Obstetrics and Gynaecology - Theses
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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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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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ItemEarly postnatal cranial ultrasonography linear measures to predict neurodevelopment at 2 years in infants born at <30 weeks’ gestation without major brain injury on sequential imaging( 2020)Background: Infants born very preterm – less than 32 weeks’ gestational age (GA) – are at risk of long-term adverse neurodevelopment related to perinatal brain injury and aberrations in brain growth and maturation. Neuroimaging within the newborn period can aid clinicians to identify high-risk infants for developmental surveillance and early intervention therapy. Cranial ultrasonography (cUS) is the most frequently used neuroimaging modality for preterm infants because it is widely available, portable, and repeatable. Whilst early and sequential cUS can reliably detect most major preterm brain injury, it remains less sensitive than brain magnetic resonance imaging (MRI) for the more prevalent, diffuse white matter injury associated with very preterm birth and adverse neurodevelopment. Declining rates of major preterm brain injury further diminishes the sensitivity of cUS to predict neurodevelopmental outcomes. In the absence of major brain injury, and where brain MRI is not readily accessible, there is a need to improve the prognostic utility of cUS, not least to better understand why some very preterm infants without major brain injury seen on cUS later develop motor and cognitive impairments. Sequential cUS, from the first weeks after birth and up to term-equivalent age (TEA), affords an opportunity to explore the prognostic utility of early postnatal brain growth as a potential marker of neurodevelopment in very preterm infants. Objectives: In my thesis, I aimed to explore the utility of early postnatal cUS linear measures of brain size and brain growth to predict neurodevelopment at 2 years in infants born at less than 30 weeks’ GA and without major brain injury. My first study aimed: (1) to assess the reproducibility of linear measures of brain tissue and fluid spaces made from cUS performed as part of routine clinical care; (2) to evaluate early postnatal brain growth using sequential cUS linear measures made from birth and up to TEA; and (3) to explore perinatal predictors of early postnatal brain growth. My second and third studies aimed to examine the associations of early postnatal cUS linear measures with (4) neurobehaviour at TEA and (5) neurodevelopment at 2 years, respectively. Methods: Participants comprised 144 infants born at less than 30 weeks’ GA, and 201 term-born controls, at a single centre between January 2011 and December 2013. Infants with major brain injury seen on cUS, or congenital or chromosomal abnormalities known to affect neurodevelopment, were excluded. Linear measures of brain tissue and fluid spaces were made from cUS performed as part of routine clinical care from the first weeks after birth and up to TEA. Perinatal data were collected prospectively. Neurobehaviour was assessed at TEA using Prechtl’s Qualitative Assessment of General Movements (GMs) and the Hammersmith Neonatal Neurological Examination (HNNE). Neurodevelopment was assessed at 2 years using the cognitive, language, and motor scales on the Bayley Scales of Infant and Toddler Development, 3rd Edition, and risk of adverse neurodevelopment at 2 years was defined by a composite of either: cerebral palsy, any developmental delay, deafness, or blindness. Assessors were blinded to the clinical course of participants. Results: 429 scans were assessed for 144 infants. Several linear measures showed excellent reproducibility. All measures of brain tissue increased with postnatal age, except for the biparietal diameter (BPD) which decreased within the first postnatal week and increased thereafter. GA greater than or equal to 28 weeks at birth was associated with slower growth of the BPD and ventricular width compared with GA less than 28 weeks. Postnatal corticosteroid administration was associated with slower growth of the corpus callosum length (CCL), transcerebellar diameter (TCD) and vermis height. Sepsis and necrotising enterocolitis were associated with slower growth of the TCD. Larger brain sizes, assessed by linear measures of the BPD at 1-week and 2-months, and CCL at 1-month, were associated with lower risk of a suboptimal HNNE and abnormal GMs at TEA, respectively. Faster positive growth rates of the CCL and TCD between birth and 1-month were related to lower risk of abnormal GMs at TEA. A larger measure of the right anterior horn width (AHW) at 1-month, and faster positive rates of increase of the left and right AHW between 1- and 2-months, were related to lower risk of abnormal GMs and a suboptimal HNNE at TEA, respectively. Larger measures of brain tissue at 1-week, 1-month, and 2-months were related to higher cognitive and language scores, and larger measures of brain tissue at 2-months were related to lower risk of adverse neurodevelopment. Faster growth of the TCD between 1-month and 2-months was related to a higher cognitive score and lower risk of adverse neurodevelopment. Unexpectedly, larger measures of fluid spaces at 1-month and 2-months were related to higher language and motor scores, larger measures of fluid spaces at 1-week and 1-month were related to lower risk of adverse neurodevelopment, and a faster increase of the interhemispheric distance between 1-week and 1-month was related to a higher language score and lower risk of adverse neurodevelopment. Conclusions: Early postnatal brain growth in infants born at less than 30 weeks’ GA can be evaluated using sequential linear measures made from routine cUS, and is associated with perinatal predictors of long-term development. Linear measures of larger brain tissue from the first weeks after birth are related to lower risk of atypical neurobehaviour at TEA and better cognitive and language development at 2 years. My thesis provides evidence to support associations of early postnatal cUS linear measures of brain size and brain growth with neurodevelopment at 2 years in infants born less than 30 weeks’ GA and free of major brain injury. The relationships between larger fluid spaces and better neurodevelopment warrant further exploration.
