Background: Polycystic ovary syndrome (PCOS) is associated with increased metabolic risk and hypertension, which may relate to enhanced sympathetic nervous system (SNS) activation. The cerebral pathways involved in this process are not known. Aims: (1) To compare blood pressure and SNS activation in response to isometric forearm contraction (IFC) between PCOS and control groups. (2) To identify and compare the neuronal signatures of this response. (3) To investigate metabolic and anthropometric correlates of SNS activation. Methods: 20 PCOS (age 29.8 yrs, BMI 26.1 kg/ m²) and 20 matched controls (age 29.7 yrs, BMI 26.1 kg/ m²; p=NS) were studied. Out-of-scanner tests: measurement of mean blood pressure (MAP) and heart rate (HR) responses to 30% IFC for 180 seconds; baseline and post-task catecholamines, and microneurography (MSNA) in a subgroup of 8 PCOS and 8 controls. In-scanner: Blood oxygen level dependent (BOLD) fMRI using an identical block paradigm design for IFC, BOLD signalcorrelating to MAP responses (threshold Z > 2.3, corrected cluster threshold p=0.05). Results: IFC elicited an increase in HR and MAP in PCOS and controls but these did not differ between groups (p=0.16[HR] and p=0.06[MAP]). Adrenaline increased significantly post-IFC in PCOS (0.68 to 1.23ng/mL p < 0.001) but not in controls (0.77 to 0.99ng/mL p=0.14). MSNA burst frequency increased by 68% in the PCOS group compared to 11.9% in controls (p=0.002). Brain activation indexed by the BOLD signal in response to IFC was significantly greater in the PCOS group compared to the control group in the right orbitofrontal cortex (p < 0.0001), left angular gyrus and lateral occipital cortex (p=0.04). When the BOLD signal was separately corrected for insulin sensitivity, BOLD signal in the right orbitofrontal cortex was no longer significant. Conclusions: PCOS is associated with enhanced SNS activation and increased regional brain activation in response to IFC. The right orbitofrontal cortex BOLD signal change in the PCOS group is associated with insulin sensitivity.

There have been phenomenal advances in the field of reproductive medicine and success rates following in vitro fertilisation have improved dramatically in recent years. The aim of this project was to improve our understanding of human preimplantation embryo development by identifying potential markers of viability that may aid us in selecting the best embryo for uterine transfer in the clinical embryology laboratory. Investigations into the distribution of cytoskeletal F-actin in human embryos demonstrated that a highly organised actin cortex is important for embryo cleavage and continued development to the blastocyst stage. Whilst they are polarised in the mouse from the oocyte to the blastocyst, the regulatory proteins leptin and STAT3 are co-localised only at the oocyte stage in humans, and are distributed within different cytoplasmic domains in human cleavage stage embryos and blastocysts. Whether polarity in humans is predetermined in the oocyte remains elusive, but none of the evidence generated in this thesis supports this idea. Leptin transiently activates STAT3 via the long form of the leptin receptor, and most significantly in the ICM of human day 6 blastocysts. Morphological features of blastocysts that can be visualised microscopically, such as a double ICM and cytoplasmic projections connecting the ICM to the TE, provide clues to their viability and may help us to choose the most suitable embryo from a cohort when deciding which to transfer. Nuclear volumes may in future contribute to this selection. Using time lapse technology to study cleavage patterns is now a routine occurrence in the clinical embryology laboratory. The results in this thesis show that distinctive patterns of divisions and the site at which blastocysts hatch can provide us with more information than a snap-shot morphological evaluation. Finally, contributing to the development of modelling software and predictive algorithms for the study of human embryos, particularly in time lapse imaging, means that our understanding of this fascinating area of medicine will continue to progress.

