Obstetrics and Gynaecology - Theses
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ItemAgeing of the placenta: potential prognostics, diagnostics and therapeutics( 2022)Abstract Ageing effects on human organs are most notable in the elderly. However, the placenta is exceptional because it shows signs of ageing in a short time span. Healthy ageing of the placenta is the process of developing and maintaining the functional ability of the placenta until term and is a feature of uncomplicated pregnancies. However, recent studies show unhealthy ageing of the placenta is a common feature of important human pregnancy disorders. Unhealthy premature ageing of the placenta is associated with pregnancy complications of preeclampsia (PE) and fetal growth restriction (FGR), whereas unhealthy placental ageing at term is associated with stillbirth. Given the emerging importance of placental ageing, the general aim of the work was to investigate potential prognostics, diagnostics and therapeutics for ageing of the placenta. Regarding prognostics and diagnostics, the sFlt-1/PlGF ratio test used to predict the risk of PE was investigated in a retrospective clinical study. The sFlt-1/PlGF ratio increased with the healthy ageing of the placenta at term. In addition, the total extracellular vesicle population isolated from the maternal peripheral blood during second trimester increased in pregnancies with a late onset PE outcome compared to a gestation matched normotensive control outcome and showed an association with the sFlt-1 concentration. These results suggest that the sFlt-1/PlGF ratio and EV population size are potential prognostic or diagnostic markers for unhealthy placental ageing-associated pathologies such as PE and stillbirth. A hallmark of ageing is damage to stem cells, which prevents them from replenishing cells in ageing organs and tissues. Pharmaceutical drugs and extracellular vesicles (EV) with anti-ageing properties secreted by healthy ageing early term decidual mesenchymal stem cells (ET-DMSC) were used to treat unhealthy premature-ageing PE-DMSC and ageing late term DMSC (LT-DMSC). As well, a microRNA present in high levels in ET-DMSC (miR-516b-5p) was also analysed. Drugs with anti-ageing properties and EV from ET-DMSC delayed the ageing phenotype of both PE-DMSC and LT-DMSC. In addition, miR-516b-5p affected stem cell survival. ET-DMSC with elevated levels of miR-516b-5p had better survivability. Finally, as described above, the total EV population size in maternal blood is a potential prognostic or diagnostic marker for unhealthy placental ageing-associated pathologies. However, many cell types, including stem cells, secrete EV into maternal blood. The final aim was to determine whether EV secreted from DMSC into maternal blood could be enriched from maternal blood. In order to achieve this, unique DMSC-EV cell surface markers need to be identified. When compared with chorionic mesenchymal stem cell derived EV (CMSC-EV), mass spectrometry analyses showed DMSC-EV and CMSC-EV have unique combinations of lipids and proteins on their surface, which could potentially facilitate the isolation of DMSC-EV and CMSC-EV from maternal blood. In addition, studies in the literature showed that changes in the levels of candidate lipids and proteins are associated with healthy ageing and in some cases with unhealthy placental ageing-associates pathologies such as PE and FGR, where the placenta shows signs of premature ageing.
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ItemBiological Pathways Used by Decidual Mesenchymal Stem Cell-Derived Extracellular Vesicles in Repairing Dysfunctional Endothelium( 2022)Preeclampsia (PE), a common pregnancy-related disorder, is characterised by endothelial cell dysfunction and oxidative stress. Oxidative stress is a consequence of reduced blood flow to the placenta and results in the secretion of anti-angiogenic factors that cause systemic damage to maternal vascular endothelial cells. Endothelial cell dysfunction culminates in the clinical symptoms of PE, including elevated blood pressure, proteinuria, and HELLP syndrome. Human cell culture models variously use damaging agents such as lipopolysaccharide (LPS), hydrogen peroxide (H2O2), and serum derived from preeclamptic women (PE serum) to model the damage to endothelial cells in PE. However, the