is a “metabolic syndrome of pregnancy” with disordered adipocyte function and metabolism, with an increased future risk of CVD in later life. Further studies on adipose accumulation, function and composition in normal and complicated human pregnancy are warranted.

No description available.

No description available.

Approximately one in ten pregnant women will have their blood pressure recorded above normal at some point during their pregnancy. Pre-eclampsia, the most common hypertensive disorder of pregnancy, affects around 5% of all first time mothers, and is an important cause of foetal and maternal morbidity and mortality worldwide. Efforts to diagnose the condition have been hampered by inability to predict which women are likely to be affected. Multiple pathways are known to be involved in its pathogenesis, and several screening tests have been suggested for its early prediction. None, however, have been sensitive or specific enough to have come into routine medical practice. The work contained in this thesis describes a study which was designed to detect biochemical and clinical markers that could improve ability to predict pre-eclampsia. Over 3900 women were recruited in early pregnancy at four maternity clinics across the West of Scotland; baseline characteristics and information on past medical and obstetric history were obtained. Women were followed up throughout their pregnancy, and information on deliveries obtained from hospital databases. One-hundred and eighty of these women, who had multiple risk factors for pre-eclampsia, attended for further sampling and vascular assessment at gestational weeks 16 and 28. The primary aim of the overall study was to examine whether a proteomic strategy could be used to identify patterns of peptides in urine that detect pre-eclampsia in the first and second trimesters. Using samples from healthy pregnant and non-pregnant women I was able to describe the normal human urinary proteome in pregnancy. By comparing these pregnancy-associated peptides between women who went on to develop pre-eclampsia and matched controls, I was able to identify a pattern of peptides, characterised by collagen fragments, fibrinogen and uromodulin that accurately predicted pre-eclampsia at week 28. No such markers were identified in the first trimester samples. A further aim of the overall study was to identify early pregnancy plasma markers that could help to identify women destined to develop pre-eclampsia. By examining samples from early pregnancy I was able to demonstrate that the angiogenic markers soluble endoglin and placental growth factor are already altered at week 12-16 in women who go on to develop pre-eclampsia. Using a multi-marker approach, I also showed that E-Selectin, an adhesion molecule expressed on endothelial cells which controls interaction between circulating leukocytes and the endothelium, is higher at week 12-16 in women who go on to develop pre-eclampsia. Experiments using samples from later pregnancy, alternative analysis techniques and samples from an independent study population all helped to confirm these novel findings. Endothelial dysfunction is known to play a key role in the development of pre-eclampsia, contributing to the hypertension, proteinuria and oedema seen in affected women. In the risk factor cohort I used vascular function studies to examine whether they supplied additional information to aid in risk stratification. Peripheral arterial tonometry, a novel non-invasive tool for the assessment of microcirculatory endothelial function, was examined in 180 women at gestational weeks 16 and 28. Reactive hyperaemia index (RHI), a measure of endothelial dysfunction calculated from vascular response to arm blood-flow occlusion, did not correlate with maternal factors such as age, BMI and blood pressure. Further, RHI did not help to identify which women would go on to develop pre-eclampsia, when examined at either week 16 or 28. I found that PAT score was negatively correlated with baseline digital pulse amplitude, suggesting that in later pregnancy, when women are more vasodilated, PAT and other techniques which rely on flow-mediated dilatation are less likely to be reliable. I used pulse wave analysis, a well-established method for measuring arterial stiffness and central pressures, to determine whether it supplied additional information about pre-eclampsia risk. This technique has been previously reported to predict pre-eclampsia in early pregnancy. In this cohort of high risk women, no difference was seen at either week 16 or 28 between those who would go on to develop pre-eclampsia and those who would have normotensive pregnancies. Although blood pressure and proteinuria return to normal after pre-eclampsia, evidence has emerged the condition has long-lasting implications; women with a history of pre-eclampsia have an increased risk of cardiovascular disease later in life, suffering stroke or myocardial infarction more frequently than women who had a healthy pregnancy. Conventional risk factors are thought to contribute, but do not fully explain this increased risk. I carried out further vascular function studies in women after pre-eclamptic pregnancy, to examine whether they had ongoing detectable endothelial dysfunction and arterial stiffness. At 6-9 months post-natally, affected women had lower baseline digital pulse amplitude but no other evidence of persistent vascular dysfunction. Taken together, these data provide information about a number of markers that may improve understanding of the pathophysiological mechanisms underlying pre-eclampsia. As well as potentially improving the early prediction of disease, this work represents a highly topical area for further studies. While vascular function analysis does not appear to provide additional information on top of risk factors, these studies also provide useful information on vascular physiology in high-risk pregnancies.

