Despite its relatively good long term survival compared to other cancers in the female, endometrial carcinoma still kills 20% of those women who develop the disease worldwide. Abnormalities in growth factor receptors have been shown to be important in both the prognosis and probably the malignant transformation of the two other oestrogen-dependent cancers, breast and ovary, which suggests that similar defects may occur in endometrial cancer. In this thesis, two of these growth factor receptors, those for the epidermal and platelet-derived growth factors, are studied in endometrial cancer at the levels of the gene and expression of the proteins on the cell surface, in an attempt to detect abnormalities which might be implicated in carcinogenesis. No amplification or rearrangement of the EGFR gene was detected on 13 tumour samples initially, or of the EGF, PDGF α- and β-subunit receptor genes subsequently on a mixture of tumour samples and cell from endometrial cancer cell lines. The size of the EGF and PDGF receptor proteins and the activation of their tyrosine kinase domains was assessed in an oestrogen responsive (Ishikawa) and an oestrogen independent (HEC-1-A) endometrial cancer cell line. Functioning receptors for EGF were demonstrated in both cell lines but no PDGF receptors were detected. EGF-binding studies were carried out and appropriate affinity constants and receptor numbers obtained in both cell lines. The growth of Ishikawa and HEC-1-A cells in culture was studied but because of the rapid growth of both lines in the absence of serum no meaningful mitogenic effects were shown by any growth factor or steroid. Phorbol ester and TGF-β inhibited the growth of HEC-1-A and Ishikawa cells, respectively. Of particular interest was that, despite the reported sensitivity of Ishikawa cells to oestrogen, no consistent stimulation was observed. The growth of both cell lines in the absence of serum suggested that the cells might be secreting growth factors and conditioned medium was obtained from long term, large scale cultures. The medium was concentrated and passed through a size exclusion column and the fractions assayed for DNA synthesis and competition for EGF-binding to Swiss 3T3 cells. No consistent effect on either of these assays was shown and no further investigation was undertaken.

The metabolic responses to trauma are well characterized and there is growing evidence supporting an important role for cytokines in its pathogenesis. Interleukin-1 (IL-1), Interleukin-6 (IL-6), and Tumour necrosis factor (TNF) are synthesized in the brain, and other tissues, in trauma. In order to test the hypothesis that these cytokines play an important role in the metabolic responses associated with trauma, the effects of chronic cerebroventricular and peripheral infusions of IL-1, IL-6, and TNF on protein metabolism, weight loss, anorexia, and pyrexia in Sprague-Dawley rats were examined. Specifically the aims of these investigations were: 1. To examine the effect of chronic central nervous system exposure to the proinflammatory cytokines IL-1, IL-6, and TNF. 2. To investigate the role of anorexia in the catabolic responses to centrally infused IL-1. 3. To investigate the effects of IL-1 on the hypothalamic-pituitary-adrenal axis. 4. To investigate the role of glucocorticoids in the metabolic effects produced by chronic central infusion of IL-1. 5. To examine the metabolic effects of increasing doses of chronic peripherally infused IL-1 and to distinguish between those responses mediated centrally and those mediated outside the blood-brain-barrier. The major findings of these studies were: 1. IL-1, but not IL-6 in the same dose, nor TNF in a lower dose, produced net protein catabolism, weight loss, and pyrexia in excess of that produced by anorexia alone. 2. Furthermore the loss of weight and nitrogen did not require sustained elevations of glucocorticoids, as IL-1 infusion in corticosterone-replaced, adrenalectomized rats resulted in similar losses of weight and nitrogen as observed in sham-adrenalectomized animals infused with IL-1. 3. Peripheral infusion of increasing doses of IL-1 mimicked the effects of increasing injury with low doses causing a mild acute phase response (as assessed by a fall in serum iron and albumin and a leucocytosis) and larger doses causing net protein catabolism and weight loss. An attempt was made to distinguish between the central and peripherally mediated metabolic effects of IL-1 using a novel model utilizing peripheral infusions of IL-1 and central infusions of IL-1 receptor antagonist. Using this model the data were consistent with the hypotheses that pyrexia is mediated inside and outside the blood-brain barrier and that leucocytosis is mediated by a direct peripheral effect, probably on the bone-marrow. In conclusion, centrally produced IL-1 may play an important role in the metabolic responses associated with trauma and this is in excess of the anorexia and adrenocortical activation produced by centrally acting IL-1. Peripherally produced IL-1 may play a role in various organs such as the bone-marrow and the liver to produce other features of the metabolic responses associated with injury.

