The mechanisms of deficient placentation in the first trimester remain poorly understood although apoptosis, hypoxia and oxidative stress have all been implicated. High uterine artery Doppler (UtAD) resistance indices (RI), indicative of poor uterine blood flow, have been shown to be predictive of placental complications of pregnancy such as pre-eclampsia (PE), fetal growth restriction (FGR) and stillbirth. We investigated these pathways in placental tissue from first trimester pregnancies characterised by their risk of developing placental complications. We provide evidence that, even in the first trimester, pregnancies with high UtAD RI demonstrate alterations in placental gene and protein expression. Illumina gene microarray analysis demonstrated that first trimester pregnancies with high RI have differentially regulated placental gene expression in pathways relating to immune and inflammatory response, cell motility, cell cycle and apoptosis compared with normal RI pregnancies. Apoptotic markers are significantly higher in high RI placental tissue as determined by western blot analysis of cleaved PARP and caspase 3. Protein expression of the trophoblast survival factor IGF2 is significantly lower. Both high RI and normal RI placental tissue show evidence of hypoxia and oxidative stress, with expression ofHIFla and 2a, HSP 70, presence ofnitrotyrosine residues and lipid peroxidation. We found no exaggerated placental hypoxia or oxidative stress associated with high RI pregnancies, in fact there was significantly lower HIFla protein in these cases. High RI placental tissue demonstrates an altered balance of antioxidant enzyme activity in response to this oxidative stress with significantly lower glutathione peroxidase activity and higher superoxide dismutase activity when compared with normal RI placental tissue. Hypoxia and oxidative stress appear to be a physiological state in early pregnancy, with our data not supporting the concept these are associated with deficient placentation in the first trimester. Higher levels of apoptosis, reduced IGF2 expression and altered antioxidant defences may contribute to abnormal placentation and the later development of pregnancy complications such as PE, FGR and stillbirth.

Human pregnancy disorders such as miscarriage, pre-eclampsia, intrauterine growth restriction and adherent placenta are thought to have their origins in defective trophoblastic invasion of the decidua. However, an elucidation of the exact mechanism of invasion is elusive because of poor understanding of early trophoblastdecidua interactions. Research is hampered by the inaccessibility of site of implantation, ethical constraints on studies of human pregnancy and lack of an adequate animal model. This research has sought to overcome many of these challenges by modelling, in vitro, events at the trophoblast-decidua interface. Human trophoblast is relatively readily available from pregnancy terminations or term pregnancies and cell lines have been produced in our laboratory. Decidua has previously proven more challenging to obtain in pure form. During this research a novel direct-vision hysteroscopic technique of decidual biopsy was developed which allowed accurate directed sampling of decidua parietalis separate from decidua basalis. Morphological and immunohistochemical studies confirmed the accuracy of the samples. Further characterisation of pure decidua parietalis versus basalis identified structural and functional changes likely to have been induced by or in response to trophoblastic invasion. For example, using electron microscopy, thicker and disrupted collagen fibrils were noted in decidua basalis versus uniform arrangement in decidua parietalis. Proteomic analysis established basic protein profiles for each type of decidua, and among the differences noted were 34 proteins with differential expression between basalis and parietalis. Consistent with the ultrastructural differences, extracellular matrix component lumican showed differences in expression between the two types of decidua. In vitro explant co- culture models were established with placental villi and pure decidua parietalis biopsies. These demonstrated early evidence of invasion of decidua parietalis by extravillous cytotrophoblast, with viability up to 96 hours. It is now possible, using the tools developed during this research, to conduct in-depth studies of early events at the materno-fetal interface.

No description available.

