Obstetrics and Gynaecology - Theses
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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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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.