Low molecular weight (LMW) proteins pass easily through the glomerular filter and are almost completely reabsorbed by the proximal renal tubule in health. An increase in their urinary excretion implies failure of reabsorption and may signal tubular dysfunction. β2-microglobulin (B2M) is a sensitive marker of tubular dysfunction, but is unstable in acid urine, whilst only limited data are available for other LMW proteins. The aim of these studies was to determine the prevalence of elevated LMW protein excretion in children with renal disease and to identify factors influencing LMW protein excretion. Secondly, these studies sought to determine conditions where individual proteins might best indicate tubular dysfunction. Enzyme-linked immunosorbent assays were used to measure B2M, α1-microglobulin (A1M) and urine protein 1 (UP1) in plasma and urine, and retinol-binding protein (RBP) in urine. Albumin, the lysosomal enzyme N-acetyl-β-D-glucosaminidase (NAG), creatinine and pH were also measured in urine. Each protein excretion was expressed as a ratio to creatinine concentration. B2M showed instability in urine with pH below 6.0, RBP was unstable in urine below pH 5.0 following frozen storage whilst A1M and UP1 were stable at physiological pH. Two groups of apparently healthy children were studied, and normal ranges were established for protein excretion in random and overnight samples. A comparison of B2M, A1M, UP1, RBP and NAG excretion was undertaken in tubular disease and in glomerular disease. A1M, UP1 and NAG were correlated with increasing albumin excretion in steroid-sensitive nephrotic syndrome, in contrast to B2M and RBP. Compared with A1M, UP1 and NAG, RBP was more closely associated with tubulo-interstitial involvement histologically. There was abnormal RBP excretion in reflux nephropathy, with levels increasing according to the degree of scarring. Increased RBP excretion was also seen in cystic disease, neurogenic bladder, allograft rejection and chronic glomerular disease. RBP excretion was inversely correlated with glomerular filtration rate in reflux nephropathy and in glomerular disease. RBP excretion was increased in diabetic children and was correlated with albumin excretion and with metabolic control. LMW protein excretion is common in children with renal problems, and may be a marker of disease severity. RBP is the most suitable marker of tubular dysfunction.

This volume contains an introduction to the topic of inflammatory arthritis, followed by Chapter 2 which is a Materials and Methods section and includes experimental protocols used for the original research detailed in Chapters 3 to 6 (Sections 2.1 - 2.4). Chapters 3 to 6 are each followed by their own Discussion section and References in much the same form as was submitted for publication.The thesis is concluded by a general discussion of the topic and the relevance of this research.

Background, hypotheses and aims Poor fetal growth is associated with increased perinatal morbidity and mortality. Recently the topic of poor fetal growth has generated considerable research interest, as numerous epidemiological studies have demonstrated that small size at birth is also associated with increased risks of adult cardiovascular diseases and non-insulin dependent diabetes Antenatal treatment aimed at improving fetal growth might therefore reduce perinatal morbidity, as well as later complications of being born small for gestational age (SGA). SGA fetuses with abnormal umbilical artery Doppler studies have placental vascular pathology. Low dose aspirin, which inhibits the production of the powerful vasoconstrictor thromboxane, might therefore increase placental blood flow and fetal growth. The hypothesis in the first antenatal study was that treatment of SGA pregnancies, with abnormal umbilical artery Doppler studies, with aspirin (100 mg) for ≥14 days, would increase fetal weight. As perinatal morbidity and mortality are increased in SGA pregnancies most obstetricians advocate a programme of regular fetal surveillance. Outpatient management with twice weekly fetal surveillance has been usual practice in our hospital in uncomplicated SGA pregnancies. These frequent checks may be unnecessary when umbilical artery Doppler studies are normal as the risk of serious perinatal morbidity is low. More conservative management in SGA pregnancies with normal umbilical artery Doppler studies has been suggested by several authors but has only been evaluated in one previous trial, where hospitalisation was usual practice. In the second antenatal study we therefore wished to test the hypothesis that the frequency of fetal surveillance could be reduced from twice weekly to fortnightly, in SGA pregnancies with normal umbilical artery Doppler studies, without increasing maternal or perinatal morbidity. Abnormal umbilical artery Doppler studies have been shown in a number of reports to be associated with increased perinatal mortality and morbidity, in SGA pregnancies. Previous investigators have not considered the potential confounding effects of both gestational age at delivery and birthweight in relation to umbilical artery Doppler status. SGA babies with normal umbilical artery Doppler studies, have a low risk of complications and have been called ‘small normal babies’ although there is little data to support this claim. In the third antenatal study the following hypotheses were tested: that abnormal umbilical artery Doppler studies would predict newborn morbidity in SGA babies, independent of birthweight and gestational age; that compared with SGA babies with abnormal umbilical artery Doppler studies, SGA babies with normal umbilical artery Doppler studies would have low rates of newborn morbidity and malnutrition; and that their mothers would be smaller, have different ethnic distribution, and