The clinical success of assisted reproductive technology (ART) is measured by the clinical pregnancies (implantation success) and the live births rates. Following ART live birth rates vary from 20-40% and are dependent upon a variety of factors. Various adjunct therapies are being used with ART to improve implantation and pregnancy outcomes. The effectiveness of these adjuvant therapies remains unclear and requires further evaluation. One group of medical adjuvant therapies widely used in clinical practice are thromboprophylactic agents, including heparin. Heparin can potentially modulate many of the mechanisms of implantation including successful apposition, adhesion and penetration of the developing embryo into the endometrium. This is independent of its anticoagulant function for which it is used routinely in clinical practice. Following completion of a literature review, it became evident that heparin could potentially improve decidualisation and implantation. It improves function of various growth factors and cytokines in the endometrium promoting and facilitating implantation in laboratory models. From this initial research, we postulated that heparin used as adjunct to ART should improve the clinical pregnancy and the live birth rates via these mechanisms described. Bleeding is a known side effect of systemic heparin due to its effect on the coagulation cascade. A systematic review and meta-analysis protocol was devised and peer-reviewed to assess the published data. The aim of this was to establish whether using the currently available evidence, peri-implantation heparin improves pregnancy outcomes in women undergoing ART. A secondary aim was to determine if there were any significant side effects. The meta-analysis was performed in accordance with the protocol. This demonstrated that peri-implantation systematic heparin does improve clinical pregnancy rates and live birth rates in these women. Nevertheless, there were only three randomised control trials (RCTs) included in the review that met the inclusion criteria and there was significant heterogeneity amongst the participants in the included studies. Systemic side effects of heparin including bleeding and bruising were also identified in this review. As the proposed mechanism of improving implantation by heparin is improvement of endometrial cytokines and growth factors. It was hypothesised that direct endometrial administration of heparin should improve decidualisation thus improving implantation. To confirm or refute this hypothesis, initially a phase 1 study is required to be undertaken for direct endometrial administration of heparin as currently it is only licenced as a systemic injectable formulation. We developed a protocol to assess the feasibility of intrauterine flushing for direct endometrial administration of low molecular weight heparin (LMWH) with a prospective randomised placebo controlled pilot study. This novel study was approved by National Research Ethics Service (NRES), Medicine & Healthcare products Regulatory Authority (MHRA), UK. Sponsorship was obtained from the University of Warwick and local Research & Development (R&D) approval was obtained. The study was undertaken at University Hospitals Coventry and Warwickshire NHS Trust (UHCW). It demonstrated the acceptability of intrauterine flushing of heparin to women. The concept of the trial was popular with patients making recruitment unproblematic. Minimal side effects were reported, no serious adverse events occurred. Most women recruited underwent ART following the study, with many achieving a clinical pregnancy and live birth. Our hypothesis for primary outcome measure, uterine natural killer (uNK) cell density, as a marker of decidualisation was refuted.

Recent research has shown an association between smoking in pregnancy and low birthweight. Many authors have concluded that the relationship is causal but some have argued that it is the smoker rather than the smoke which is responsible. This thesis examines the relationship between the smoking habit in pregnancy and birthweight corrected for gestational age using data from the St. George's Hospital Birthweight Study. Adjustment is made for confounding factors so that the effect of smoking can be estimated. The statistical problem of adjusting birthweight for gestational age when very early births are included is discussed and a solution presented in the form of a birthweight ratio. The relationship is examined between birthweight ratio and many socioeconomic and psychological factors and shows that few are associated with reduced birthweight. Those associations which are observed can be explained by smoking. Alcohol and caffeine are only related to birthweight in smokers. When the smoking habit is analysed in terms of quantity and constituents, a threshold is observed whereby women smoking a low number of low yield cigarettes have mean birthweight similar to non-smokers. For women smoking higher numbers of cigarettes but a low yield brand mean birthweight is reduced by the same amount (6% or more) as women smoking high yield brands. The effect on birthweight of alcohol and caffeine in smokers only is adjusted for smoking by using this threshold. This shows that smoking, alcohol and caffeine are all associated with reduced birthweight. For alcohol and caffeine consumption this relationship is strongest in early pregnancy and weakest near delivery. The association between smoking and birthweight is not explained by any of the wide range of confounding factors examined. This provides evidence that the relationship is a causal one.

No description available.

No description available.