three methods have not been compared. Several studies reported that decidual mesenchymal stem cells (DMSCs) and the extracellular vesicles derived from them (DMSC_EVs) have beneficial effects on the cell growth profile of endothelial cells. However, the biological pathways by which DMSC_EVs exert their beneficial effects are poorly understood. The damaging agents (LPS, H2O2, and PE serum) were used to treat human umbilical vein endothelial cells (HUVEC), which are a common model for human endothelial cells. Damaged HUVEC were treated with or without DMSC_EVs. HUVEC growth profiles were measured by xCELLigence real-time functional assays. HUVECs were collected by centrifugation (i.e. cell pellets) as was as the conditioned medium (CM) containing secreted proteins. The general hypothesis for the study was that DMSC_EVs repair endothelial cell damage in human cell culture models of PE, and that the biological pathways involved in repair can be identified. The first aim determined the optimal concentration of each damaging agent and xCELLigence assays showed that all damaging agents had a detrimental effect on HUVEC growth phases of attachment and proliferation. Following exposure of HUVEC to the damaging agent and treatment with various concentrations of DMSC_EVs (0, 50, 100, 150 microgram/ml), the growth phases of HUVEC were assessed by xCELLigence real-time analysis. In each of the three models of endothelial cell damage, adding 100 microgram/ml DMSC_EVs to damaged HUVECs had a consistent stimulatory effect compared to untreated cells with respect to both cell attachment (LPS P<0.01, H2O2 P<0.001, PE serum P<0.01) and proliferation (LPS P<0.001, H2O2 P<0.001, PE serum P<0.001). PE serum was chosen for subsequent analyses as this damaging agent most reflects the in vivo situation in PE. Following xCELLigence analysis, cell pellets and conditioned media were collected from PE serum-damaged HUVEC treated with, or without, 100 microgram/ml DMSC_EVs. These were assessed by mass spectrometry to determine which biomolecules were involved in the effect of DMSC_EVs on PE-serum damaged HUVEC attachment and proliferation. Following DMSC_EV treatment of PE serum-damaged HUVEC, the most abundant and differentially expressed HUVEC proteins during the attachment phase were NADH-ubiquinone oxidoreductase chain 4 (1.91 fold) and Interferon-induced transmembrane protein 3 (-2.14 fold), while in the proliferation phase, cellular proteins YIF1B (3.58 fold) and Brain acid soluble protein 1 (-2.15 fold) were the most abundant and differentially expressed. The most abundant and differentially expressed secreted proteins after DMSC_EV treatment of PE serum-damaged HUVEC treatment in the attachment phase were Cytochrome c1 (6.54 fold) and Midasin (-4.21 fold), while in the proliferation phase, Collagen alpha-1(IV) chain (6.6 fold) and cAMP-dependent protein kinase catalytic subunit alpha (-3.76 fold) were the most abundant and differentially expressed. Analysis of relevant biological processes revealed the most affected processes were cell organization and biogenesis. Kegg pathway analysis revealed that DMSC_EV treatment altered pathways relevant to cell attachment included focal adhesion, crosslinking of collagen fibrils and extracellular matrix organization. Kegg pathway analysis showed cell proliferation pathways of extracellular matrix organization and degradation were altered by DMSC_EV treatment. Finally, literature searches showed many of the proteins that displayed the highest abundance and differential expression levels following DMSC_EV treatment were implicated directly or indirectly either in the pathogenesis of the PE placenta or showed altered levels in PE patient blood. This study shows that in a cell culture model of PE, treatment with DMSC_EVs had beneficial effects on important cell growth phases of damaged endothelial cells (i.e. attachment and proliferation), that relevant biological processes and biological pathways were altered and that many of the proteins involved were also altered in human PE. This work lays the groundwork for future PE animal model studies to determine whether DMSC_EVs improve endothelial cell function and whether the biological processes and pathways of endothelial repair identified in the human cell culture models of PE in this study are relevant in vivo.
