Obstetrics and Gynaecology - Theses
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Determinants of uterine receptivity
‘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.
Cognitive and brain aging and the influence of amyloid-beta
Models of cognitive and brain aging suggest that some cognitive decline and cortical volume loss is normal in aging beyond age 60; however, these models have failed to account for the presence of Alzheimer’s disease (AD) neuropathological markers such as amyloid-B (AB). The inadvertent inclusion of older adults who later progress from being cognitively normal (CN) to mild cognitive impairment (MCI) or dementia, or who have elevated levels of cerebral AB (AB+) may lead to inflated estimates of cognitive decline or cortical volume loss that may not be entirely due to the process of aging. The overarching aim of this thesis was to characterize the cognitive and brain morphological changes associated with normal aging, defined as the cognitive and neurobiological changes that occur with the passage of time independent of neuropathological processes. Changes in cognition and brain morphology were examined in up to 599 CN older adults from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Flagship Study of Ageing who underwent repeated clinical, neuropsychological and neuroimaging (MRI and AB PET) assessments over up to 8 years. The presence of AB+ in otherwise CN older adults was associated with greater risk of future clinical disease progression to MCI or dementia, and faster rates of cognitive decline and cortical volume loss compared to those who were AB-. These results remained consistent when examined with respect to the SuperAging construct, and suggest that trajectories of cognitive and brain morphological changes in AB+ are not reflective of normal aging processes. This thesis challenges widely-accepted models of cognitive and brain aging by showing that aging is characterized by preserved cognitive function and minimal cortical volume loss, and concludes that cognitive decline is not inevitable in aging.
The role of decidual mesenchymal stem/stromal cell ageing in labour
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.
Midpregnancy prediction of preeclampsia
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.
The response of normal tissues to synchrotron microbeam radiotherapy
This thesis presents multidisciplinary and collaborative research aimed at translating microbeam radiation therapy (MRT) to clinical trials. MRT is a novel pre-clinical synchrotron-based radiation therapy modality that is a potential substitute for conventional radiotherapy (CRT) modalities due to its improved therapeutic index facilitated by remarkable tolerance of normal tissues with successful tumour ablation. One of the main limitations of CRT is that while tumour ablation is dependent on radiation dose, the maximum tumour dose is limited by the radiation tolerance of the surrounding healthy normal tissues. A key attribute of MRT is the normal tissue sparing effect that is thought to occur due to the physical geometry of the MRT beam. MRT involves synchrotron-generated X-rays that are spatially fractionated to generate an array of wafer-thin parallel microbeams, known as peaks, separated by hundreds of microns, known as valleys. The valleys typically receive 2-4% of the peak dose. Presently there is no conclusive evidence that can fully explain the efficacy of MRT compared to synchrotron BB radiotherapy (a radiation modality that resembles CRT). Various hypotheses have been proposed to explain MRT effects including: radiation- induced bystander/abscopal effects (RIBE/RIAE), microvascular effects, increased tumour cell migration, increased cell proliferation and stem cell survival in normal tissues, and changes in gene regulation and immune response in tumours and normal tissues. My contribution in our multidisciplinary group is to investigate the biological mechanisms associated with synchrotron MRT. To establish if radiation parameters influence the RIAE, a range of variables including beam modality, dose and size of irradiation field were tested in vivo at 1 and 4 days post irradiation following local synchrotron X-ray BB and MRT. DNA damage, apoptosis and local and systemic immune responses were monitored in directly irradiated tissue and out-of-field tissue. We demonstrated that irradiation dose and size of irradiation field do not substantially influence RIAE in out-of-field tissues and a short pulse of MRT and BB irradiation was sufficient to induce persistent systemic and genotoxic effects in mice. While immune responses have been proposed as a possible mechanism that facilitates RIAE, it has never been demonstrated before. To identify which immune response components are involved in RIAE