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ItemDeterminants of uterine receptivity( 2020)‘Uterine receptivity’ refers to the status of the uterus when the endometrium is available to accept the embryo for implantation. Optimal endometrial receptivity leads to normal embryo implantation processes that serve as a foundation for a healthy pregnancy. Over the last three decades, there have been many advances in embryology that have resulted in significant improvements in embryo viability and overall in vitro fertilization (IVF) success rates. It is believed that implantation failure due to reduced endometrial receptivity is one of the major remaining impediments to higher IVF pregnancy rates. In a normal ovulatory cycle, the receptive endometrium develops following sequential exposure to sex steroids – estrogen and progesterone, secreted by the ovaries during follicular development, ovulation and formation of a corpus luteum. Successful implantation will occur when the development of endometrium is in synchrony with the growing embryo. Both asynchronous and pathological endometrium can lead to reduced receptivity and implantation failure. With the rapid development of transcriptomic technologies in the recent years, gene expression studies of endometrium have advanced our understandings of endometrial physiology. Clinical tests based on gene expression profiles of endometrium have also been developed. Unfortunately, there are still significant knowledge gaps in endometrial receptivity research. Expression quantitative trait loci (eQTL) analysis seeks to identify genetic variants – single nucleotide polymorphisms (SNPs) that affect the expression of genes. Identification of eQTLs has proven to be a powerful tool in the study and understanding of various diseases. We hypothesized that an eQTL approach to identify DNA variants (SNPs) which influence the expression level of genes in the peri-implantation human endometrium would have significantly more power to identify genes and gene pathways that influence uterine receptivity than simple endometrial gene (or protein) expression profiling experiments that have been undertaken previously. The overall aim of this thesis is to improve the current understanding of uterine receptivity through transcriptomic and genomic (eQTL) approaches. In the second half of the thesis, we also evaluate current practices for improving uterine receptivity. It has been well established that endometrial gene expression is influenced by stage of the menstrual cycle. Adequate normalization for cycle stage is crucial in differential gene expression research of endometrium. However, no such normalization technique exists. In order to address this issue, we have developed a molecular ‘day of cycle’ model through precise histology dating and endometrial gene expression data. This model serves as an accurate and reliable way to date endometrial samples for subsequent studies. The main body of this thesis involves transcriptomic and genomic studies of endometrium from women suffering from recurrent implantation failure (RIF), in search for markers of uterine receptivity. Differential gene expression studies of endometrial samples after normalization for cycle stage and false discovery rate correction did not show any significant difference between control and study groups. In order to gain enough power for identification of eQTLs, we have combined our endometrial samples with a larger cohort for endometrial eQTL analysis. This analysis identified novel endometrial cis- and trans-eQTLs. However, eQTL for RIF genes could not be detected due to absence of any significant differential gene expression between RIF and control groups. In the recent years, various methods and strategies have been proposed to improve uterine receptivity and embryo implantation. These include freeze-all cycles to reduce the impact of controlled ovarian hyperstimulation on endometrium and to improve embryo-endometrial synchrony. We performed a cross sectional study of more than 10,000 cycles comparing clinical outcomes between fresh and frozen embryo transfer cycles of cleavage stage embryos. Our results suggested that any potential gains due to improved endometrial receptivity and embryo-endometrial synchrony following FET are lost, presumably due to freeze-thaw process related embryo damage This thesis also investigates whether any genes are differentially expressed in endometrium following endometrial scratch, which is a common practice by many IVF groups for improvement of uterine receptivity. The results from this study showed no statistically significant gene expression differences before and after endometrial ‘scratch’. However, endometrial gene expression profiles before and after endometrial ‘scratch’ were very similar in most of the subjects, suggesting that endometrial gene expression remains consistent from cycle to cycle. In conclusion, this thesis has explored the molecular mechanisms underlying uterine receptivity. We have developed a molecular ‘day of cycle’ prediction model as a useful tool for determining ‘biological’ day of cycle for endometrium. The model was subsequently used to normalize endometrial samples for cycle stage in differential gene expression studies. Differential gene expression studies did not identify transcriptomic markers for RIF. The failure to identify RIF-related genes is not surprising given the multiple causes that could contribute to RIF, coupled with the possibility that some subjects in the RIF group could have undetected co-morbidities. This work has advanced our knowledge on uterine receptivity and provided several new avenues for ongoing research. There is still a need for better clinical tests for uterine receptivity. Hopefully the work in this thesis will provide a foundation for that to occur.