have lower rates of vascular problems in pregnancy than mothers of SGA babies with abnormal umbilical artery Doppler studies. After birth approximately 20% of SGA babies fail to show catchup growth and remain short at two years of age. Persisting short stature has been associated with later psychological difficulties, abnormal neurodevelopmental testing in childhood, poor school performance and hypertension in childhood and adult life. Most catchup growth occurs in the first six months after birth and most children who are small at six months will not show further catchup growth. Early identification of children who will remain small may enable interventions aimed at improving later outcomes. There are no previous reports of the influence of perinatal variables on size at six months. In the first postnatal study in this thesis we therefore chose to investigate the perinatal factors associated with small size at six months of age. We tested the hypotheses that children who were small at six months would: have been diagnosed SGA earlier in pregnancy; be more likely to have abnormal umbilical artery Doppler studies; have smaller body proportions at birth; and be more likely to have normal ponderal indices at birth. Previous studies, mostly of children born in the 1960s and 1970s, have reported more abnormal neurodevelopmental test results in children who were SGA at birth compared with appropriate weight for gestational age children. There are very few published studies of neurodevelopmental outcome in SGA children born in the 1990s, who have had the advantage of recent advances in antenatal and especially newborn care. The aims of the second postnatal study were therefore to: assess neurodevelopmental outcome in a cohort of SGA children at 18 months and compare results to a reference population; determine whether one or more of the following perinatal variables might be predictive of abnormal neurodevelopmental test results: early recognition of SGA antenatally, abnormal antenatal Doppler results, lack of participation in the antenatal studies in SGA pregnancies, gestation at delivery, head size at birth, head growth from birth to six months, and ponderal index at birth.

The primary object of this thesis was to investigate aspects of empirically-measured, anaesthesia-induced electroencephalographic (EEG) changes that could be explained by network models of cortical interactions. The thesis consists of a collection of various theoretical and clinical papers in three sections: (a) A background section summarizing previous relevant published works on the molecular actions of anaesthetic agents, and the origin and problems with the acquisition of the EEG. (b) A second section dealing with the development of various theoretical / computer models of general anaesthetic action. (c) A third section of various clinical and EEG studies of anaesthesia and sleep. These were done to confirm and clarify some of the theoretical results from the models. However there are many different ways of numerically capturing the information contained within the EEG. Because this problem needed to be overcome before the primary aim of the thesis could be accurately handled; the clinical studies tended to be diverted into this secondary "signal-acquisition" aspect of the EEG analysis in anaesthesia and sleep. Because the thesis has a constellation of different interlocking threads of investigation, I have summarized the various themes in the following table. (A) Background Section The relevant conclusions from the background section were: 1) The transition from aesthesia to anaesthesia involved the whole animal, and thus may not be entirely measurable by the EEG. In contrast the transitions from consciousness to coma - and even more specifically - from mnesis to amnesis were predominantly observable by changes in the activity of the cortex and in the resultant EEG signal. 2) The EEG signal arose largely from the summation of coherent post-synaptic potentials in the cortical pyramidal cells. These were, in turn, mainly controlled by cortical layer-l modulation - which was subject to ascending neuromodulatory systems. 3) The main artifacts in the EEG signal were eye-movements, blinks, and frontalis EMG. 4) The Bispectral index (BIS) consisted of three subcomponents. The first two (BetaRatio and SynchFastSlow) probably reflected the loss of high-frequency activity that is associated with the transition from consciousness to unconsciousness. 5) The state of the midbrain reticular formation projecting via the non-specific thalamocortical connections can profoundly alter the oscillatory state of the cortex and thence the EEG. Direct or indirect actions of general anaesthetic agents on these structures may be a major cause of the observed EEG changes. These changes are qualitatively similar to the changes observed during natural sleep. 6) Although there was variation between different agents, the most consistent action of commonly-used general anaesthetic agents was to prolong the fast inhibitory postsynaptic potential> 50% by GABAergic potentiation. This action was consonant with the observed EEG effects of anaesthesia - which are (1) an increase in EEG spectral power and frequency followed by, (2) a decrease in dominant frequency and overall power with increasing concentrations: the so-called "biphasic response" There were also a number of more specific features: namely loss of alpha band activity, an initial increase in relative beta activity, followed by a shift to lower frequencies, spindles - and in deep anaesthesia - burst suppression patterns. Other contributions to anaesthetic action may include: (1) inhibition of NMDA receptor function, (2) reduction of nicotinic acetylcholine modulation, and (3) a direct hyperpolarizing action by opening potassium channels. Anaesthetic agents disrupted cortical function at lower doses than those required for inhibition of brain stem function. (B) Theoretical Models The fact that the cerebral cortex consisted of, effectively, a two-dimensional interconnecting neural network was of overriding importance. All the theoretical models were constrained by this topological fact, and showed a remarkable degree of similarity. Regardless of whether the model was continuous (appendix 2) or discrete (cellular automata), some robust and consistent features emerged: 1) Some form of abrupt decrease in information flow within the network occurred when the interaction between the network elements was interrupted greater than certain critical threshold amount - the order/disorder phase transition. 