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.

Lymphovascular invasion, including both blood and lymphatic vessel invasion, is an important step in tumourigenesis and is a prerequisite event in the complex process of metastasis. In breast cancer, data suggest that lymphatic vessels plays a significant role by being the major route for lymphatic vessel invasion (LVI) and that inflammatory cells, may be involved in its regulation. Macrophage, a major component of inflammatory infiltrate, has been shown to be importance in tumour growth and metastasis. The biomolecular mechanisms by which macrophage can mediate dissemination of tumour cells through the lymphatic compartment are, however, not yet fully understood. In the first part of this project, CD68 (total) and CD163 (M2 subtype) macrophages were examined in two consecutive sections of 89 full-face invasive breast cancer samples. The density and localisation of macrophages were assessed using the Chalkley-grid counting method and their association with clinicopathological variables and clinical outcome was identified. A high count of CD68 macrophages was associated with the presence of lymphatic vessel invasion (LVI) and a high microvessel density (MVD), and tumour with high microvessel densities had high expression of CD163 macrophages, confirming a role for macrophage in mediating the process of angiogenesis and metastasis via lymphatic vessels. CD68 and CD163 macrophages were not associated with disease-free or breast cancer-specific survivals. In order to elucidate previous conflicting data by our lab, suggesting that recombinant, and macrophage-induced IL-1β can mediate in vitro LVI, with patient based IHC data showing that high expression of tumour IL-1β was associated with improved disease specific survival and was not associated with LVI, it was of interest to study the expression of IL-1β signalling-related proteins including caspase-1, IL-1R I, IL-1R II, IL-1RacP, and IL-1RA. This was conducted in 1902 early stage invasive breast cancer patients, with long-term follow-up, using IHC. In addition, serum IL-1β concentration was measured in a matched subset of patients using enzyme-linked immunosorbent assay (ELISA) and expression levels examined for associations with the above proteins as well as clinicopathological criteria and patient prognosis. Although pre-requisites for IL-1 signalling pathways are present in breast tumours, tumour IL-1β expression was not strongly correlated with the expression of caspase-1, IL-1R I, IL-1R II, IL-1RacP and IL-1RA. Caspase-1, IL-1R I, IL-1R II, IL-1RacP and IL-1RA were not independent prognostic factors. The analysis of the markers stained in the current study could not provide any further elucidation of the conflict between previous in vitro and IHC studies regarding the role of IL-1β in regulating LVI. The third part of the study dealt with IHC and in vitro investigations of macrophage-associated cytokines (IL-6 and IL-10) to assess their role in regulating LVI in breast cancer. Scratch wound migration assays were conducted to examine the influence of L-6 and IL-10 on breast cancer cell migration with results showing that low concentrations of IL-6 induced migration, whereas high concentrations inhibited it. Similarly, a high concentration of IL-10 also inhibited breast cancer cell migration. In addition, the effect of these cytokines, IL-6 and IL-10, on the tumour- endothelial (blood and lymphatic) adhesive process was also studied. Static adhesion assays showed that the adhesion patterns of breast cancer cell lines to the endothelial cells did not change following pre-stimulation of either blood or lymphatic endothelial cells with either IL-6 or IL-10. Thus, in vitro data suggest that both cytokines may not play a significant role in regulating LVI other than their effect on migration. The important of IHC expression of IL-6 and IL-10 were examined in a large cohort of early stage invasive breast cancer patients. Data showed that high expressions of IL-6 and IL-10 in breast cancer tissues were not associated with the presence of LVI. A significant association was found between high expression of IL-6, and longer disease-free interval, but it was not associated with improved disease-free survival. However, high IL-10 expression was not associated with improved disease-free survival, and breast cancer-specific survival. In the final part of the project, the correlation between macrophage-associated cytokines including IL-1β, IL-6, and IL-10 and their downstream signalling elements, and target genes was assessed using Spearman’s rank correlation test. Expression data for downstream signalling elements was provided via collaboration with the Breast Pathology group. No strong correlations were observed between these cytokines. In addition, their expressions in breast cancer tissues were not strongly correlated with downstream signalling compartments, or target genes. In conclusion, macrophages seem to play an important role in regulating LVI, with such LVI being almost entirely invasion of lymphatic vessels. Macrophage and macrophage-associated cytokines (IL-6 and IL-10) have been found to potential play role in breast cancer progression, with preliminary in vitro data suggesting that this may be via tumour cell migration rather than influencing tumour-endothelial cell interaction and LVI. This project has shed some light on the role of macrophage-associated cytokines in regulating LVI however more studies are needed to determine the mechanisms whereby IL-1β, IL-6, and IL-10 regulate the progression and prognosis of breast cancer.