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ItemThe role of metzincins and TIMPs in ovarian cancer( 2022)Epithelial ovarian cancer has the highest fatality-to-case ratio of all the gynaecological malignancies. In Australia, only 46% of patients survive 5 years after diagnosis of epithelial ovarian cancer. The metzincin family of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in matrix remodelling, regulation of signal-transduction pathways, angiogenesis and a range of diseases including cancers. This thesis investigated the role of metzincins and TIMPs in ovarian cancer providing insights in progression, chemoresistance and recurrence in patients. It revealed that TIMP-2, specifically, plays a role in serous epithelial ovarian cancer progression and regulates chemosensitivity of ovarian cancer cells through pathways that regulate cancer stem cell (CSC) phenotypes. In chapters 3 and 4, the expression patterns of metzincins and TIMPs were assessed in (a) FIGO stages, Silverberg grades and WHO types of primary serous ovarian carcinomas; and (b) ascites-derived tumour cells collected from chemo-naive and chemotherapy-treated recurrent patients and fluids. The expression of TIMP-2 and TIMP-3 was significantly upregulated in high-grade serous ovarian tumours compared to benign tumours. Moreover, TIMP-2 mRNA was regulated in the chemotherapy-treated, ascites-derived epithelial tumours compared to chemo-naive samples. TIMPs and MMPs were differentially expressed in fourteen ovarian cancer cell lines. Chemotherapy treatments (cisplatin or paclitaxel) modulated TIMPs and MMPs expression in association with increased expression of genes related to chemoresistance. Overall, the findings indicated that TIMP-2 was a likely candidate to be involved with ovarian tumour progression and chemoresistance. In chapter 5, TIMP-2 gene expression was transiently knocked-down by siRNA (T2-KD) in three high grade serous ovarian cancer cell lines (JHOS-2, OVCAR4 and OVCAR5) and a control cell line derived from normal oviductal epithelium (FT282). Overall, TIMP-2 downregulation significantly downregulated the expression and activation of MMP-2, but enhanced: proliferation, invasion, MMP-14 expression, induced EMT and increased sensitivity to cisplatin and paclitaxel. Chemotherapy treatments significantly enhanced the expression of TIMP-2, MMP-2, MMP-14, chemoresistant and CSC genes and induced activation of STAT3 in control cells, but remained unchanged in T2-KD cells. These results demonstrate that TIMP-2 plays a role in ovarian cancer progression and regulates chemosensitivity of ovarian cancer cells through pathways that regulate CSC phenotype. In chapter 6, TIMP-2 was edited in OVCAR5 cell line using CRISPR/Cas9 using two different guide RNAs. This resulted in two clonal cell lines, with gRNA2 (63% TIMP-2 knock-down) cells having similar characteristics as siRNA T2-KD cells. However, the functional outcome from gRNA1 (80% knock-down) was partially different. This cell line had similar MMP-2 and MMP-14 profile as gRNA2 but was resistant to paclitaxel. In addition, gRNA1 cells produced greater tumour burden than control cells in mice and a significantly shorter survival response compared to gRNA2 cells which produced small tumour burden and significantly longer survival. Collectively, these results demonstrate a complex role of TIMP-2 in ovarian cancer progression and chemoresistance. It is not only the reduction of TIMP-2 expression but the degree of reduction that influences the physiological/biological end points. Findings in this thesis have furthered understanding of TIMP-2 in ovarian tumorigenesis and laid the foundation for TIMP-2 mediated targeted therapy.
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ItemHuman amnion epithelial cells and bronchopulmonary dysplasia in preterm infants( 2021)Bronchopulmonary dysplasia (BPD) is a frequent and important morbidity for extremely preterm infants. The lung injury that gives rise to BPD occurs during the late canalicular and early saccular phases of lung maturation and is induced by various agents including hyperoxia, ventilation associated injury and infection. The result is arrest of alveolar development. Consequently, infants with BPD are more likely to have poorer respiratory health, not just in infancy, but through their lifetime. Sufferers of BPD are at greater risk of neurodevelopment challenges such as cerebral palsy and cognitive impairment. Treating, or better still, preventing this multifactorial disease is challenging. Cell therapy, with its capacity to modulate the inflammatory response, support angiogenesis and protect endogenous progenitor cells, is a unique and promising therapy for BPD. Human amnion epithelial cells (hAECs) are a type of cell therapy derived from the amniotic membrane of term placentae. The mechanisms of hAEC action and evidence of their therapeutic potential in BPD-like injury are evaluated here. Following this examination of the pre-clinical evidence, this thesis outlines the development and implementation of a phase 1 dose escalation study of hAECs in infants at significant risk of developing BPD. This work builds on an earlier, first in human study, of a modest dose of hAECs in infants with established severe BPD. The tolerability of larger doses of hAECs given to less mature infants in our study called ‘RENEWAL; Reducing Neonatal Lung Disease With Amnion Cells’ is reported. In an important and novel contribution to the field of neonatal cell therapy we have highlighted and resolved deficiencies in bedside cell therapy infusion protocols. Established infusion protocols were discovered to delivered less than 20% of the intended dose of hAECs during the early phases of the RENEWAL study. We solved this challenge with simple measures using equipment that was readily available in the neonatal unit. Finally, the strengths and limitations of the RENEWAL study are examined, and future directions for neonatal cell therapy are discussed. The RENEWAL study represents an important milestone in neonatal cell therapy. The RENEWAL study has detected and resolved important knowledge gaps. With its completion anticipated in the next 18 months, the RENEWAL study will add significantly to the body of evidence for the safety of cell therapy in extremely preterm infants. Importantly, the tolerable dose of hAECs in extremely preterm infants reported here provides a foundation on which to build the next series of cell therapy trials.