2) This critical point was manifest as (i) a large change in the simulated mean soma potential (the order parameter), (ii) divergence of the spectral power of the simulated pseudoEEG signal, and (iii) decrease in frequency content (in the cellular automaton models). Unfortunately the order parameter for the EEG is probably unmeasurable in clinical practice. The first derivative is measurable, but not completely reliable as a proxy order parameter. 3) All models required some form of noisy external (subcortical) input to drive them. Variations in this input could explain some of the anomalies observed in the clinical section of the thesis. (C) Clinical Papers The clinical papers were a series of papers recording the clinical data, which then influenced the development of the theoretical models. The single most popular clinical measure of anaesthetic depth in present practice is the Bispectral Index (BIS). Many of these studies explore the strengths and weaknesses of this index in various clinical situations. The main disadvantage of the BIS was that crucial aspects of its formulation are not in the public domain for commercial reasons. We attempted to separate out the changes in the various subcomponents of the BIS in patients under general anaesthesia, and natural sleep. 1) In the first paper we described how the EEG changed during induction and recovery from general anaesthesia with propofol and isoflurane. We compared EEG vs heart rate variability changes as monitors of anaesthetic depth. This study served to evaluate a clinically accepted measure of anaesthetic depth, as well as gaining practical EEG experience in collecting experimental data, both using the ASPECT EEG monitor, and in devising the best clinical methods of quantifying levels of consciousness. The main results were that the BIS was relatively reliable in steady-state conditions, and better than the raw 95% spectral edge frequency, and the approximate entropy of the EEG. 2) Auditory recall and response-to-command during recovery from propofol anaesthesia. In this paper we were able to collect data in a very controlled situation, somewhat different from that pertaining to clinical practice. From this we were able to identify separate components of consciousness and mnesis and how they correlated with EEG changes. We were also able to compare frontal and parietal EEG data. We established that there is a clear divergence of spectral power around the time of loss-of-consciousness when the propofol was administered as a slow infusion. This is good evidence supporting the basis of the theoretical models. This experiment also established that it is possible for a patient to be conscious, not paralyzed, but not responding to verbal command. 3) Electroencephalographic measures of depth of anaesthesia: the importance of the gamma band and the electromyogram signal. This study was concerned with different technical aspects of EEG signal acquisition and processing. It suggested that usually the EMG is not a significant problem, and that the gamma (40-60Hz) frequency band is important in distinguishing the awake from the anaesthetized state. 4) The Bispectral Index: A Measure of Depth of Sleep? Because the EEG changes of general anaesthesia are very similar to those of sleep, this study was a simple observational study collecting some information about changes in the BIS with natural sleep. 5) The Bispectral Index and Sleep Stage: A Polysomnographic study. In this study we formally compared the BIS with a full polysomnogramnographic sleep staging. It confirmed the results of the previous study, and demonstrated that the subcomponents of the BIS (the BetaRatio and the SynchFastSlow) changed in a manner very similar to those observed under general anaesthetic. Conclusions The observed changes in the EEG on induction of anaesthesia can be explained by changes in relatively simple theoretical network models. These changes can be reliably reduced to univariate Parameters

This volume contains a series of linking chapters that follow the evolution of percutaneous intervention from conventional balloon angioplasty in the early to mid 1990s to stent implantation which has now become standard practice. It describes contributions to the extension of clinical situations in which percutaneous intervention has been found to be efficacious as well as refinements in practice and technique. I was fortunate to have been a fellow and then staff member of the Cardiology department of the Thoraxcenter, Rotterdam from 1993-1995, which was then, and remains, at the forefront of the development of percutaneous coronary interventional techniques. My continued links with the Thoraxcenter as well as new projects have allowed me to continue my research and complete this thesis. Part 1 contains two chapters on balloon angioplasty which was standard clinical practice for suitable obstructive coronary lesions until the mid 1990s. The first chapter describes an audit of immediate clinical outcome and thus rates of procedure related complications of contemporary practice in a high volume