Reproduction is a crucial process, required for bringing the next generation into the world. In preparation for pregnancy, and throughout pregnancy itself, reproductive tissues recruit specific populations of immune cells that are thought to contribute in a variety of ways to successful reproduction. Pregnancy culminates in parturition, an inflammatory process characterised by an influx of inflammatory cells into reproductive tissues. Effective healing of reproductive tissues in the post-partum period is vital for continued reproductive success, and it too is thought to involve specific populations of immune cells. For leukocytes to effectively perform their functions in the reproductive system and elsewhere, migration to the right place at the right time is crucial. Key regulators of leukocyte homing are the chemokine family of chemoattractants and their G-protein coupled receptors. The chemokine network is complex and controls migration of leukocytes from the Bone Marrow (BM) into the blood and from the blood into tissues. Chemokines influence leukocyte position within tissues, and orchestrate their departure. Very little is known about the types of leukocytes present within reproductive tissues in the post-partum period, or the chemokines and receptors that could be involved in their migration. Exploring these processes is critical for an understanding of how tissues are repaired in readiness for subsequent pregnancies. In this thesis I have examined the leukocyte populations in reproductive and peripheral tissues of mice during the post-partum period and compared them to those found in Non-Pregnant (NP) mice. This analysis has encompassed a range of myeloid cell types, and also the complex populations of CD3+ cells that exist in reproductive tissues. I was also interested in how these cells are instructed to enter reproductive tissues, and in particular on the role of CC chemokine Receptor 2 (CCR2), a receptor associated with the recruitment of monocytes and T cells into tissues. My work has clearly identified cells expressing CCR2 both in reproductive tissues and elsewhere, and defined the impact of the genetic deletion of this receptor on leukocyte populations during the post-partum period. These experiments exploited a variety of standard techniques including histology, quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR), Enzyme-Linked ImmunoSorbent Assay (ELISA) and Luminex, but they also required the development of challenging multiparameter flow cytometry protocols that allowed the simultaneous analysis, and definitive identification, of several leukocyte populations in various tissues and at specific reproductive time-points. Chapter 3 describes detailed experiments that focussed on characterising the myeloid cell populations across a variety of tissues in NP, 1 Day Post Partum (DPP) and 7DPP mice. Most strikingly, this revealed a profound accumulation of several myeloid cell populations in reproductive tissues at 1DPP, including inflammatory Ly6Chigh (hi) monocytes and neutrophils. Moreover, many of these myeloid cells expressed active CCR2 and remarkably CCR2 deletion was associated with a dramatic reduction in myeloid cell abundance in the uterine horn one day after birth. Thus, CCR2 appeared to be required for myeloid cell recruitment to the post-partum uterine horn. Chapters 4 and 5 describe changes in CD3+ cell populations over the post-partum period. Interestingly, the main finding from reproductive tissues was that the large majority of CD3+ cells lacked expression of CD4 and CD8, and were thus termed CD3+ Double-Negative (DN) cells. Three main CD3+ DN cell populations were described. CD3+CD25+NK1.1+TCRβ+ DN cells, likely to be Natural Killer T (NKT) cells, which were mainly found in reproductive tissues and blood. All tissues studied were found to contain CD3+NK1.1-TCRβ+ DN cells, likely to be ‘true’ DN T cells and CD3+NK1.1-TCRβ-TCRγδ+ DN cells, which were consistent with a γδ T cell phenotype. CD3+ DN cells were also found to increase in number at 1DPP, compared to NP tissues, driven by an increase in DN T cells. In contrast to myeloid cells CCR2 was not required for this change. However, at 1DPP there was a CCR2-dependent increase in the proportion of CD3+ DN cells in the blood. Finally, in Chapter 6, hormonal influences on leukocyte populations in reproductive and peripheral tissues were considered. This work had two major components: analysing sex differences in myeloid and T cell populations and exploring the effect that lactation has on these cell subsets over the post-partum period. Females were found to have an increased proportion of eosinophils in their blood, whereas males had a higher proportion of monocytes. I also found that female and male reproductive tissues, as well as peripheral tissues, have very similar CD3+ DN cell populations, suggesting that these cells serve roles in reproductive tissues that are not unique to one sex. Finally, CD3+ cell populations in the post-partum period were found to be controlled to some extent by lactation. Collectively, this work has significantly extended our understanding of leukocytes in various tissues in the post-partum period, and revealed the importance of chemokines in the regulation of these cells. It has laid the groundwork for future investigations aimed at dissecting the functions of these cells in reproductive tissues in the post-partum period.