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ItemNasal high flow therapy in neonates: new evidence and a novel application( 2021)Background Newborn infants may require respiratory support in the first days of life. Preterm infants receiving respiratory support are susceptible to a chronic lung disease called bronchopulmonary dysplasia (BPD). Non-invasive respiratory support reduces rates of BPD, compared with ventilatory support via an endotracheal tube. Continuous positive airway pressure (CPAP) is the most common form of non-invasive respiratory support in neonates. Nasal high flow therapy (nHF) is a novel alternative to CPAP. It comprises two small, tapered nasal prongs through which heated, humidified, blended air and oxygen are delivered at high flow rates. There are currently two common indications for nHF in neonates: primary respiratory support soon after birth for the prophylaxis or treatment of respiratory distress syndrome, and post-extubation support following a period of mechanical ventilation. The evidence for the use of nHF is evolving, and systematic evaluation of this evidence is important. There may be novel applications of nHF. In older children and adults, nHF is used during endotracheal intubation (the placement of a breathing tube in the trachea to support breathing with a mechanical ventilator). Neonatal endotracheal intubation is a procedure that is technically difficult and is occurring less frequently over time; hence success rates on the first attempt are low. In addition, oxygen desaturation during endotracheal intubation is common. Interventions to improve the likelihood of first attempt intubation success, as well as infant stability during the procedure are therefore needed. Based on adult and paediatric data, nHF use during endotracheal intubation may prolong the time to desaturation. There have previously been no studies of the use of nHF during neonatal intubation. Aims This thesis aims (i) to synthesise existing evidence for nHF use in neonates and (ii) to evaluate the innovative application of nHF during neonatal endotracheal intubation. Methods For this thesis, I summarised the literature on invasive and non-invasive neonatal respiratory support, focusing on preterm infants. I also summarised the difficulties associated with neonatal endotracheal intubation. I then undertook the following original research: 1. A Cochrane systematic review and meta-analysis of the use of nHF for primary respiratory support in preterm infants, compared with other non-invasive respiratory support modalities; and 2. A multicentre, randomised controlled trial of nHF during oral neonatal endotracheal intubation, in which neonates undergoing intubation were randomly allocated to either nHF during the first intubation attempt, or to standard care. Results of original research 1. When compared with CPAP, the use of nHF for primary respiratory support in preterm infants (<37 weeks’ gestation) was associated with no difference in the outcomes of death, BPD or the combined outcome of death or BPD. The rate of treatment failure at 72 hours after trial entry was higher in infants managed with nHF, compared with CPAP. However, there was no difference in the rate of mechanical ventilation between groups. When compared with non-invasive positive pressure ventilation (CPAP with superimposed pressure rises to a set peak pressure), the use of nHF was associated with no difference in the outcomes of death, BPD, the combined outcome of death or BPD, treatment failure, or mechanical ventilation. 2. The use of nHF during neonatal endotracheal intubation increased the likelihood of successful intubation on the first attempt without physiological instability. Infants managed with nHF were more likely to be intubated successfully on the first attempt, and fewer infants desaturated during the procedure. Whilst there was no evidence of a difference in effect in the prespecified subgroups of postmenstrual age and use of premedication, the beneficial effect of nHF during intubation was greater when intubation was performed by inexperienced operators. Nasal HF was quick to apply and there was no difference in serious adverse events between groups. Conclusions These findings suggest that the use of nHF for primary respiratory support in preterm infants may result in little to no difference in the clinically important outcomes of death or BPD, compared with CPAP or NIPPV. Nasal HF is associated with a higher rate of treatment failure than CPAP. However, provided surfactant and/or CPAP are available in the event of nHF treatment failure, there is no difference in the rate of mechanical ventilation. The use of nHF during neonatal endotracheal intubation increases the likelihood of successful intubation on the first attempt and improves physiological stability of the infant. Using nHF during neonatal endotracheal intubation is a simple, easily applicable technique which may improve neonatal care and aid acquisition of a critical procedural skill.