propagation, various RIAE endpoints (including DNA damage and apoptosis) were tested in a range of out-of-field tissues of immune-deficient mice treated with MRT. We demonstrated that the abscopal effect relies on a functional immune response, particularly the involvement of macrophages, monocyte chemotactic protein 2 (CCL2) and TGFβ in order for RIAE to be transmitted to out-of-field tissues. Due to the complex dose profile of MRT, MRT dosimetry is problematic using current physical dosimeters. An alternative is to use γH2AX (a biomarker of DNA damage) as a biodosimeter to measure the biologically equivalent valley dose following MRT. A range of BB doses were used to generate a BB standard curve, which was used to extrapolate the biologically equivalent valley dose of a range of MRT peak doses. The results revealed that γH2AX may not be an appropriate biodosimeter to predict the biological valley dose of MRT due to the non-linear dose response observed in MRT- irradiated mouse skin and human fibroblast cells. The response of gastrointestinal tract (GIT) to MRT was also investigated since no normal toxicity data for GIT response to MRT exists. We investigated alterations in gene expression of 6 radiation responsive genes at 4 and 48 hrs post-irradiation in male and female mice using qRT-PCR following partial body irradiation of the abdomen using MRT and BB. The results showed that MRT and BB are able to induce significant modulations in gene expression in directly irradiated GIT tissues. MRT may induce more oxidative stress and reduce macrophage infiltration in GIT compared to BB, due to the significant downregulation of inflammation and DNA repair genes. Both modalities induced a protective response in the GIT by upregulating MDM2 and IL-10 at 4 hrs post-irradiation. MDM2 was most abundant in the colon compared to the upper GIT tissues. Unlike MRT, BB induced differential inflammation response via NFκβ2 expression in female mice compared to male mice, indicating that gender-specific mechanisms may play a role in response to BB irradiation. These studies provide further information on the biological mechanisms that contribute to the normal tissue sparing effects of MRT and could assist in optimisation of MRT treatment protocols. Further knowledge in mitigating or reducing normal tissue toxicities and increasing tumour control would greatly benefit cancer patients that require radiation therapy.
The role of decidual mesenchymal stem/stromal cells and their secreted extracellular vesicles in ageing of the human placenta
Ageing is an inevitable process associated with age-related diseases. Unhealthy ageing is a major risk factor for many chronic diseases including cardiovascular and neurodegenerative diseases. The human placenta, despite being a short-lived and transient organ, does not escape the effects of ageing and aberrant placental ageing is a feature of important placental pathologies including preeclampsia (PE) and fetal growth restriction (FGR). Recent evidence shows placental tissues undergo significant molecular age-related changes in a short period, between early-term (ET) (37 0/7– 38 6/7 weeks gestation) to late/post-term (L/PT) (41 0/7 weeks gestation and beyond), after the due date of the pregnancy has been reached. Moreover, there is a dramatic increase in the risk of unexplained antepartum stillbirths in pregnancies that progress into the L/PT period, which may be linked to increased ageing of placenta. The role of ageing and the underlying mechanisms that regulate aberrant placental ageing during this period remain unclear. The maternal-fetal interface develops between the chorionic placenta to the maternal uterine wall and is vital for the maintenance of a healthy pregnancy. The decidua is the maternal tissue between the placenta and the muscular uterine wall. Age-related tissue and cellular changes are evident throughout the decidua and placenta. The decidua basalis tissue, which is the focus of this study, and underlying myometrium are continually exposed to damaging reactive oxygen species (ROS) and inflammatory molecules secreted primarily by the placenta. The decidua basalis must withstand harsh physical conditions associated with changes in blood flow into the intervillous space during uncomplicated pregnancy. However, the extent of decidual ageing in uncomplicated pregnancies that reach term, and how decidual ageing affects the placenta, are not well understood. Stem cell exhaustion and dysfunction are major contributors to the decline in tissue and organ functionality associated with ageing. The placenta and decidua are rich sources of stem cells, particularly mesenchymal stem/stromal cells (MSCs) which are highly active and involved in placental development and growth. However, the role of stem cells and their