Obstetrics and Gynaecology - Theses
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ItemUnderstanding placental and cardiovascular adaptations in pregnancy: implications for therapeutic development for fetal growth restriction and preeclampsia.( 2021)Preeclampsia and fetal growth restriction are among the most serious obstetric conditions worldwide. Though they affect so many, we still do not completely understand their pathogenesis, nor have good ways to detect or treat them. In this thesis, I aimed to: improve our understanding of placental and vascular adaptations in preeclampsia and growth restriction, assess the ability of candidate therapeutics to mediate vascular dysfunction associated with preeclampsia, and to explore new models of preeclampsia and the long-term impacts on maternal cardiovascular health. DAAM2 and NR4A2 transcripts are elevated in the circulation of individuals whose pregnancies are complicated by fetal growth restriction (with or without preeclampsia). In Chapters 2 and 3 of this thesis, I identified these transcripts are expressed in the placenta, but their expression in either growth restricted or preeclamptic placenta does not mirror the increased expression of DAAM2 and NR4A2 in the maternal circulation. Thus, they are unlikely to originate from the dysfunctional placenta. However, their expression in the placenta throughout gestation, and clear regulation under hypoxia suggest they have roles in normal placental development and placental dysfunction. LOX-1 is elevated in the maternal vasculature in preeclampsia. In contrast to this, I identified in Chapter 4 that LOX-1 expression is reduced in the preeclamptic placenta. Furthermore, its expression is reduced in trophoblast under hypoxia. Treatment of trophoblasts with candidate preeclampsia therapeutics, esomeprazole and lansoprazole, (proton pump inhibitors) increased LOX-1 expression. These findings suggest that LOX-1 has a distinct role in the placenta compared to the vasculature. In Chapters 5 and 6, I assessed the ability of statins and new generation antiplatelets to mitigate preeclampsia-associated vascular dysfunction. In a model of endothelial dysfunction, pravastatin and simvastatin reduced secretion of vasoconstrictor, endothelin-1 and anti-angiogenic factor, sFLT-1. The new generation antiplatelet agents clopidogrel, prasugrel and ticagrelor reduced vasoconstriction of pregnant human omental (healthy and preeclamptic) and mouse mesenteric arteries through three different vasoconstrictors. Therefore, these candidate therapeutics can mitigate a key aspect of the pathogenesis driving preeclampsia. In Chapter 7, we established a model of preeclampsia in our laboratory through the blockade of nitric oxide synthesis (using L-NAME) to induce vasoconstriction. This led to elevated blood pressure, impaired fetal growth and elevated circulating levels of ‘toxic’ factors associated with preeclampsia. We were able to use this model to assess the effects of the new generation antiplatelet prasugrel, as a therapeutic, finding that prasugrel administration alongside L-NAME could reduce maternal blood pressure. I followed the dams post-delivery to investigate whether this model could simulate the long-term effects of preeclampsia on maternal cardiovascular health. I found that blood pressure and circulating toxic factors recovered as soon as 1 week post-delivery. At 10 weeks post-delivery, mice administered L-NAME during pregnancy demonstrated altered vascular reactivity, and increased expression of genes associated with inflammation in both the heart and kidney. However, these changes did not model the breadth of effects we anticipated, based on what is seen clinically post-preeclampsia. Overall, this thesis has added critical new knowledge regarding placental development, placental dysfunction, and vascular dysfunction. It provides further insight into the capability of novel candidate therapies for the prevention and treatment of preeclampsia, and provides new models of preeclampsia that can be used to enhance both our understanding of disease, and to assess future therapeutic potential.