No description available.

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.

My three papers evaluate the effectiveness and cost-effectiveness of clinical and policy strategies to improve women’s health, focusing on human papillomavirus (HPV) vaccination in the U.S. and maternal health care in a developing country context. Paper 1 presents a claims-based econometric analysis of the U.S.’s Patient Protection and Affordable Care Act provision requiring the elimination of cost-sharing for recommended preventive care. I evaluate the effect of this value-based insurance design intervention on HPV immunization rates among girls and young women enrolled in private insurance plans. My regression approach uses variation in the intensity and timing of the intervention across plans to distinguish policy effects from background trends. I find that the policy was associated with modest increases in age-specific vaccination rates. Increases in vaccination per dollar reduction in cost-sharing were notably larger among beneficiaries in socioeconomically disadvantaged areas. Nevertheless, vaccination rates under free preventive care were well below federal targets, highlighting the need for additional interventions to increase HPV vaccine coverage. In Paper 2, I undertake a comparative effectiveness analysis of HPV vaccination by dose level within a U.S. cohort of adolescent girls and young women. Rates of screening-detected cervical abnormalities in claims are compared among recipients of zero, one, two, or three doses, using a marginal structural model approach to adjust for a broader set of potential confounders than would be possible with conventional regression methods. Findings from these analyses complement prior evidence from immunogenicity trials, and although protective effects appear greatest with three doses, support the value of HPV vaccination even when incomplete. Vaccine effect estimates are largest with respect to high-grade lesions that are precursors to cervical cancer. Using primary data from a randomized experiment, Paper 3 examines the cost-effectiveness of pay-for-performance interventions among obstetric care providers in rural Karnataka, India. I construct a decision analytic model to quantify incremental costs and life years under alternative policy scenarios, combining obstetric complication outcomes and program expenditures from the trial with published evidence on complication-related mortality and medical costs. Results suggest that an incentive program based on input quality is not cost-effective in its current form, but could become economically attractive if program activities can be adjusted to reduce costs while maintaining similar health effects. Performance data collection costs were substantial in this resource-limited setting and represent a key barrier to cost-effectiveness. / Health Policy

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No description available.

The evidence suggests that alteration of the endometrial environment in women with previous failed in vitro fertilization (IVF) or intra-cytoplasmic sperm injection (ICSI) by inflicting endometrial injury improves the outcome of the subsequent treatment cycle. The selected population assessed in the studies supporting this statement prevents from generalizing this to all women undergoing IVF or ICSI treatment. The mechanisms responsible for alteration of the endometrial environment following biopsy remain still unclear. If proven effective, endometrial injury could serve as a beneficial adjunct for all couples undergoing assisted reproductive treatment (ART). The hypothesis forming the basis of the work reported in this thesis was that endometrial injury in the cycle directly preceding an embryo transfer cycle, be it fresh or frozen, improves the outcome of that treatment irrespective of previous reproductive history. In order to support or refute this hypothesis, a clinical trial of endometrial biopsy prior to IVF or ICSI treatment has been designed. Additional objectives that allow examining the mechanisms responsible for the beneficial effects of the biopsy on the endometrium included utilization of two-dimensional, three- dimensional and Spatio-Temporal Image Correlation (STIC) ultrasound and assessment of the predictive value of any sonographic indices on ART outcome. Same sonographic modalities were used to determine the effect of endometrial injury on endometrial and subendometrial blood flow. Similarly, an examination of the value of uterine natural killer (uNK) cell numbers and expression of selected molecular markers of endometrial function as a predictors of ART outcome was explored. In this work it was not possible to clearly demonstrate a benefit of routine endometrial biopsy in all women undergoing ART on treatment outcomes, though clues, as to which population might benefit from the procedure, were identified. Extensive sonographic analysis of endometrial factors did not produce results allowing for unequivocal non- invasive identification of a receptive endometrial milieu. Triple layer endometrial pattern at oocyte collection was correlated with positive outcome and endometrial biopsy. Spatio-Temporal Image Correlation did not fulfil expectations as a non-invasive marker of endometrial receptivity and was not able to identify women that would go on to have a successful ART outcome. In a small number of patients, STIC indices were able to predict first trimester miscarriage with relatively high sensitivity and specificity. uNK cell numbers were not associated nor predictive of ART outcome, and as such not useful as a routine diagnostic tests prior to ART. An observed significant decrease in uNK cell levels following endometrial biopsy indicates a possible mechanism of action of this intervention. Limited (3) molecular cues were not able to differentiate between a receptive and non-receptive endometrium. This work, however extensive, indicates that the endometrium is a complex microenvironment requiring further investigation in order to understand and influence the mechanisms related to pregnancy establishment and development.