association with ageing of the chorionic placenta and maternal decidua basalis, especially in the short period between ET and L/PT, has not been investigated. The work of this thesis focuses on maternal decidua basalis derived MSCs (DMSCs). The first aim of this study was to compare ageing/senescence functions of ET DMSCs (ET- DMSCs) from (37-39 gestational weeks) with L/PT DMSCs (L/PT-DMSCs) (41-42 gestational weeks). The second aim was to screen for differentially expressed known and novel small RNAs between ET-DMSCs and L/PT-DMSCs. The third aim was to analyse and characterise ET-DMSCs and L/PT-DMSCs secreted extracellular vesicles (EVs). EVs are nanosized particles secreted by MSCs, and they potentially regulate health and the ageing processes. The final aim of this work was to treat ET-DMSCs, L/PT-DMSCs and PE-DMSCs with a low dose of aspirin, which is an anti-aging and anti-inflammatory drug. ET, L/PT, and PE-affected placentae were collected with informed consent. DMSCs were successfully isolated from decidua basalis of ET, L/PT, and PE placentae using an enzymatic digestion method and then characterised for their stem/stromal cell properties. The phenotypic characterization of DMSCs met the minimum criteria of MSCs. L/PT-DMSCs, when compared to ET-DMSCs, showed significantly lower cell proliferation and a significant higher level of cell apoptosis. Using a quantitative Aldefluor assay, ET-DMSCs showed a significantly higher resistance to oxidative stress compared with L/PT-DMSCs. There was a significant decrease in antioxidant capacity of L/PT-DMSCs compared with ET-DMSCs. Western blot analysis revealed increased expression of the stress-mediated p38MAPK protein in L/PT- DMSCs. These data provide the first evidence of advanced ageing and loss of important stem cell functions in L/PT-DMSCs. Sequencing of small RNAs and validation by qRT-PCR demonstrated miR-516-5p, which is a member of the chromosome 19 miRNA cluster (C19MC), was present at significantly lower levels in L/PT-DMSCs. This miRNA was also contained in EVs isolated from ET- and L/PT-DMSCs and the difference in levels of this miRNA in whole ET- and L/PT-DMSCs was also maintained in their respective EVs. This demonstrated that EV miRNAs are origin-dependent and their differences in levels reflected those in the ET- and L/PT-DMSCs that they were secreted from. EVs from both groups were further analysed using a novel method resonance enhanced atomic force microscopy and infrared spectroscopy (AFM-IR) which revealed a distinct fingerprint of biomolecules between these two groups. There were substantial differences in peak intensity of EVs contents between ET- and L/PT-DMSCs, particularly those associated with lipid and protein content. The combination of conventional assays and AFM-IR provide the first evidence of advanced age-related, and other changes, in EVs from L/PT decidua MSCs. Finally, low dose aspirin treatment improved PE-DMSCs functionality by a novel mechanism via increasing cell adhesion. Aspirin treatment also increased antioxidant capacity and decreased inflammatory markers in PE-DMSCs. Aspirin had no similar effect on ET- and L/PT-DMSCs.
Improving the detection of risk factors for stillbirth
Stillbirth is a global tragedy, claiming over 2.6 million lives annually. In Australia, seven in 1000 babies are stillborn - a figure that has remained static over the last three decades. The majority of stillbirths after 24 weeks occur late in pregnancy, after 34 weeks’ gestation, with the risk increasing exponentially at term. This is a time where, if delivery were expedited, a healthy newborn is almost invariable. Fetal growth restriction due to uteroplacental insufficiency has consistently been identified as a major risk factor for stillbirth. Small-for-gestational-age (<10th centile) fetuses are known to have a three-four fold increased stillbirth risk, yet current antenatal care detects only a minority of these fetuses. Improved antenatal detection of fetuses destined to be born small is recognised internationally as a leading priority to reduce stillbirth risk. Where the fetus is suspected to be small, stillbirth risk is halved, through a combination of increased surveillance and timely delivery. Fetuses may also be growth restricted yet not small-for-gestational-age. While being small is a leading risk factor for stillbirth, the relationship between birthweight centile and perinatal death is continuous. Fetuses between the 75th and 97th centiles are at the lowest risk for stillbirth, with a stepwise increase in risk at every centile below the 50th. This body of work investigates novel blood-based biomarkers, and applications of existing ultrasound techniques, to better identify the growth restricted fetus. The first aim of our study was to identify