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ItemIdentifying biomarkers for preeclampsia: from bedside to benchtop( 2021)Preeclampsia is a hypertensive disorder of pregnancy which claims the lives of thousands of mothers and babies every year. It is a major cause of both maternal and fetal morbidity and mortality throughout the world. There is no cure for preeclampsia but maintaining tight blood pressure control from the earliest possible gestation, alongside frequent medical reviews to identify progression of the disease, allows timely delivery to obtain the best outcome for both mum and baby. Further, the recently discovered benefits of instituting aspirin therapy before 16 weeks to prevent the development of the disease clearly demonstrates the need for a test to predict which patients are most likely to develop preeclampsia in their pregnancy. The current means sought to identify patients at risk of future preeclampsia is based on maternal risk factors. However, there is growing interest in detecting predictive biomarkers in the maternal circulation that are released as ‘distress signals’ from organs affected by preeclampsia: the placenta or the maternal endothelium. It is widely believed that the pathophysiology of preeclampsia originates in early placentation. Poor implantation very early in the pregnancy leads to a hypoxic placental environment. The hypoxic placenta then releases antiangiogenic factors into the maternal circulation which cause widespread maternal endothelial dysfunction. Examples of biomarkers for preeclampsia in current literature include soluble Flt1 (sFlt1) and placental growth factor (PlGF). There has also been significant investigation toward understanding the mechanisms by which such biomarkers may contribute to disease pathogenesis - which is key in allowing better understanding of the underlying pathophysiology. The theme of this PhD is biomarker discovery and ‘reverse translation’: potential biomarkers will be identified in blood samples collected from patients who participated in a large prospective cohort. The biomarkers were then studied in the laboratory, taking the samples from bedside to benchtop. The first aim of this PhD was to investigate potential predictive biomarkers originating from dysfunctional maternal endothelial cells. I have investigated the mechanisms by which the biomarkers are altered, carrying out functional studies to observe how placental factors influence the expression of these markers of endothelial cell dysfunction, and whether there are any specific implicated placental factors. Indeed, I identified that mRNA for the transcription factor GATA2 is differentially expressed in the maternal circulation up to 12 weeks before the clinical diagnosis of preeclampsia. Further, the vasoactive peptide hormone adrenomedullin is similarly dysregulated. While GATA2 is likely endothelial in origin, adrenomedullin may originate from maternal endothelium or the placenta. The second aim of this PhD was to investigate the possible use of circulating microRNAs (miRs) to predict preeclampsia. Their use in distant cell signalling has been investigated for prediction and detection of cancer and other diseases. The C19MC miRNA cluster is a primate specific cluster of miR genes that is highly expressed in the placenta. Using microarray technology, I have identified miRs from this cluster, alongside those involved in endothelial cell dysfunction, which are altered in preeclamptic placentas and in the blood of women destined to develop preeclampsia. Of note, two microRNAs which are involved in regulating endothelial cell function (miRs 363 and 149), I demonstrated are dysregulated in the circulation and placentas of women with diagnosed preterm preeclampsia, as well as in the blood at 36 weeks’ gestation preceding term preeclampsia onset. In the third aim of this PhD, I have investigated the mechanisms by which placental growth factor (PlGF) may be regulated. PLGF is a proangiogenic molecule which is highly expressed in placental trophoblast cells and is vital for angiogenesis. Circulating PlGF has been implicated as a potential predictive biomarker of disease especially when used in combination with sFlt1 in the sFlt1:PlGF ratio. However, the pathways involved in the regulation of PlGF are still poorly understood. Due to the close relationship between sFlt1 and PlGF, I have assessed the impact of known regulatory pathways of sFlt1, on the secretion of PlGF. I have identified molecules in the EGFR pathway which regulate PlGF secretion and ruled out the mitochondrial electron transport chain as a means of enhancing PlGF production. In conclusion, this thesis has demonstrated that endothelial produced biomarkers (GATA2, Adrenomedullin and miRs363 and 149) hold perhaps the greatest potential as predictors of term preeclampsia. Although also dysregulated in the blood of women with established preterm disease, whether they might also be predictive of preterm preeclampsia remains to be assessed. While circulating levels of placental specific miRNAs were unchanged in term disease, their potential as biomarkers for preterm preeclampsia also remains unexplored. Notably this work also identified the EGFR superhighway as a negative regulator of PlGF production and suggested that future identification of therapeutics that reduce EGFR signalling, may hold potential for enhancing PlGF production and release in preeclampsia. Overall, it is my hope that this work has significantly contributed to the future development of novel screening tests that will lead to improved outcomes for women and their babies.