unit following 970 balloon angioplasty procedures over a one-year period. Although the rate of adverse events appeared to remain static when compared to the previous decade, the requirement for emergency bypass surgery appeared to be diminishing and it is suggested that may be due to the increasing availability of stents for "bail-out". The second chapter studies the long-term outcome with respect to major adverse clinical events in 856 consecutive patients treated by balloon angioplasty from 1980-1985. Although the long-term outcome was good and comparable to results of coronary bypass surgery, most patients suffered a further cardiac event during the 10 years following balloon angioplasty, most of which were repeat revascularisation. Part 2 contains four chapters that describe and document contributions to the evolution of percutaneous coronary interventional practice from balloon angioplasty to stent implantation. Chapter 1 is a review of the literature that describes the evolution of stenting from early animal experiments through early human studies to current practice and on to a vision of the future. Chapter 2 describes follow-up of patients enrolled in the Benestent I study, which was pivotal to the introduction of stenting into clinical practice. Patients, in many centres world-wide, were randomised to balloon angioplasty or stent implantation and underwent 6-month clinical and angiographic follow-up. We report the extended one-year clinical outcome. Chapter 3 documents the landmark Benestent II study. This was a randomised comparison of 827 patients in many centres worldwide (including Auckland) who were allocated to balloon angioplasty or a heparin-coated stent plus treatment with the anti-platelet agent ticlopidine. Patients randomised to stent implantation suffered fewer adverse events and less restenosis but this treatment was found to be more costly than balloon angioplasty. Chapter 4 analyses in more detail some data from the Benestent II study. As well as being randomised to balloon angioplasty or stent implantation, patients were also sub-randomised to 6 month clinical only or clinical and angiographic follow-up. We therefore set out to assess whether management and outcome were influenced as a result of a follow-up angiogram. We found that more interventions were undertaken in those who underwent angiography with no adverse effect on outcome. As a result of the above and other studies, stenting has become routine clinical practice with most patients (approximately 90%) undergoing percutaneous coronary intervention receiving a stent. Part 3 describes several studies which have expanded our knowledge of stenting in various clinical situations and conditions, and also incorporates studies that have added refinement to the technique of stenting. Chapter 1 studies the intermediate term clinical outcome of stent implantation in degenerated saphenous vein grafts. Although the immediate outcome is good, with respect to adverse clinical events, the long-term benefit is limited indicating that with the available technology and medication percutaneous treatment of vein graft is a palliative procedure. Chapter 2 describes the results of a prospective multicentre study assessing the outcome of stenting long native coronary artery lesions. The rates of restenosis were acceptable. The study also provides us with an opportunity to analyse, using quantitative coronary angiography, the location of and give insights into, the mechanism of the restenosis process. Chapter 3 studies the outcome of stenting smaller coronary arteries. Its unique design, in that patients were required to have a lesion in a "small" and a "large" vessel, has provided us with the opportunity to compare both the rates and nature of the restenosis process in the same patients. In this way we abolished clinical and procedure related variables that confound the results when using historical comparisons. Chapter 4 describes the introduction of a new stent, the MultiLink stent, into everyday clinical usage. As with all new devices and minor variations of contemporary devices, which slowly undergo small progressive changes, a careful assessment and audit must be undertaken to ensure it has resulted in improvement in, or at least no detriment to patient care. This chapter audits the clinical outcome of a new stent implanted in the first 126 patients in Auckland and Monash Medical Centre, Melbourne. Chapter 5 is a study that tests a refinement in interventional technique. With refinement and miniaturisation of angioplasty equipment including the profile of balloons and stents, the possibly of implanting a stent without predilating the lesions with a balloon was raised. In this study 81 patients with suitable coronary artery lesions were randomised to stenting with and without predilatation. It was found that in selected patients a strategy of direct stenting was feasible, faster and more rapid than stenting after predilatation. Chapter 6 describes a study which analyses data from 10 studies co-ordinated and managed by the Cardialysis core laboratory in Rotterdam The inclusion of risk factors allows the opportunity to perform meta-analyses looking for factors that may influence outcomes. In this case we attempted to identify clinical and angiographic factors that influence asymptomatic restenosis following percutaneous coronary intervention. Chapter 7 gives an overview of angioplasty numbers and practice in New Zealand by summarising data from the National Angioplasty Registry for the years 1995-1998. This period saw a steady growth in patients with coronary artery disease treated by percutaneous intervention and the number of patients who received stents rose from 23% in 1995 to 84% in 1998. This data attests to the data and results of the earlier studies reported in this thesis.