A lack of effective therapies continues to impair attempts to prevent preterm birth associated with inflammation. This study aimed to broaden the scope of candidate agents available to address this clinical problem. Using live cell confocal microscopy, Western immunoblotting, cell toxicity assay, and quantitative polymerase chain reaction techniques this study investigated the ability of a novel class of peptide vectors, termed Cell Penetrating Peptides (CPPs), to deliver cargo to human uterine and placental cells. It examined the ability of a CPP-linked peptide cargo: the Nemo Binding Domain (NBD) peptide, to inhibit inflammatory Nuclear Factor Kappa B (NFκB) signalling in uterine cells; comparing this response to a group of small molecule inhibitors of inflammatory pathways. Three CPP derived vectors were able to deliver fluorescent cargo to uterine myometrial and placental amnion cells within one hour over a concentration range of 1-10μM. Similar uptake kinetics in uterine cells were observed with the use of a fluorescently-tagged CPP conjugated to NBD. The NBD peptide, conjugated to a CPP derived from antennopaedia protein (Pen-NBD), was able to inhibit cytokine-stimulated cyclooxygenase-2 (COX2) protein induction at four hours; an effect that was not seen with other CPP-NBD conjugations, nor with NBD-mutant or unconjugated peptide controls. Data derived from both Western blots and gene arrays indicated that the anti-inflammatory effects of Pen-NBD were comparable to non-peptidic small molecule inhibitors of NFκB. However, Pen-NBD peptide did not prevent the cytokine-induced degradation of Inhibitor of Kappa B Alpha (IκBα) protein; nor did it inhibit the cytokine-induced expression of NFκB pathway genes, thus the precise targeting of NBD peptide within uterine cells remains uncertain and may be distinct to the canonical NFκB pathway. This research demonstrates the proof of concept that CPPs can enter human utero-placental cells and can deliver bioactive cargo to exert an anti-inflammatory effect. It provides a framework by which future research can examine CPP mediated delivery of a broad variety of potential cargo into uterine cells and thus offers a novel approach for the development of treatments aimed at preventing preterm birth.

The maternal immune system is unique and complex due to the influence of maternal and fetal compartments on each other. Researchers have postulated mechanisms that enable the feto-placental unit to escape maternal immune recognition and maternal adaptations to protect the fetus. Despite these, pregnant women can mount strong immune responses to infecting organisms. In order to profile the changes observed in uncomplicated pregnancies, longitudinal peripheral blood including delivery and postnatal samples were obtained. In a separate cohort, labour peripheral blood, cord blood and myometrial samples were obtained. Combination of IFN-γ, IL-10, IL-4 and granzyme B ELISpot data, flow cytometry, and lymphoproliferative and multiplex assays were analysed. Collectively the data indicates an activated immune system (CD38, IFN-γ) during pregnancy, with simultaneous negative regulation of responder T cells (IL-10) that reverses in late pregnancy. Parturition is accompanied by a loss of Treg mediated tolerance with suppression of immune activation. In a further study patients receiving progesterone or RU486 treatment in pregnancy were recruited to study the immunological effects of progesterone. Data revealed progesterone reduces IFN-γ and granzyme B T cell responses in vitro. Achieving this by a combination of altered memory T cell antigen sensitivity (effector memory/EM, and terminally differentiated effector memory/TEMRA subsets), leukocyte migration (CCR3, CCR6), and negative regulation of apoptosis and exhaustion (CD95, PD-1, CD57). Advancing pregnancy may be associated with an inherent loss of sensitivity to progesterone. Finally, to understand the effects of immunisation, pregnant and control subjects immunised against influenza were recruited. Ex vivo antibody and in vitro antigen specific pregnancy response to immunisation are comparably potent in both. However, this is achieved through different mechanisms of T cell, CTL and APC regulation. Thus, the maternal immune system is partly regulated by progesterone, which has suppressive quality. However, immuno-modulation in pregnancy is complex and antigen responses vary when compared to non-pregnant individuals.