Introduction: Preterm birth is the leading cause of perinatal morbidity and mortality and rates are rising. The UK now has the highest rate of premature birth in Europe with 5.3% of overall births in Scotland occurring spontaneously before 37 weeks gestation (1, 2) .Preterm babies have higher rates of perinatal mortality and morbidity and those that survive are at risk of multiple conditions including respiratory distress syndrome, central nervous system abnormalities, necrotising enterocolitis and sepsis. The mechanisms of preterm birth are poorly understood. Preterm birth can be spontaneous or induced and spontaneous preterm labour has multiple aetiologies. Current evidence suggests that prolonged treatment with progesterone and 17 α-hydroxyprogesterone caproate (17OHPC) may reduce the incidence of premature delivery in high risk patients with a history of spontaneous preterm birth (3) or with a short cervix. However, progesterone is not uniformly effective in preventing preterm labour and at present its principal mode of action on myometrium is unknown. I aimed to determine some of the specific mechanisms of action of progesterone. Aims: I hypothesised that progesterone has a direct inhibitory effect on spontaneous myometrial contractility, induces increased sensitivity to tocolytic agents and decreases sensitivity to contractile agonists such as oxytocin. I also hypothesised that progesterone has inhibitory effects on endogenous uterine stimulants, stimulatory effects on endogenous uterine relaxants, induces upregulation of endogenous receptors that inhibit uterine contractions and inhibits contraction associated proteins both in vitro and in vivo. Methods: I recruited women already enrolled in the STOPPIT (a double blind randomised placebo controlled study of progesterone for the prevention of preterm birth in twins) who were given vaginal progesterone, or placebo and who were scheduled for caesarean section. I also recruited women with healthy twin or singleton pregnancies undergoing elective caesarean section. Myometrial biopsies were obtained from the upper border of the lower uterine segment incision during caesarean section. Samples were divided and used for contractility measurements, or subsequent mRNA, protein and immunohistochemical analysis. Myometrial strips were cut and suspended under resting tension within organ baths. Concentration-response curves were carried out in response to oxytocin, levcromakalim, nifedipine and ritodrine to ascertain any reduction in effect by progesterone on oxytocics or enhancement of tocolytic effects. I also carried out concentration-response curves to progesterone alone and in the presence of potassium channel blocking agents. I then assessed ex vivo, the inherent contractility of the placebo versus progesterone groups from myometrium sampled from the STOPPIT cohort of patients. I carried out cell culture experiments on myometrium from healthy singleton women who were not in labour at the time of sampling. Myometrial explants were placed in cell culture medium, cultured for 1, 4 and 24 hours, and the supernatants were then analysed using Bio-Plex array technology to ascertain cytokine release. I selected time points and concentrations conditions to incubate myometrial tissue using progesterone and 17OHPC and was able to assess cytokine release. The myometrial explants were used for subsequent molecular studies. I performed real time-polymerase chain reaction (RT-PCR) (Abi,Taqman) to quantitate endogenous inhibitors of uterine contractility (cGRPR, EP2,NOS), cytokines (interleukins- IL6, IL8, IL1β), uterine stimulants COX-2 and gap junction components ( connexin 26 and connexin 43) expressed relative to housekeeping gene 18s. Lastly, I analysed the STOPPIT cohort of myometrial samples for to determine the in vivo effect of progesterone. We carried out RT-PCR (Abi,Taqman) to quantitate endogenous inhibitors of uterine contractility (cGRPR, EP2,NOS, PGDH), cytokines (IL6, IL8, IL1β) and gap junction components (connexin 26 and 43).I performed immunohistochemistry, staining for localisation of pro-inflammatory cytokines. I then carried out protein expression analysis using Western blot for contraction associate protein, connexin 43. The project was approved by North Glasgow University Hospitals Research Ethics Committee ref no. 05/S0705/18. All patients gave written informed consent to participate. Results: I found that progesterone, exerted consistent, rapid and sustained inhibition of the amplitude of spontaneous myometrial contractions in vitro at high concentrations however, this affect was not achieved through the principal potassium channels. Levcromakalim, a KATP channel opener, produced a concentration-dependent inhibition of the amplitude and frequency of spontaneous contractions. These effects were antagonised by the KATP channel blocker, glibenclamide. In contrast, glibenclamide had no effect on the progesterone-induced inhibition of myometrial contractility. Charybdotoxin 10 nM (which blocks IKCa, BKCa and Kv channels), iberiotoxin 100 nM (which blocks BKCa channels) and apamin 100 nM (which blocks SKCa channels) failed to affect the ability of progesterone to inhibit myometrial contractility. In contrast, 17OHPC did not exert any inhibitory effect on myometrial activity in vitro. Results indicated, at the selected pharmacological doses used in vitro that progesterone did not increase sensitivity to tocolytic agents tested. There was no decrease in sensitivity to the uterotonin oxytocin. Lastly, from our STOPPIT patient cohort I demonstrated no difference between the progesterone and placebo groups in either spontaneous contractility, response to tocolytics as above or response to oxytocin. One main conclusion of this arm of the study is that in vivo progesterone therapy to prevent pre-term labour does not appear to modify contractility ex vivo. I demonstrated that administration of progesterone but not 17OHPC for up to 24 hours in vitro does not appear to modify mRNA expression of uterine stimulants such as cytokines, COX-2 or endogenous uterine relaxants such as NOS and PGDH. Progesterone but not 17OHPC inhibited production of gap junction component connexin 43. This modification of contraction associated protein is in agreement with other literature presented on human myometrial data in vitro (4) . I used STOPPIT patients as a potential example of the myometrial effects of progesterone in vivo with a placebo treated control group. Prolonged maternal administration of progesterone appeared to inhibit expression of gap junction components connexin 26 and 43 in myometrium. Connexin 43 importantly, was also modified in vitro within the progesterone treated arm. However, ex-vivo assessment of the functional impact on human myometrium does not demonstrate a long-term inhibitory impact on myometrial function with down regulation of endogenous contractile inhibitors such as eNOS and EP2. The connexins play an essential role in regulating synchronous myometrial contractions. If progesterone has been of benefit in those at risk of preterm labour with a history of spontaneous preterm birth, it is possible therefore that this is by reducing connexin expression, which prevents the development of these synchronous contractions whilst on progesterone therapy. In summary, I have demonstrated putative mechanisms by which progesterone (and its analogue 17OHPC) might prevent preterm birth. Further studies characterising these pathways might inform the design of other agents which could provide additional efficacy in preventing preterm delivery.