novel biomarkers of term fetal growth restriction. To do so, we collected over 2000 blood samples from pregnant women at 28, and at 36 weeks’ gestation. We systematically screened the 36 week blood samples to identify potential biomarkers that could predict infants destined to be born small-for-gestational-age using a nested case-control approach. We focussed on proteins originating from genes highly expressed in the placenta relative to other tissues. We found four proteins that were significantly altered in the circulation of women destined to birth a small-for-gestational-age infant. In particular, we identified serine peptidase inhibitor Kunitz type 1 (SPINT1) as a novel biomarker of uteroplacental function. SPINT1 demonstrated better predictive performance (measured by area under the receiver operator characteristic curve) than placental growth factor, the current best biomarker of late pregnancy fetal growth restriction. Importantly, the predictive accuracy of low SPINT1 was unaltered when measured in the entire 2000 samples. We also found SPINT1 levels to be lower in the maternal blood at 28 weeks’ gestation among pregnancies destined to deliver a small-for-gestational-age infant. SPINT1 levels showed a biological gradient across all birthweight centiles, mirroring the observed continuum of perinatal risk. SPINT1 showed significant correlations with birthweight centile, neonatal lean mass, placental weight, and ultrasound measurement of uterine perfusion. Together these data suggest that SPINT1 has potential as a novel biomarker of uteroplacental insufficiency and fetal growth restriction. Our findings suggest that SPINT1 shows stronger correlations with indicators of uteroplacental function than any other biomarker reported to date. Our second aim was to determine whether we could add to existing ultrasound parameters to improve detection of the growth restricted fetus in late pregnancy. We investigated Doppler ultrasound measurement of maternal, placental and fetal vessels which demonstrate altered resistance in the face of uteroplacental insufficiency. In early-onset fetal growth restriction (<32-34 weeks), ultrasound Doppler parameters tend to deteriorate according to a well-established pattern, beginning with increasing umbilical artery resistance. This is followed by decreasing cerebral vascular resistance and finally abnormal waveforms of precordial veins such as the ductus venosus. Umbilical artery Doppler measurement has been found to reduce the risk of perinatal death in high risk pregnancies. Furthermore, decisions regarding timing of delivery in early fetal growth restriction are well informed by Doppler parameters. In late pregnancy fetal growth restriction (>32-34 weeks) optimal ultrasound assessment of uteroplacental function has not been determined. Routine Doppler ultrasound in low-risk populations has not been found to confer benefit. Moreover, most adverse outcomes in late pregnancy occur in the presence of a normal umbilical artery pulsatility index. In this study, we interrogated all fetoplacental parameters individually, and in combination, for their ability to identify fetal growth restriction in late pregnancy. We recorded the resistance of the maternal uterine arteries, the umbilical artery, and the fetal middle cerebral artery, aortic isthmus, ductus venosus, and renal arteries. We calculated the well-described cerebroplacental ratio (middle cerebral artery pulsatility index divided by that of the umbilical artery). We also examined the reproducibility of the fetal left myocardial performance index. We first screened each of the vessels’ Doppler measurements for their ability to predict a small-for-gestational-age infant. Mean uterine artery pulsatility index and the cerebroplacental ratio demonstrated the highest areas under receiver operator characteristic curves. This led us to describe for the first time, a novel Doppler combination, the cerebral-placental-uterine ratio (CPUR). This new combination, the cerebroplacental ratio divided by the uterine artery pulsatility index, proved to be advantageous. The CPUR predicted birthweights <10th centile, <5th centile or <3rd centile with greater sensitivity than either of its constituent parameters. It also significantly correlated with all other antenatal and neonatal indicators of uteroplacental function measured – third trimester fetal growth velocity, neonatal body fat percentage and ponderal index. Importantly, CPUR also demonstrated a biological gradient mirroring the continuum of adverse perinatal outcome associated with uteroplacental insufficiency. Together these data suggest the CPUR is a superior ultrasound measure of uteroplacental function, with higher sensitivity for fetal growth restriction in late pregnancy than previously described