Obstetrics and Gynaecology - Theses
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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 decidual mesenchymal stem/stromal cell ageing in labour( 2019)Ageing and parturition share many common pathways, but whether these two processes are associated, or even dependent on each other, is poorly understood. This study focuses on the decidua, where there is evidence that decidual cells undergo ageing as parturition approaches term, and these ageing-related changes of the decidua may trigger labour. Mesenchymal stem/stromal cells (MSCs) are a type of stem cell that reside in the decidua. Ageing of decidual MSCs (DMSCs) may contribute to the functional and molecular changes in decidual tissue required for spontaneous onset of labour (SOL) at term. The objective of this study was to determine whether DMSCs from patients experiencing SOL show evidence of molecular and functional loss and/or changes compared with DMSCs from patients, not in labour (NIL), undergoing Caesarean section (C-section) delivery. Placentae were collected from full-term (39-40 week gestation), SOL (n = 8) and NIL (n = 7) patients with non-complicated pregnancies. DMSCs were isolated from decidua basalis, which remains attached to the placentae following delivery. DMSCs were characterised to ensure their decidua-origin and stem cell properties. Important cell functions, including cell proliferation, cell migration, cell death, lipid peroxidation, aldehyde dehydrogenase (ALDH) expression, and pro-inflammatory cytokine secretion were then compared between SOL- and NIL-DMSCs. SOL-DMSCs demonstrated a significant increase in necrosis, lipid peroxidation, migration, and IL-6 production compared with NIL-DMSCs (p < 0.05). SOL-DMSCs also showed an increase in apoptosis as well as a decrease in proliferation, ALDH expression and IL-8 production compared with NIL-DMSCs, however, the differences were not statistically significant (p > 0.05). These findings suggested that SOL-DMSCs underwent advanced ageing at term. The lipid contents of DMSCs from both patient groups were extracted using Folch’s method, separated using High Performance Liquid Chromatography (HPLC) and identified using tandem mass spectrometry (MS/MS). The quantity of each lipid species from DMSCs of both patient groups was measured and compared. SOL- and NIL-DMSCs contained the same lipid species, however the expression level of particular lipid species significantly differed. DMSCs may undergo changes in lipid metabolism during labour at term, possibly to provide the energy source for the labour process. In conclusion, this study provides novel evidence that DMSCs undergo ageing-related functional and molecular changes associated with labour and these changes may play a significant role in promoting spontaneous labour at term.
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ItemMidpregnancy prediction of preeclampsia( 2018)AIM: Placental biomarkers soluble Fms-like tyrosine kinase-1 (sFlt1) and placental growth factor (PlGF), when tested at midpregnancy, may predict preeclampsia. This thesis investigates testing PlGF and the sFlt1/PlGF ratio at midpregnancy, both in isolation and as part of a multivariable algorithm. The performance of three immunoassay platforms for testing these biomarkers will be compared. METHODS This prospective study included singleton pregnancies 19-22 weeks gestation. Maternal history, mean arterial pressure (MAP), uterine artery pulsatility index (UAPI) and maternal blood were collected at recruitment. Preeclampsia was the outcome measured. Inter-assay comparison was performed using Intraclass Correlation Coefficient and Bland-Altman plots. Screening performances for biomarker raw data and MoM values were evaluated using receiver operating characteristic (ROC) curves, with clinical characteristics calculated using selected cut-off values. Maternal factors, MAP, UAPI, PlGF MoM and sFlt1 MoM values for prediction of preterm preeclampsia were entered into the Fetal Medicine Foundation (FMF) algorithm and screening performances evaluated using selected cut-off values from ROC curves. RESULTS: 512 patients were included. Results for PlGF and the sFlt1/PlGF ratio from the three platforms were well correlated, with R-values 0.896-0.949 (p<0.0001). Consistent differences between raw data values obtained between the three platforms was noted and confirmed on Bland-Altman analysis. MoM values proved equivalent between platforms. PlGF levels were lower at midpregnancy in patients who developed preterm and early onset preeclampsia (p<0.05), but not term preeclampsia. PlGF raw data values using the early onset preeclampsia cut-off performed best, with AUC 0.92-0.93, sensitivity 100%, specificity 77.8-80.75%, PPV 2.59-2.97% and NPV 100%. Patients who developed early onset preeclampsia had significantly higher sFlt1/PlGF ratio raw data and MoM values (p<0.05), and patients who developed preterm preeclampsia had significantly higher sFlt1/PlGF ratio MoM values (p<0.05), with no significant difference in patients who developed term preeclampsia. The sFlt1/PlGF ratio using raw data values and the cut-off for early onset preeclampsia performed better than PlGF raw data values, with AUC 0.97, sensitivity 100%, specificity 95.87%, PPV 12.5% and NPV 100%. Using the cut-off for preterm preeclampsia, PlGF MoM and sFlt1/PlGF MoM performed similarly, with AUC 0.71-0.74, sensitivity 62.5%, specificity 82.34-89.29%, PPV 5.32-8.47% and NPV 99.28-99.34%. The multivariable FMF algorithm, incorporating maternal factors, MAP, UAPI and PlGF MoM performed superiorly to testing with biomarkers alone, with AUC 0.983-0.984, sensitivity 100%, specificity 94.25-95.04%, PPV 21.62-24.24% and NPV 100%. sFlt1 MoM did not further improve predictive performance. CONCLUSION: While MoM values appear equivalent between platforms, specific reference ranges should be used for raw data values. sFlt1/PlGF ratio raw data values using the cut-off for early onset preeclampsia performed best, with fewest false positives. PlGF MoM and sFlt1/PlGF MoM using the cut-off for preterm preeclampsia performed similarly. The multivariable FMF algorithm gives superior performance over screening using biomarkers alone but requires more resources to undertake. In conclusion, PlGF, the sFlt1/PlGF ratio tested in isolation and PlGF MoM incorporated into a multivariable algorithm are all effective and feasible options for prediction of preeclampsia at midpregnancy. Implementation within different healthcare services would depend on resources available and require cost-benefit analysis prior to implementation.