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ItemCharacterising the impact of chemotherapy on the ovary( 2021)Cancer survivorship has risen, but in females, DNA-damaging treatments deplete the ovarian reserve of primordial follicles (PMFs), causing infertility and ovarian endocrine failure. It has previously been shown in mice that elimination of the pro-apoptotic BH3-only protein, PUMA, protects oocytes from gamma-irradiation-induced apoptosis. However, the role of such pathways following chemotherapy has not previously been studied and the underlying mechanisms of chemotherapy-induced PMF loss have been unclear, hampering efforts to develop effective pharmacologic ovarian protection agents. This thesis had three main aims: 1. To determine whether PUMA elimination prevents loss of primordial follicles following gonadotoxic chemotherapy; 2. To identify the underlying mechanisms of PMF loss following cisplatin and cyclophosphamide, including their intraovarian cellular targets and the time course over which depletion occurs; 3. To investigate the DNA repair capacity of PMFs following cisplatin- and cyclophosphamide-induced damage and identify repair pathways used. To address these aims, three original studies were devised, using mouse models. In Chapter 2, unbiased stereology was used to quantify the effect of cyclophosphamide and cisplatin on PMF numbers in wild type (WT), TAp63-/-, and Puma-/- female mice. In WTs, cyclophosphamide depleted the ovarian reserve by 96% and cisplatin by 78%. In striking contrast, Puma-/- females in all treatment groups retained all their PMFs, as did TAp63-/- mice treated with cisplatin. Interestingly, cyclophosphamide-treated TAp63-/- females were only partially protected, indicating mechanistic differences in Puma induction pathways. Fertility trials showed that both drugs reduced fertility in WTs. In contrast, all fertility parameters were completely preserved in both TAp63-/- and Puma-/- groups, regardless of treatment; offspring appeared healthy. Together, these data demonstrate that PUMA is the critical trigger of apoptosis in PMFs following these chemotherapies and that its elimination protects the ovarian reserve and preserves fertility. Chapter 3 investigated the underlying mechanisms of PMF depletion following cisplatin or cyclophosphamide treatment. Immunofluorescence for phospho-histone family, member X (gamma-H2AX) showed that oocytes within primordial, transitional, and primary follicles sustain DNA double strand breaks (DSBs), but in secondary and antral follicles, granulosa cells are damaged; apoptosis was detected in the corresponding cell types using TUNEL. Given that most oocytes in reproductively young mice are stored within PMFs, this demonstrates that direct PMF oocyte damage is the primary mechanism of ovarian reserve depletion in this setting. Therefore, pharmacological strategies for ovarian protection during cancer treatment must specifically prevent PMF oocyte apoptosis. Chapter 4 investigated DNA repair pathways employed by PMFs after cisplatin or cyclophosphamide treatment. Immunofluorescence for RAD51, an essential protein in the high-fidelity, homologous recombination (HR) pathway, indicated that PMF oocytes initiate HR within 8 hours of treatment with cisplatin or cyclophosphamide, coinciding with high levels of DSBs as shown by gamma-H2AX. Immunofluorescence for DNA-PKcs, part of the lower fidelity, non-homologous end joining (NHEJ) pathway, showed that NHEJ is seldom utilised in PMF oocytes following these chemotherapies. Collectively, these studies represent a body of work which enhances our mechanistic understanding of chemotherapy-induced ovarian damage, explains how common cancer treatments cause female infertility, and identifies targets for future pharmacological strategies to preserve ovarian function in female cancer patients.
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ItemMaternal ophthalmic ultrasound in the assessment of preeclampsia( 2021)The neurological complications of preeclampsia, including eclampsia and intracerebral haemorrhage, are responsible for most of its maternal morbidity and mortality. Treatments are aimed at preventing these cerebral sequelae, and include antihypertensive agents (to prevent intracranial haemorrhage) and magnesium sulphate (for seizure prophylaxis). It is likely that these agents have a direct effect on the maternal cerebrovasculature, although their precise mechanisms of action remain incompletely understood. Ophthalmic ultrasound makes use of the eye’s direct connection to the brain: the ophthalmic artery, which is similar in size and embryological origin to smaller calibre intracerebral vessels, can undergo Doppler interrogation, while the optic nerve sheath diameter (ONSD) can be measured as a marker of intracranial pressure. This thesis aims to assess the utility of ophthalmic ultrasound in the assessment and prediction of preeclampsia, which hitherto has not been performed in an Australian population. In so doing, sonographic assessment of the optic nerve sheath diameter and Doppler variables of the ophthalmic artery was performed in three discrete cohorts: healthy pregnant women (n = 51), pregnant women with suspected but excluded preeclampsia (n = 50), and women with established preeclampsia receiving treatment with the antihypertensive agent labetalol (n = 20) and the anticonvulsant magnesium sulphate (n = 1). In the cohort of healthy pregnant women, the ocular sonographic variables demonstrated no relationship with maternal mean arterial pressure (MAP), gestational age, or each other. Postpartum analysis identified an apparent increase in cerebrovascular resistance – a novel finding. In the cohort of women with suspected but excluded preeclampsia, ocular