parameters. Our third aim was to determine whether reduced third trimester growth velocity is a measurable indicator of uteroplacental insufficiency among appropriate-for-gestational-age fetuses. Although being small-for-gestational-age is a major risk factor for stillbirth, 50% of stillbirths (and 70% of term stillbirths), occur among ≥10th centile fetuses. Low antenatal growth velocity has previously been found to identify fetuses at high risk of adverse outcome among the small-for-gestational-age. The significance of growth velocity among fetuses born appropriate-for-gestational-age was previously unknown. We measured the change in estimated fetal weight centile, and the change in abdominal circumference centile, over eight weeks in fetuses born appropriate-for-gestational-age. We correlated the change in third trimester fetal growth centiles with antenatal, intrapartum and postnatal indicators of uteroplacental insufficiency. Fetuses that exhibited a fall in estimated fetal weight or abdominal circumference of >30-35 centiles were at increased risk of all indicators of uteroplacental insufficiency. These were: (i) adaptive brain sparing at 36 weeks, as evidenced by an abnormal cerebroplacental ratio; (ii) neonatal acidosis on cord blood samples from fetuses who underwent the hypoxic challenge of labour; and (iii) low neonatal body fat percentage. We suggest that reduced antenatal fetal growth velocity is associated with uteroplacental insufficiency regardless of fetal size. This may allow clinicians to detect a previously unrecognised cohort potentially at increased risk of stillbirth. In conclusion, this thesis has identified novel blood-based biomarkers and obstetric ultrasound parameters of uteroplacental insufficiency and fetal growth restriction. These are: (i) protein biomarker SPINT1; (ii) the CPUR Doppler combination; and (iii) reduced third trimester fetal growth velocity in appropriate-for-gestational-age fetuses. These measurable indicators each have the potential to be validated and clinically translated for more effective identification of fetuses at risk of late pregnancy stillbirth.
Synchrotron radiation therapy for the treatment of cancer
Despite advances in radiation oncology there remain numerous clinical scenarios where radiation therapy, in conjunction with other cancer therapies, is unable to significantly improve disease prognosis. Advanced lung cancer, pancreatic cancer, and aggressive paediatric brain tumours such as Diffuse Intrinsic Pontine Glioma are examples of incurable diseases with an extremely poor prognosis. Synchrotron-based radiation therapy modalities challenge classical radiobiology paradigms and could address these unmet clinical needs. The two synchrotron radiation therapy modalities investigated in this thesis are microbeam radiation therapy (MRT) and high dose-rate synchrotron broad-beam radiation therapy (SBBR). The brilliance and minimal divergence of synchrotron-generated radiation gives MRT and SBBR physical properties that are distinctly different to conventional radiation therapy (CRT). Pre-clinical animal studies demonstrate the potential of these unique physical characteristics to both control tumours and reduce radiation-induced damage to healthy tissue. The purpose of this thesis was to present radiobiological data that would inform future veterinary and clinical trials of MRT and/or SBBR. Specific objectives were to: 1) produce systematic toxicity data for organs of the head, thorax and abdomen, 2) characterise dose-equivalence between MRT, SBBR and CRT based on both in vitro and in vivo techniques, 3) describe the differential effects of MRT and broad-beam radiation therapy on healthy tissue at a molecular level, and, 4) identify optimal clinical scenarios were MRT could be applied, considering the limitations imposed by normal tissue toxicity. Based on total and partial body dose-escalation studies in a murine model, MRT peak doses of approximately 120 Gy and 260 Gy were equivalent to approximately 7 Gy and 12.5 Gy, respectively. The MRT valley dose was a better predictor of normal tissue toxicity than the MRT peak dose, and for SBBR, a normal tissue sparing effect (ie. a ‘FLASH’ effect) could not be detected at a dose-rate of 35-40 Gy/s. Based on a treatment planning study using clinical datasets, small recurrent glioblastomas and head and neck tumours demonstrated the most favourable MRT dosimetry. This thesis includes the first in vivo dose-equivalence data for synchrotron radiation therapy compared to conventional radiation therapy and the first MRT toxicity data for total body, abdominal and thoracic irradiation. These data are essential for designing safe treatment regimens for future veterinary trials and ultimately, the first human clinical trial of MRT and/or SBBR.