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ItemCan dietary polyphenols prevent adverse pregnancy outcomes?( 2020)GDM and spontaneous preterm birth are significant pregnancy complications that impact up to 20% of pregnancies worldwide. Despite current interventions, these rates are increasing. Both GDM and preterm birth leave a legacy of negative health consequences on maternal and fetal health post-pregnancy. Further, both conditions create significant economic burden on the healthcare system that continues beyond pregnancy. GDM and spontaneous preterm birth are seemingly disparate conditions; however, both exhibit a maternal inflammatory response that drives their pathophysiology. For example, inflammation is positively correlated with insulin resistance associated with GDM. Additionally, GDM pregnancies are characterised by increased oxidative stress. Inflammation is also increased in pregnancies that culminate in spontaneous preterm birth, with several studies identifying a contributing role for pro-inflammatory cytokines to myometrial activation and fetal membrane weakening. Current interventions, however, target only the downstream symptoms of GDM and preterm birth, and fail to adequately target the underlying inflammation. Given the role of inflammation in their pathophysiology, there is an urgent need for anti-inflammatory therapeutic that can prevent the development of GDM and preterm birth. Recent epidemiological evidence suggests that plant-based diets, rich in polyphenols, are associated with reduced incidence of GDM and preterm birth. Polyphenols are bioactive plant compounds found in fruits, vegetables, nuts and legumes and are classified according to four categories (flavonoids, phenolic acids, stilbenes and lignans). Importantly, polyphenols possess anti-inflammatory properties. However, the specific effect of polyphenols in the context of GDM or spontaneous preterm birth is unknown. Thus, this thesis employs a series of in vitro and in vivo models to determine the effect of dietary polyphenols on the expression of mediators involved in the development of GDM and preterm birth. To determine the effect of polyphenols as therapeutic interventions for GDM, three studies in this thesis investigated the effect of flavonoids (naringenin and nobiletin) and phenolic acids (punicalagin and curcumin) in in vitro and in vivo models of GDM. In vitro, human placenta, adipose tissue (visceral and subcutaneous) and skeletal muscle were treated with or without the pro-inflammatory cytokine TNF. In vivo, pregnant heterozygous leptin receptor deficient db/+ mice were used to model GDM. These chapters found that naringenin, nobiletin and the phenolic acids exerted anti-inflammatory effects in placenta and adipose tissue in vitro and in vivo. Naringenin and nobiletin were also found to improve human skeletal muscle glucose uptake, as well as lower fasting blood glucose of pregnant GDM mice. Naringenin and the phenolic acids also reduced oxidative stress associated with GDM by regulating antioxidant mRNA expression and reducing hydrogen peroxide levels in placenta and adipose tissue in vitro and in vivo. Interestingly, using a proteomic approach, nobiletin was found to differentially regulate metabolic signalling pathways in the placenta, pancreas, adipose tissue and skeletal muscle of pregnant GDM mice. The findings from these chapters indicate that polyphenols can target inflammation, insulin resistance and oxidative stress associated with GDM. To determine the effect of polyphenols as therapeutic interventions for spontaneous preterm birth, two studies in this thesis investigated the effect of the phenolic acids gallic acid and punicalagin in in vitro and in vivo models of intrauterine inflammation associated with spontaneous preterm birth. In vitro, primary cells were isolated from myometrium and fetal membranes (decidua, amnion mesenchyme and amnion epithelia) and stimulated with or without either pro-inflammatory cytokine IL1B or TNF. In vivo, pregnant mice were treated with LPS via intraperitoneal injection on gestational day 16.5 to induce intrauterine inflammation associated with preterm birth. These chapters found that gallic acid and punicalagin exerted anti- inflammatory effects in primary human myometrial, decidual and amnion cells. Gallic acid and punicalagin also downregulated the expression of prostaglandin pathway proteins PTGS2 and PGF2a in both myometrium and decidua in vitro and in vivo. Notably, using an in situ 3D collagen gel assay, gallic acid and punicalagin inhibited cytokine-stimulated myometrial cell contractility. Both phenolic acids also differentially regulated MMP expression associated with fetal membrane weakening in vitro. Using a proteomic approach, gallic acid was also found to differentially regulate expression of collagen proteins and cytoskeletal proteins associated with cell contractility. Altogether, these findings indicate