Epidemiology has linked preeclampsia (PET) to vitamin D deficiency. To date, studies have focused upon serum 25-hydroxyvitamin D3 (25(0H. )D3) alone as the marker of vitamin D status. We provide strong evidence comprehensive analysis of vitamin D metabolites in pregnancy is highly informative, particularly within the context of PET. Uniquely, analysis of maternal urinary metabolites provides a novel insight into vitamin D and the kidney, with lower 25(0H)D3 and 24,25(0H)2D3 excretion early indicators of a predisposition towards PET. Since vitamin D is a potent regulator of immune function, and the decidua appears a key extra-renal site for vitamin D metabolism, we investigated effects of 1 ,25(0H)2D3 upon decidual uterine natural killer cells and macrophages. We show both express a functional vitamin-D system and demonstrate differential sensitivity to 1 ,25(0H)2D3 compared to their peripheral counterparts. To understand the functional impact of vitamin D, whole transcriptomic analysis of 1,25(0H)2D3-mediated effects upon uNK and macrophages was performed. We show the actions of vitamin D extend far beyond simple immuno-regulation, targeting major cellular functions including migration, adhesion and apoptosis. In particular, our data support effects highly relevant to decidualisation. We anticipate these findings to be highly relevant within the context of vitamin D deficiency, mal placentation and PET.

Objective: Mastitis is a common and painful condition experienced in up to 33% of breast-feeding women (Cusack & Brennan, 2011; Jahanfar, Ng & Teng, 2009; Scott, Robertson, Fitzpaterick, Knight, & Mulholland, 2008; Spencer, 2008). The period following having a baby may be emotionally and physically demanding due to the physical, hormonal and lifestyle changes a woman undergoes (Cusack & Brennan, 2011). Developing mastitis during this time may therefore be debilitating and has been found to be a common reason for discontinuing breastfeeding. This study aimed to explore women’s experiences of breast-feeding and mastitis, the formal and informal support that was available to them when they had mastitis, and the impact it had on whether they continued or discontinued breastfeeding (Amir, Forster, Lumley & McLachlan, 2007; Cleminson, Oddie, Renfrew & McGuire, 2015). Method: Grounded theory was used to analyse the data. Sixteen women who had experienced mastitis were included in the study. Results: All of the women intended to breast-feed. However, they often experienced problems early on (i.e. engorgement and nipple damage) that were unexpected and left them feeling exhausted and emotional. These problems precipitated mastitis. Mastitis was for most, a very difficult experience sometimes leading to discontinuation of breast-feeding. Delaying help-seeking negatively affected health outcomes. Determination and receiving good advice and support were fundamental factors in breast-feeding continuation. Conclusion: Improved support, communication, and advice with breast-feeding from the outset would reduce the risk of problems occurring and persisting, and potentially reduce the risk of mastitis developing. Early diagnosis and treatment of mastitis once it has developed is very important. Understanding that breast-feeding is a skill that often encompasses both ups and downs may reduce the pressure women put on themselves when they feel like they are failing because it is not going as well as they expected it to. Once mastered, women found breast-feeding to be a lovely bonding experience that exceeded their expectations.

The effects of undertaking a regular exercise programme during and following pregnancy were investigated with healthy primigravid women within Ayrshire Central Hospital, Irvine. A randomised control trial was used with subjects being randomly assigned to either a control group who continued with the existing antenatal education programme or an exercise group who had the addition of participating in an aerobic exercise programme. In early pregnancy, no significant differences were found between the groups in relation to activity levels or mean scores of psychological variables with the exception of the control group having significantly more positive scores for perceptions of body image. During and following pregnancy, the exercise group maintained their scores on all psychological variables i.e. perceptions of coping assets (positive psychological well-being), coping deficits (negative psychological well-being), physical well-being, body image, somatic symptoms experienced, attitudes to marital relationships, sex and pregnancy. In contrast, the control group tended to have significant reductions in perceptions of the ability to cope (positive psychological well-being), physical well-being, body image, somatic symptoms experienced, attitudes to marital relationships, pregnancy and sex during pregnancy in addition to an increase in perceived coping deficits (negative psychological well-being).The exercise group participated in a significantly higher number of episodes of physical activity sessions than the control group. No significant relationship was noted between frequency of physical activity and responses to psychological indictors in post pregnancy. In conclusion, women who participated in regular physical activity tended to have a protection against a reduction of psychological well-being as measured by a variety of psychological constructs. The maintenance in psychological well-being was experienced both during and following pregnancy and there was no indication of any risk to the pregnancy or the baby. This was in contrast to the significant reduction in psychological well-being experienced by the women in the control group during the same period.