Targeting Epidermal Growth Factor to treat pregnancy complications
Epidermal growth factor receptor (EGFR) signaling is one of the most avidly studied signaling networks in mammalian biology, with critical roles in cellular growth and survival. The EGFR is also vital for normal placental formation and development. This body of work examines the role and potential of targeting the EGFR cascade in the pregnancy complications preeclampsia and ectopic pregnancy. In the first arm of this PhD, we investigated EGFR signaling in preeclampsia, a major pregnancy complication associated with shallow placental invasion and placental insufficiency. Anti-angiogenic factors are then released from the preeclamptic placenta into the maternal circulation causing endothelial dysfunction (a hallmark of the disease), hypertension and organ injury. Firstly, we examined whether administering EGF peptide (the natural EGFR ligand) to primary endothelial cells affects endothelial dysfunction in-vitro. From this, we found EGF peptide to significantly reduce a number of markers of endothelial dysfunction in-vitro, potentially suggesting this natural ligand might offer a therapeutic approach to treat the endothelial dysfunction characteristic of preeclampsia. Over the last two decades the understanding of this disease has increased greatly with the discovery of the anti-angiogenic molecules soluble Fms-like tyrosine kinase-1 (sFLT-1) and soluble endoglin (sEng). Theses factors are released at excessive levels from the preeclamptic placenta and cause the widespread maternal endothelial dysfunction that gives rise to the multi-organ injury that occurs in preeclampsia. However, upstream mechanisms regulating the release of these molecules, while avidly sought, are still poorly described. In this thesis, we describe a series of studies that demonstrate EGFR is overactive in preeclampsia, and is a positive upstream regulator of sFLT-1 and sEng. We first characterized EGFR signaling in a large cohort of severe preterm preeclamptic placentas, compared to gestation matched normotensive controls. We found not only EGFR signaling to be significantly increased in preeclamptic placenta, but also confirm that there is increased signaling of the key downstream EGFR adaptor molecules: ERK1/2, Akt and STAT-3. We then proceeded to functional experiments in primary trophoblast (placental) cells to investigate whether manipulating EGFR (and its downstream molecules) affected the release of sFLT1 and sEng. Indeed, we found activating EGFR signaling in placenta increased sFLT-1 secretion and conversely, inhibiting multiple points of the EGFR signaling cascade using either small molecule inhibitors or siRNAs significantly and dose-dependently reduced primary trophoblast sFLT-1 and sEng secretion. We progressed with an in-vivo experiment and confirmed that administration of gefitinib, a small molecule EGFR inhibitor, to pregnant mice significantly reduced circulating levels of sFLT-1. We previously identified sulfasalazine, esomeprazole and statins decrease sFLT-1 secretion, but were yet to uncover the molecular mechanism by which these drugs were affecting sFLT-1 secretion. We therefore investigated EGFR signaling in primary trophoblasts after treating with sulfasalazine, esomeprazole and statins, and found these drugs that reduce sFLT-1 secretion also significantly down regulate EGFR and downstream adaptor molecule expression. Together, this body of work suggests EGFR signaling positively regulates sFLT-1 secretion. The clinical implication is that blocking placental EGFR signaling may be a novel therapeutic approach to treat preeclampsia. The second arm of this thesis was to investigate therapeutics for ectopic pregnancy, and is relevant to the theme of this thesis given EGFR inhibitors have become a leading therapeutic candidate to treat this condition. Ectopic pregnancies arise from implantation of an embryo outside of the uterus, most commonly in the Fallopian tubes. They represent a medical emergency as they can rupture, causing fatal internal bleeding. The majority of ectopic pregnancies are surgically excised, in part owing to the inefficacy of the only available medical treatment, methotrexate. Previous work has shown combining methotrexate with the EGFR inhibitor, gefitinib to be additive at resolving ectopic pregnancy, however this combination is yet to prove successful for large ectopic pregnancies, which still require surgery. Therefore, in an attempt to improve the medical management of ectopic pregnancy, within the second arm of this study we investigated therapeutics, either as single agents or in combination with gefitinib (an EGFR inhibitor) that could provide superior efficacy to methotrexate or combination methotrexate and gefitinib to treat ectopic pregnancy. We screened a number of chemotherapeutics for their ability to induce placental death in-vitro, beyond that of methotrexate and the combination of methotrexate and gefitinib (which is currently been assessed in a stage III clinical trial). From this screening we identified vinorelbine, a well tolerated chemotherapeutic, to be 100 to 1000 times more potent than either methotrexate or combination methotrexate and gefitinib at inducing placental cell death. Importantly, vinorelbine is a tablet, which makes it a highly attractive drug option to treat ectopic pregnancy. Thus, we further investigated vinorelbine in-vivo using a mouse placental xenograft model and found vinorelbine to be significantly more