sonographic variables were not found to differ between those with transient and gestational hypertension on the day of assessment. In contrast to the first cohort, a positive relationship was identified between maternal MAP and the ophthalmic artery peak ratio, and a negative relationship between MAP and the ophthalmic artery pulsatility index. This novel finding is suggestive of greater cerebrovascular susceptibility to changes in systemic blood pressure in women with hypertensive disorders of pregnancy. These two cohorts were combined to assess the utility of ocular sonographic variables in predicting pregnancy outcome. Univariate analysis identified the ophthalmic artery pulsatility/resistive indices and peak ratio as being significantly different in those who went on to develop a sustained hypertensive disorder, whereas the ONSD performed better in multivariate analysis. This was the first study to examine the utility of the ONSD in predicting preeclampsia. Finally, women with established preeclampsia had lower pulsatility indices and higher peak ratios in the ophthalmic artery, and greater dimensions of the optic nerve sheath, than either healthy pregnant women or those with other hypertensive disorders of pregnancy. In a novel finding, administration of labetalol resulted in a shift in the ONSD and Doppler variables of the ophthalmic artery toward those exhibited by healthy pregnant women, which appeared to be independent of labetalol’s effect on systemic blood pressure. Magnesium sulphate appeared to have a similar effect. These observations may permit more precise and individualised care of women with preeclampsia, and thus a reduction in the neurological burden of this disease.
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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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ItemCharacterisation of endometriotic lesions( 2021)Endometriosis is a common gynaecological disease where “endometrial-like” tissue forms ectopic lesions, most commonly in the pelvis. The disease affects 11% of reproductive-aged women within their lifetime, and presents with a range of symptoms, often including pelvic pain. Surgical and medical treatments aimed at reducing symptoms can have limited effectiveness, side effects and patient-to-patient variability. While some patients respond well to treatment there is no ‘one shoe fits all’ approach to managing endometriosis. Endometriotic lesions are the characteristic feature diagnostic of endometriosis and variable macroscopic and microscopic lesion appearances have been observed. Despite this heterogeneity, features beyond the presence/absence of endometrial-like glands and/or stroma are not factored into diagnosis. Additionally, existing endometriosis classification systems are based on surgical observations and do not predict treatment responsiveness or disease prognosis. This is unlike other pathologies (e.g. gynaecological cancers) where an array of disease characteristics are factored in to diagnosis to guide treatment decisions and predict outcomes. The identification of distinct endometriotic lesion subtypes that relate to clinical outcomes would be invaluable to improving treatment for endometriosis patients. The objective of this thesis was to characterise specific microscopic features of superficial peritoneal endometriotic lesions including histological and immunohistochemical characteristics such as collagen, smooth muscle, leukocytes, steroid receptors, proliferative markers, innervation, and vasculature. Studies aimed to determine if distinct subtypes of lesions exist based on these features. These studies also aimed to determine if endometriotic lesions exhibit patterns in these features based on menstrual cycle phase and if these patterns reflected those of matched eutopic endometrium. Based on the histological features and tissue and cell types analysed, there was considerable heterogeneity in the appearance of superficial peritoneal endometriotic lesions, even within individual patients and single biopsies. For example, the presence and/or morphology of collagen, smooth muscle, and immune cells adjacent to these lesions was diverse and, in some cases, varied based on biopsy location. The heterogeneity in lesion appearances, particularly within an individual biopsy or single patient, presents a challenge to identifying distinct lesion subtypes that could aid improvements to endometriosis classification and patient stratification. Only a subset of endometriotic lesions displayed histological and immunohistochemical features associated with the menstrual cycle phase. Where there were menstrual cycle-related patterns in histological features and steroid receptor expression, these diverged from those of the matched eutopic endometrium. There may be few similarities between endometriotic lesions and the endometrium beyond the observation of “endometrial-like” glands and stroma. Consequently, future endometriosis studies should shift focus and be wary of simply comparing endometriotic lesions with eutopic endometrium when it may be more appropriate to study endometriotic lesions independently. To improve patient treatment outcomes, future studies should investigate the contribution of endometriotic lesions to disease pathophysiology and potential therapeutic targets. The findings of this thesis suggest research should focus on determining if the diversity of lesion appearance relates to variable steroid hormone responsiveness, stages of disease progression, lesion location and other potential aspects of disease pathophysiology.
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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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