New generation T cell assays offer hope in the diagnosis of Mycobacterium tuberculosis infection and disease. We assessed the ELISPOT assay using cross-sectional and longitudinal studies and a natural gradient of M. tuberculosis exposure by sleeping proximity to a tuberculosis (TB) case in The Gambia. Two antigens, ESAT-6 and CFP-10 (EC), were compared to purified protein derivative (PPD) by ELISPOT and to the PPD skin test in 735 TB contacts. All three tests responded to the exposure gradient, the PPD skin test most dramatically. Inter-test comparison showed that the EC ELISPOT provided improved specificity in the diagnosis of M. tuberculosis infection, but at the cost of some sensitivity. Increasing discordance, particularly between PPD ELISPOT and PPD skin test results, down the exposure gradient to 105 community controls was identified. In 693 children, the EC ELISPOT was slightly less sensitive than the PPD skin test in the diagnosis of M. tuberculosis infection from recent exposure; neither test was confounded by prior BCG vaccination, even in the very young. A fusion protein of EC compared favourably with their respective peptides by ELISPOT assay in 488 TB contacts, a combined test result offered improved sensitivity. Quantitative ELISPOT and PPD-skin test responses were assessed in 1052 TB case contacts, according to an ELISPOT response to EC. Only the ELISPOT count was sensitive to the exposure gradient (p=0.009), revealing a positive dose-response relationship. In the longitudinal assessment, both ELISPOT and PPD skin test conversion occurred over time. PPD skin test reversion occurred in 10% of individuals after 18 months, ELISPOT reversion occurred in 39% at 3 months. In conclusion: the EC ELISPOT offers increased specificity in the diagnosis of M. tuberculosis infection in The Gambia, at the cost of some sensitivity; the PPD skin test appears to be down-regulated in the community; neither test is confounded by prior BCG vaccination; a fusion protein in combination with EC peptides offers optimal ELISPOT sensitivity; the quantitative ELISPOT response in specific-antigen-positive TB case contacts reflects the infectious load of M. tuberculosis; and significant early reversion of the ELISPOT test suggests it is unreliable in M. tuberculosis dormancy.