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ItemNovel technology for the measurement of newborn and infant heart rate( 2017)Background: Monitoring heart rate in newborns and infants is crucially important in guiding resuscitation and medical care. Established methods for heart rate assessment of these children have inherent drawbacks. In recent years, novel methods for assessing neonatal and infant heart rate have been developed, with varying levels of evaluation conducted. Digital stethoscopes may provide a better means of heart rate assessment for newborns and infants. Aim: The aim of this thesis was to comprehensively review existing established and novel technologies used to monitor newborn and infant heart rate, and compare new digital stethoscope technology with the gold standard, electrocardiogram (ECG). Methods: This thesis (a) outlines the definition and importance of heart rate in medicine, presented in the context of a review of cardiac anatomy and physiology relevant to understanding this vital sign and aspects of its measurement in neonates and infants; (b) presents a narrative review of established methods for monitoring heart rate; (c) expands the scope of this review from established to emerging methods for monitoring heart rate with a systematic literature review of novel methods for newborn and infant heart rate assessment; (d) describes original research using a prototype digital stethoscope attached to a smart device containing software for detecting and displaying heart rate in real-time that was conducted on infants in the neonatal intensive and special care setting, as well in the delivery room setting using an improved version of the device and software. Results: A review of the literature analysing methods of assessing neonatal and infant heart rate found strengths as well as significant weaknesses in the various methods in clinical use or in development. In the neonatal unit, a prototype digital stethoscope and smartphone device for assessing heart rate had a mean difference (±2 standard deviations) of 7.4 (48.5) beats per minute (bpm) when compared to the gold standard of electrocardiography. The mean (interquartile range) time to first digital stethoscope heart rate display was 4.8 (1 to 7) seconds, and the device failed in 12.3% of use attempts. Repeating the comparison in the delivery room setting using an updated algorithm and new hardware, Bland-Altman analysis revealed a smaller mean difference (±2 standard deviations) between the digital stethoscope and electrocardiography of 0.2 (-18 to +18) bpm including crying periods (Figure 23), and 1.0 (-11 to +12) bpm excluding crying periods. The improved digital stethoscope took a median (interquartile range) of 7 (5 to 11.5) seconds after application to display a heart rate. It failed to detect heart rate in 37% of cases, all of which were in crying infants. Conclusion: A digital stethoscope and smart device with software can rapidly detect neonatal and infant heart rate. In the delivery room, device failure primarily occurred during infant crying, with improved accuracy during non-crying periods.
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ItemMy kite will fly: development of a program for working with parents with life threatening illness and their dependent children (3-9 years)( 2015)Recent Australian estimates suggest that approximately 30,000 parents are diagnosed with cancer each year (Thursfield et al., 2013). Patient families evidence acute distress and vulnerability following a cancer diagnosis, and difficulties in communication around the realities of life threatening illness are commonplace. Prior research has identified the need for a brief resource tool for parents and children where there is accelerated malignancy (Turner et al., 2007, Turner et al., 2009, Turner et al., 2008). This study reports on the progress of the My Kite Will Fly program (MKWF), devised to offer multi-disciplinary staff a toolbox for families living in households with young children enduring parental cancer. The present study aimed to (i) replicate previously established findings that families enduring parental cancer suffer significant distress, vulnerability and disruption to family roles and routine, and (ii) determine whether the design template built for the MKWF appropriately addressed the needs of these families. Participants were a purposive sample of n=36 children (24 girls, 12 boys) drawn from 19 families who enrolled in the program either shortly after diagnosis, during treatments, or at palliative care. Data collection was divided into three distinct phases with assessment at diagnosis, treatment, and palliative care, though few families participated in all phases of data collection due to the variable demands of disease progression. Families completed a series of semi-structured interviews, MKWF workbook tasks, and art activities, at each of the three key phases. Each family member actively contributed to the compilation of commemorative hard copy and e-books. Program evaluation data was gathered from a subset of participants who evaluated the commemorative hard copy and e-books, and by exit interview. Quantitative and qualitative data were triangulated and analysed in compliance with descriptive case study methods. Six family case studies (n=13 children) were used alongside cross-case analyses of data gathered from all 19 families comprising 36 children in the study. Results from cross-case analysis highlighted five key themes encapsulating patient family concerns: (i) A new diagnosis – what happens next? Vulnerability and the experience of uncertain personal health; (ii) Consequences for others, how younger children understand illness developmentally, and reinforcing family wellbeing when children see too much; (iii) The valuing of roles and routine, planning ahead, and making the My Kite Will Fly contents a legacy; (iv) How to help when mum gets sick again and the hospital just can’t make it better; and (v) End of life decision and saying goodbye – how young is too young? Although program evaluation was preliminary with a small study population, findings suggest the MKWF program is a helpful resource for families living with advanced parental illness. The present study makes a significant contribution to Psycho-Oncology research by illustrating the concerns of parents and children subsequent to parental cancer diagnosis. It integrates theoretical knowledge and empirical findings in developing an innovative and efficacious therapeutic resource for parents and children facing accelerated parental malignancy. Despite the challenges associated with family participation and data collection, the present study provides a platform for future evaluative research of the program in patient family populations.