that polyphenols can target pro- inflammatory and pro-labor mediators involved in myometrial contractility and fetal membrane weakening associated with spontaneous preterm birth. Taken together, these studies demonstrate that polyphenols exert anti-inflammatory properties in in vitro and in vivo models of GDM and preterm birth. Additionally, polyphenols may target other pathological features, including insulin resistance and oxidative stress associated with GDM; and myometrial activation and fetal membrane weakening associated with spontaneous preterm birth. Polyphenols may represent alternative therapeutic options to prevent GDM and spontaneous preterm birth. Further research is warranted to investigate the effect of polyphenols on maternal and fetal health post-pregnancy.
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ItemThe role of placental heparan sulphate proteoglycans in the pathogenesis of pre-eclampsia( 2015)Introduction: Uncomplicated pregnancies represent a hypercoaguable state. However, placental thrombosis is rare in these pregnancies which suggests that thrombin generation must be tightly regulated. In contrast, pregnancy disorders such as pre-eclampsia not only demonstrate an exaggerated increase in procoagulant activity which may contribute to the thrombotic lesions observed in the uteroplacental circulation of these pregnancies, but there is evidence of substantial cell growth, differentiation and angiogenic functional defects. Proteoglycans are abundantly expressed within the placenta compared to other human tissues. Particularly of interest are heparan sulphate proteoglycans which have important anticoagulant, anti-inflammatory and angiogenic properties. Hypothesis: Altered abundance, structure or function of placental heparan sulphate proteoglycans contributes to the development of pre-eclampsia by: increasing placental thrombin generation, interfering with cellular growth, differentiation, disrupting normal angiogenesis and altering growth factor interactions. Aims : 1) To determine the mRNA expression, protein abundance and cellular localisation of placental heparan sulphate proteoglycans from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. 2) To investigate the functional consequence of reduced HSPGs in placental cells 3) To determine the differences in the abundance and structure of heparan sulphate proteoglycans and heparan sulphate GAGs from placentae obtained from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. Methods: 1) The mRNA, protein abundance and cellular localisation of placental heparan sulphate proteoglycans was determined using real-time PCR, western immunoblotting and immunofluorescence, respectively. 2) To investigate the functions of reduced heparan sulphate proteoglycans, a cell culture model using short interference RNA was used. Cellular growth will be assessed using the xCELLigence system, thrombin generation using the calibrated automated thrombogram system and angiogenesis will be determined using a matrigel based assay. Cellular differentiation and apoptosis will be determined using real-time PCR. Growth factor signalling will be determined using a real-time PCR growth factor array. 3) To isolate proteoglycans from placenta, anion exchange chromatography will be utilised. Enzymatic digestion will be used to isolate the glycosaminoglycans, and enzyme-linked immunosorbant assays will be undertaken to determine the abundance of PGs and GAGs. Results: The mRNA expression of heparan sulphate proteoglycans, Syndecan 1, Syndecan 2, Glypican 1 and Glypican 3 are significantly reduced in the placentae of women whose pregnancies are complicated by pre-eclampsia. A cell culture model was utilised to determine the functions of heparan sulphate proteoglycans in the placenta. Successful downregulation of heparan sulphate proteoglycans was achieved in the cell lines using short interference RNA treatment, and a number of functions were significantly altered as a result. The downregulation of Syndecan 1, Glypican 1 and Glypican 3 resulted in significant alterations in the downstream growth factor targets. Reduced Syndecan 2 expression resulted in a significant reduction in the thrombin generation potential of endothelial cells. Investigation into the abundance and structure of glycosaminoglycans within the placenta, demonstrated heparan sulphate glycosaminoglycans to be significantly reduced in pregnancies complicated with pre-eclampsia compared to gestation matched controls. Conclusion: The reduction in heparan sulphate proteoglycans expression observed in pregnancies complicated with pre-eclampsia may be responsible for the altered growth factor interaction commonly observed in preeclamptic pregnancies. This study has provided us with a greater understanding of the biological role of heparan sulphate proteoglycans within the human placenta and its potential implications in the pathogenesis of pre-eclampsia.