efficacious at resolving placental xenografts than either methotrexate or combination methotrexate and gefitinib. Importantly, vinorelbine did not impact upon subsequent fertility in mice. Lastly, we assessed the potential of combining low dose vinorelbine with the EGFR inhibitor, gefitinib. This combination proved additive, causing placental cell death beyond either drug alone. In conclusion, this thesis has explored the role of EGFR signalling and potential of targeting this cascade in preeclampsia and ectopic pregnancy. Notably, we describe EGFR signalling to positively regulate placental sFLT-1 secretion and may therefore present as a novel molecular target to identify therapeutics for preeclampsia. Furthermore, we have uncovered vinorelbine, an orally available and well tolerated therapeutic, for ectopic pregnancy that has potential to be clinically translated and be a more effective treatment of ectopic pregnancy (either alone, or in combination with the EGFR inhibitor gefitinib). It is my hope that this body of work will further the field of obstetrics and gynaecology and lead to improved maternal outcomes.
Impact of new criteria for the diagnosis of gestational diabetes: a maternal and neonatal health outcome and economic analysis in a large tertiary level maternity centre
Background: The development of diabetes mellitus during pregnancy, referred to as gestational diabetes (GDM), is associated with adverse maternal and fetal outcomes. Routine screening for this condition is therefore undertaken in all pregnant women. In 2014, gestational diabetes underwent a more liberal diagnostic update, which has been widely adopted in Australia but not universally accepted internationally. Concerns around a rapidly increasing annual incidence and implications for costs and workloads have been raised, but they are assumed to have been offset by improvements in clinical outcomes. As one of the only large Western countries to have adopted universal screening under the new criteria, Australia is uniquely poised to assess the value of such a system change. Hypothesis: That the change in diagnostic criteria for gestational diabetes has resulted in improved clinical outcomes and equitable health economics. Aim: To compare an entire cohort of women and babies diagnosed with gestational diabetes in 2014 (under the original criteria) with those diagnosed in 2016 (under the updated criteria) and, in particular, to assess any improvements in outcomes, to attribute costs to the increased incidence and to assess any overall economic benefit. Methods: All women diagnosed with gestational diabetes in 2014 and in 2016 were included as cases. Control groups in each year were defined as those who underwent screening and had a negative test (pre-existing diabetes was thus an exclusion criteria). Women with multiple pregnancies were excluded from both cases and controls. Demographic data were collected from all groups. Maternal outcomes and fetal outcomes were selected to represent those reported in the studies upon which the new criteria are based. Three broad groups of outcome analyses were undertaken. Firstly, all women in 2014 were compared to all women in 2016 to determine whether the change in policy has caused a hospital-wide improvement in outcomes. Secondly, women with GDM in 2014 were compared to controls in 2014 and women with GDM in 2016 were compared to controls in 2016. Finally, women with GDM in 2014 were compared to women with GDM in 2016. In each analysis, women with GDM were examined as a whole and subdivided into diet-controlled and insulin-controlled. For the economic analysis, models of care for routine pregnancy, GDM diet-controlled and GDM-insulin controlled were costed using average-occasions-of-service for clinical reviews, pharmacy fees for medications and consumables, and Medicare Benefits Schedule item numbers for ultrasound services. Cost-savings were assessed using modelling of adverse outcome avoided based on relative-risk reductions published in the studies upon which the new criteria were based. Results: There was an increase in annual incidence for GDM from 6.0% to 10.4% with gross costs of care increasing by approximately $900 000 and nett costs of care by approximately $560 000. There was a small hospital-wide reduction in very large babies (>95% for birth weight) from4.31% to 3.61% with no other significant differences between 2014 and 2016. Women with GDM remain a higher risk cohort in both demographics and outcomes than those without GDM, but in 2016 women with GDM that is controlled by dietary measures alone represent a cohort with similar outcomes to women without GDM. Modelling for adverse outcomes avoided by the change in criteria did not reveal a cost-saving in the short-term. Conclusions: The new criteria for diagnosing GDM has resulted in a marked increase in annual incidence (73% relative, 4.4% absolute) without a significant improvement in maternal and neonatal outcomes. While small numbers of adverse outcomes are likely avoided, it is unlikely the potential short-term savings would outweigh the increase in costs if applying a high-risk model of care to all women with GDM. The new criteria may lead to long-term improvements in health of the mother and/or baby that are cost-effective but further research is required to substantiate this possibility. Future randomized controlled trials into different systems of diagnosis and less expensive models of care for mothers with GDM and their babies are also warranted.