Background: Obesity is an emerging public health concern in Sub-Saharan Africa and adult women are the population group most likely to be obese. Previous research conducted in high-income countries has demonstrated that high body mass index (BMI) has an adverse effect on a large number of reproductive outcomes; however no study has investigated this in Sub-Saharan Africa. Methods: Cross-sectional data of women (15-49 years) from twenty-seven countries in Sub- Saharan Africa, collected through the Demographic and Health Surveys, were used to examine the effect of BMI on female fecundity and adverse pregnancy outcomes. Underweight women (BMI <18.5kg/m2) were excluded. Multivariable regression models were used to examine four outcomes: (i) subfertility; (ii) time to first conception; (iii) caesarean delivery; (iv) neonatal death. The fecundity results were supplemented with a systematic review on the effect of BMI on waiting time to conception. Results: Increased BMI was associated with significantly increased odds of subfertility (Obese OR: 1.51; 95% Cl: 1.35-1.69). Five years after first marriage, 17% of non-contracepting obese (BMI Z30kg/m2) and 12.5% of pre-obese women (BMI 25-29.9kg/r2) had yet to conceive compared to 9.3% of optimal women (BMI 18.5-24.9kg/r2); however this difference was not statistically significant (p=0.5378). Each unit increase in maternal BMI (kg/m2)w as significantly associated with a 7% increase in the odds of caesarean delivery (OR:1 .07;9 5%C l: 1.06-1.08).M maternal obesity as significantly associated with increased odds of neonatal death (OR:1 .52;9 5%C I:1 .18-1.97). The association was strongest or deaths occurring in the first week of life to infants delivered Vaginally (OR: 2.43; 95% Cl: 1.43-3.88). Conclusions and Implications: Increased BMI is associated with adverse reproductive outcomes in the Sub-Saharan context. The risk is particularly strong or outcomes occurring around the intrapartum period. Overweight/obesity should be recognised as a risk actor for adverse reproductive outcomes i n Sub-Saharan Africa.

As less developed countries experience lower fertility, the age/parity distribution of pregnancies may shift. While these shifts may affect maternal mortality levels, their exact impact remains largely unknown. The aim of this thesis is to quantify the impact of fertility changes on maternal mortality. First, the literature was systemically reviewed for the strength of association between maternal age/parity and the maternal mortality ratio. Second, a retrospective cohort study utilised data from Matlab, Bangladesh to investigate the relationship between maternal age/gravidity and the pregnancy-related mortality ratio (PRMRatio) using logistic regressions. Lastly, the impact of observed (in Matlab) and theoretical shifts in childbearing composition on pregnancy-related mortality indicators was modelled using a compartmental model. The systematic review, including 62 studies, found that the risk of maternal death was higher for very young adolescents, older women and nulliparas. However, it was difficult to disentangle the confounding effect of age and parity. The retrospectivec ohort study found that the odds of pregnancy-relatedd eath was four times higher for women at the extreme maternal ages, even after adjustment for confounders, including gravidity. Nulligravidas were at increased risk of pregnancy-related death (adjusted OR=1.63, Cl: 1.24-2.16), but multigravidas were not. The adverse effect of first pregnancies was more pronounced for older women. The compartmental model suggests that the fertility decline in Matlab between 1983-1993 and 2000-2005 accounted for a 30% reduction in the pregnancy-related mortality rate (PRMRate). However, it made no contribution to the reduction in the PRMRatio observed during this period. Reducing or eliminating pregnancies at extreme ages and high gravidity could reduce the PRMRatio by 1-17% and the PRMRate by 1-50%. If all women had a maximum of one pregnancy each, the PRMRate would decrease by 74%. However, the PRMRatio would increase by 32% due to higher risk of first pregnancies. Fertility changes have limited impact on maternal mortality ratios, but can have substantial effect on the maternal mortality rate.

With a view to strengthening systems for maternal health, this study presents a comprehensive analysis of determinants of uptake of care and pregnancy-related mortality, with the main emphasis on distance to care. Data on geographical positioning (GIS), socio-demographic information, birth histories and deaths in women of reproductive age were collected during a household census in five rural districts of Southern Tanzania in 2007. Deaths reported as pregnancy-related were followed up by verbal autopsies. Health facility census information collected in the same area in 2009 was used. Data limitations included 30% either missing or low quality GIS data and missing birth histories for 9% of women. The analysis included 507 pregnancy-related deaths and 64,098 live births. Major deficiencies in quality of care provided in health facilities were identified. Although 75% of women lived within a distance of 4.6km to a facility providing delivery care, overall institutional delivery was low with 29% of all births in hospital and 11% in first-line facilities. Seventy-two percent of women living <5km away delivered in hospital and levels declined rapidly thereafter with no evidence of confounding. In contrast, less than 30% of women delivered in a first-line facility even if they lived less than 1km away. Overall pregnancy-related mortality was high at 712 deaths per 100,000 livebirths (95% Confidence Interval 652-777), with 32% due to haemorrhage. There was weak evidence of higher mortality with increasing distance to hospital, which was accentuated if the analysis was restricted to direct maternal deaths. Sensitivity analysis restricting analysis to the 70% of households with good quality GIS data did not alter conclusions. There was no evidence that low uptake of care at first-line facilities was explained by distance or socio-demographic factors. Deficiencies in quality of care influence both care uptake and mortality suggesting that investments in quality should be prioritized.