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ItemIdentification of novel biomarkers in the cervicovaginal fluid to predict preterm birth( 2013)INTRODUCTION: Preterm birth is associated with major perinatal morbidity and mortality. Despite the many advances in modern obstetrics the rate of preterm birth is increasing. The discovery of novel biomarkers that could reliably identify women who will subsequently deliver preterm may allow for timely medical intervention and therapeutic treatments aimed at improving maternal and fetal outcomes. The cervicovaginal fluid (CVF) provides a rich source for the discovery of putative biomarkers of pathophysiological disorders of pregnancy. It is hypothesised that the biochemical alterations that occur in the cervix and the overlying fetal membranes with labour may be reflected in the CVF proteome. Given the multifactorial aetiology of preterm birth, women may present with different clinical presentations that lead to preterm birth. This thesis has investigated the CVF proteome of three clinical groups of women in order to discover putative novel biomarkers of preterm birth in: (i) asymptomatic women at risk of preterm labour (PTL); (ii) women with symptoms of threatened PTL; and (iii) asymptomatic women who subsequently experienced preterm premature rupture of the fetal membranes (preterm PROM). A functional proteomic approach was used to detect putative novel biomarkers of PTL or preterm PROM using two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry. Validation of these differentially expressed proteins was performed using enzyme-linked immunosorbant assay (ELISA) or Western blot on an independent cohort. MAIN FINDINGS: Chapter 3 contains work that has been published in Reproduction. IL-1ra and thioredoxin were significantly decreased in asymptomatic women with subsequent PTL and predictive modelling found these biomarkers to be effective predictors of spontaneous preterm birth. Chapter 4 contains work that has been published in PLoS One. This study investigated the temporal changes in vitamin D binding protein (VDBP) in the CVF with approaching term and PTL. VDBP was increased in the CVF with approaching term and PTL. Predictive modelling analysis also found VDBP to be a reliable predictor of spontaneous term and PTL. The study presented in Chapter 5 identified a number differentially expressed protein in the CVF proteome of women presenting with threatened PTL. IL-1ra, IL-1α, IL-1β, VDBP, thioredoxin and albumin were significantly altered in the CVF of symptomatic women in threatened PTL with subsequent preterm birth. Modelling analysis using albumin & VDBP was superior to fetal fibronectin in predicting preterm birth. The study presented in Chapter 6 has been published in Reproduction and describes the proteomic analysis of CVF samples collected from asymptomatic women who subsequently experienced preterm PROM. Western blot analysis confirmed IL-1ra, annexin A3 and cystatin A to be significantly altered in the women who later experienced spontaneous preterm PROM. CONCLUSION: This thesis has identified a number of novel biomarkers for the prediction of preterm birth and has demonstrated that multi-marker models have improved predictive utility compared to individual biomarkers alone (Chapters 3 and 5). The proteomic analysis of the CVF has confirmed that labour is a complex physiological process involving tissue remodelling, oxidative stress and inflammation. The discovery of IL-1ra, thioredoxin and VDBP to be differentially expressed with preterm labour in both asymptomatic and symptomatic women supports the theory of a final common pathway of labour.