Cancer stem cell-associated pathways regulating chemoresistance in ovarian cancer
Epithelial ovarian cancer (EOC) is the most lethal of the gynaecologic malignancies, with an overall 5-year survival rate of only <30%. This poor prognosis is due to the advanced-stage disease at initial diagnosis in approximately 70% of women. Despite good initial response to chemotherapy treatment, almost 80% of these patients will relapse and ultimately die within 5 years due to the development of chemoresistant recurrent disease. Emerging evidence suggests that a subpopulation of cancer stem cells (CSCs) that possess stem cell-like self-renewal and pluripotency properties are responsible for drug resistance and disease recurrence. The results from this thesis demonstrated that the expression of JAK2, STAT3, Src and EGFR activation was significantly higher in the ascites-derived tumour cells of recurrent serous EOC patients previously treated with chemotherapy compared to that of untreated chemo-naïve patients. STAT3 is a major regulator in stem cell regulation, and is predominantly activated by upstream JAK2 tyrosine kinase. Hence, this thesis aimed to explore whether targeting the JAK2/STAT3 pathway is sufficient in suppressing the development of ovarian CSCs which consequently would overcome chemoresistance-associated recurrence, and improve the disease-free survival period of EOC patients. To achieve the aim, in the first instance, this thesis investigated the in vitro suppression of paclitaxel-induced JAK2/STAT3 pathway activation with Momelotinib (a potent, small molecular inhibitor of JAK2) and paclitaxel-induced Src/STAT3 activation with dasatinib (a potent, small molecular inhibitor of Src family kinases) on the development of CSC-like phenotypes and tumour cell viability in HEY high-grade serous carcinoma and TOV21G clear cell carcinoma cell lines. The in vitro results demonstrated that suppression of JAK2/STAT3 with Momelotinib and paclitaxel was relatively more effective at reducing CSC-like markers and cell viability than suppression of Src/STAT3 with dasatinib and paclitaxel. Next, an in vivo mouse xenograft model was included to determine the effect of combination treatment with paclitaxel and Momelotinib on the sustained suppression of CSC-like emergence, tumour burden and disease-free survival of mice. Intraperitoneally injected HEY cells in Balb/c nude mice that received a combination of weekly paclitaxel and daily Momelotinib treatment survived the longest and produced the smallest tumour burden that exhibited significant reduction in paclitaxel-induced JAK2/STAT3 activation and CSC-like (Oct4 and c-Kit) tumour staining, but enhanced Src activation. However, termination of combination treatment resulted in the re-activation of JAK2/STAT3 and an increased in CSC-like tumour staining, but reduced Src activation. This thesis is the first to show that maintenance therapy with Momelotinib in a group of mice pre-treated with paclitaxel and Momelotinib further reduced the overall tumour burden and extended the overall disease-free survival period. Collectively, the results presented in this thesis supports the role of JAK2/STAT3 activation, in CSC-mediated chemoresistance and recurrence in EOC, while the activation of Src pathway compensated for the effects of Momelotinib. As we are now entering an era of personalized therapy, the findings in this thesis have increased our understanding of the resistance mechanisms and the complexity underlying successful clinical implementation, which have profound impact in developing a more effective treatment paradigm for EOC patients.
Novel technology for the measurement of newborn and infant heart rate
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.