Introduction: Malaria and curable sexually transmitted and reproductive tract infections (STIs/RTIs) are important causes of adverse birth outcomes (ABO) and are both prevalent in most parts of sub-Saharan Africa. From a public health perspective, control of these infections requires interventions that are part of an integrated antenatal care package. The extent to which there may be coinfection increases the importance of such an integrated approach to reduce ABO. A systematic review and meta-analysis published in 2012 showed that the prevalence of malaria and curable STIs/RTIs among antenatal attendees in sub-Saharan Africa is considerable. However, the prevalence of malaria and curable STI/RTI coinfection has not been reported in any epidemiological setting. The primary objective of this thesis is to address this knowledge gap by estimating the prevalence of malaria, curable STIs/RTIs and their coinfection and to highlight the importance of an integrated approach to control malaria and STIs/RTIs in pregnancy. Secondary objectives of the study were to: (1) determine risk factors for malaria, curable STIs/RTIs and their coinfection; (2) estimate the prevalence of ABO and identify risk factors for ABO; (3) measure the in vivo efficacy and the prophylactic effect of sulphadoxine-pyrimethamine (SP) in pregnant women, and (4) characterise the molecular markers associated with parasite resistance to SP among pregnant women. Methods: A prospective cohort study of 1,086 antenatal attendees was conducted in Nchelenge District, Zambia. Consenting women visiting two health centres for their first antenatal care (ANC) visit were screened for malaria and curable STIs/RTIs (Chlamydia, gonorrhoea, trichomoniasis, bacterial vaginosis [BV] and syphilis). Socio-demographic data and maternal characteristics were also collected at enrolment. Sulphadoxine-pyrimethamine was administrated as intermittent preventive treatment to eligible women and they were followed up at day 28 for a second 13 malaria screening to determine the therapeutic and prophylactic failure of SP. At delivery participants were screened for placental malaria and data on birth outcomes were recorded. Univariate and multivariate analyses were conducted to determine the association between the potential risk factors for infection and ABO. Results: Of the 1086 women recruited 729 were successfully followed to delivery. The prevalence of malaria infection measured by microscopy was 31.8% (95% CI, 29.1-34.6) and by PCR was 57.8% (95% CI, 54.9-60.8). The risk of malaria infection was higher among pregnant women recruited from Nchelenge health centre compared to those attending the Kashikishi health centre (adjusted odds ratio [aOR] = 1.81; 95% CI, 1.38-2.37, P < 0.001), and HIV-infected women across health centres had a greater risk of malaria infection compared to HIV-uninfected women (aOR = 1.46; 95%, 1.00-2.13, P = 0.045). Infection with at least one STI/RTI was observed in 64.8% (95% CI, 61-67.4) of the participating women. With the exclusion of BV the prevalence of infection with at least one curable STI was 34.5% (95% CI, 31.7-37.4). Infection with at least one STI was associated with BV. In comparison to uninfected women, women infected with BV were at a higher risk of being infected with at least one STI (aOR 1.44; 95% CI, 1.08-1.92, P = 0.012). HIV-infected women had a higher risk of infection with BV than HIV-uninfected women (aOR 1.87; 95% CI, 1.24-2.83, P = 0.003) and women infected with at least one STI had a higher risk of BV (aOR 1.40; 95% CI (1.07 -1.84, P = 0.01). Among participants with complete results (n=1071), 38.7% (95% CI,35.7-41.6) were coinfected with malaria parasites and at least one STI/RTI; 18.9% (95% CI, 16.5-21.2) were infected with malaria parasites only; 26.0% (95% CI, 23.5-28.8) were infected with at least one STI/RTI but no malaria parasites, and 16.4% (95% CI, 14.1-18.6) had no infection. The risk of malaria and curable STI/RTI coinfection was higher among HIV infected women than HIV-uninfected women (OR; 3.59 [95% CI, 1.73-7.48], P < 0.001). The prevalence of composite ABO was 35.1%. Women shorter than 1.5m were at a higher risk of experiencing at least one ABO (aOR 1.55; 95% CI, 1.10-2.18, P = 0.02). The risk of having ABO among para II was less than half of the risk observed in 14 primiparous women (aOR 0.41; 95% CI, 0.27-0.61, P < 0.001) and much lower among multiparous women (aOR 0.32; 95% CI, 0.22-0.48, P < 0.001). Having taken two or more doses of SP during pregnancy was protective against ABO (aOR 0.47; 95% CI, 0.31-0.72, P = 0.001). None of the infections (malaria, curable STIs/RTIs and their coinfection) diagnosed at first ANC were associated with ABO. The prevalence of highly resistant quintuple mutant was 68.8% among first ANC attendees. Despite the moderate prevalence of the quintuple mutant among pregnant women, SP cleared parasitaemia in 86% of the asymptomatic malaria cases among HIV-negative women Conclusion: The prevalence of malaria, STI/RTI and their coinfection at first ANC in this study population was considerable. However, no association was found between ABO and infection with malaria or STI/RTI or their coinfection. This lack of association is partially a result of interventions within the ANC package including treatment of some STI/RTI, intermittent preventive treatment in pregnancy with SP and iron and folic acid supplementation. Sulphadoxine-pyrimethamine retains partial efficacy against P. falciparum malaria in this area with moderate prevalence of the quintuple mutant. While continuing the policy of offering intermittent preventive treatment with SP during pregnancy, an alternative preventive therapy that is effective against both malaria and STIs/RTIs needs to be considered.

Hyperglycosylated human chorionic gonadotrophin (HhCG) is a glycoprotein hormone that reportedly has biological functions different from those of hCG. It is produced by invasive cytotrophoblast cells at the time of implantation and in gestational trophoblastic disease. The invasion of cytotrophoblasts and their regulated proliferation are major determinants of pregnancy outcome: a shift of these controlling mechanisms has been associated with adverse pregnancy complications. Altered levels of HhCG may mirror placental dysfunction or impaired placental differentiation. This observational cohort study was undertaken to correlate various pregnancy outcomes with serum and urinary concentrations of HhCG and other hCG molecular forms, in order to determine whether HhCG is a helpful predictor of adverse pregnancy outcome. The first cohort included 287 women with spontaneous conceptions who attended the Obstetrics and Gynaecology Department of Mafraq Hospital in the United Arab Emirates. Paired serum and urine samples were collected on a single occasion from singleton pregnancies between 6 and 24 weeks of gestation. Patients were followed up until the pregnancy outcome was available. HhCG levels in pregnancies with uneventful outcomes and those with pregnancy complications were compared. Significantly lower HhCG levels were observed for pregnancies resulting in miscarriage as compared with late pregnancy complications. In the second cohort of 128 patients undergoing in vitro fertilisation or intracytoplasmic sperm injection at the Assisted Conception Unit in Edinburgh, the usefulness of serum HhCG levels on Day 14 of oocyte retrieval was assessed. HhCG levels were found to be significantly lower in pregnancies ending in spontaneous miscarriages and biochemical pregnancies. The potential diagnostic and prognostic utility of HhCG was confirmed by a receiver operating characteristic curve plot. The sugar chain heterogeneity of hCG from various sources was also investigated by SDS-PAGE and immunoblot analyses of pregnancy urine samples to assess their potential value as new diagnostic tools for predicting pregnancy outcome. Using monoclonal antibodies and a panel of lectins that can separate N- from O-linked sugar chains, variations in the hCG glycosylation patterns during different stages of pregnancy and pregnancy complication were studied. While the differences in the hCG glycosylation profiles reported here are interesting, whether they can helpfully contribute to the prediction of pregnancy outcome requires further study.