Objectives This thesis had the following objectives: 1) To assess the existing evidence base for the effectiveness of tubal flushing as a treatment for infertility (Section II, Chapters 4 and 5). 2) To assess current practice and prior beliefs amongst Australasian fertility specialists concerning the role of tubal flushing as a treatment for infertility (Section III, Chapter 6). 3) To investigate the possible mechanism of the fertility enhancing effect of the oil soluble contrast medium lipiodol, and specifically whether there is an effect on the endometrium (Section IV, Chapter 7). 4) To generate definitive evidence from a randomised controlled trial for the effectiveness of lipiodol flushing as a treatment for infertility (Section V, Chapters 8 and 9). 5) To evaluate the adoption of lipiodol flushing as an innovative treatment into clinical practice (Section VI, Chapter 10). Methods The work undertaken in this thesis was based on prospective study protocols using the following research methodologies: Systematic reviews and meta-analyses of treatment efficacy to meet objective 1. Two types of structured survey questionnaires with a Bayesian analysis to meet objective 2. A randomised animal study involving 60 Swiss white mice, combined with genital flushing procedures under anaesthesia, some of which involved microsurgical techniques, followed by genital tissue harvesting, tissue preparation and immunohistochemistry studies to meet objective 3. An open, parallel group, single centre, randomised controlled trial involving 158 women with unexplained and endometriosis-related infertility to meet objective 4. A survival analysis of women in the lipiodol flushing randomised trial to meet objectives 4 and 5. A prospective observational study of the first 100 women to undergo lipiodol flushing in clinical practice to meet objective 5. A clinical hysterosalpingogram procedure to meet objective 4 and 5. Results 1) Eight randomised controlled trials involving 1,971 women were identified and included in the systematic review. Tubal flushing with oil soluble contrast media versus no intervention was associated with a significant increase in the odds of pregnancy (Peto odds ratio [OR] 3.57, 95% confidence interval [CI] 1.76 to 7.23) but there were no data for live birth. There were no data from RCTs to assess tubal flushing with water-soluble media versus no intervention. Tubal flushing with oil soluble contrast media was associated with a significant increase in the odds of live birth versus tubal flushing with water soluble contrast media (OR 1.49, 95% CI 1.05 to 2.11) but the odds of pregnancy showed no significant difference (OR 1.24, 95% CI 0.97 to 1.57) and there was evidence of statistical heterogeneity for these two outcomes. The addition of oil soluble contrast media to flushing with water soluble contrast media (water plus oil soluble contrast media versus water soluble contrast media alone) showed no significant difference in the odds of live birth (OR 1.06, 95% CI 0.64 to 1.77) or pregnancy (OR 1.16, 95% CI 0.78 to 1.70). 2) Nineteen Australasian fertility specialists returned survey questionnaires. Eighteen of the 19 specialists believed that lipiodol flushing was more likely to be beneficial than harmful. The most widely held prior belief, reflected in both textual and numerical responses, was that lipiodol was likely to produce a small beneficial response. The credible limits of this belief were compatible with a reasonable fertility benefit, as more than 50% believed that a 1.5-fold increase in pregnancy rate was plausible. The two surveys found that a 1.2-fold or 1.4-fold increase in pregnancy rate was the median expected level of benefit at which clinicians would have been inclined to recommend lipiodol flushing to their patients (combined range 1.1 to 2.3-fold). Individual and collective equipoise was justification to proceed with a definitive randomised controlled trial. 3) The mean number of cluster determinant (CD) 205+ uterine dendritic cells decreased significantly in mice following lipiodol treatment compared to sham treated and saline treated mice, particularly in endometrial and subendometrial tissues. The mean number of CD1+ uterine dendritic cells increased significantly following lipiodol treatment compared to shamtreatment. No significant differences were found in the mean number of total leukocytes or macrophages in the murine uterus between the three treatment groups. 4) Six month follow up of the randomised trial of 158 women showed that lipiodol flushing resulted in a significant increase in pregnancy (48.0% versus 10.8%, RR 4.44, 95% CI 1.61-12.21) and live birth (40.0% versus 10.8%, RR 3.70,.95% CI 1.30-10.50) rates versus no intervention for women with endometriosis (n=62), although there was no significant difference in pregnancy (33.3% versus 20.8%, RR 1.60, 95% CI 0.81-3.16) or live birth (27.1% versus 14.6%, RR 1.86, 95% CI 0.81-4.25) rates for women with unexplained infertility without confirmed endometriosis (n=96). Survival analysis up to 24 months showed a significant benefit in overall pregnancy rate following lipiodol treatment (hazard ratio 2.0, 95% confidence interval [CI] 1.3 to 3.2) for the combined endometriosis and unexplained infertility populations. Amongst women with endometriosis, the benefit in pregnancy rate seen in the first 6 months following lipiodol (hazard ratio 5.4, 95% CI 2.1 to 14.2) was not present at 6 to 24 months following lipiodol (hazard ratio 0.6, 95% CI 0.2 to 2.1). There was a more consistent effect of lipiodol on fertility throughout the 24 month follow up amongst women with unexplained infertility (hazard ratio 2.0, 95% CI 1.1 to 3.5). 5) Six month follow up in the observational study of 100 women undergoing lipiodol flushing as an innovative treatment in clinical practice showed an overall pregnancy rate 30% and live birth or ongoing pregnancy rate 27% six months after the procedure. For women under 40 years old, a 32% pregnancy rate and 25% live birth or ongoing pregnancy rate was seen in women with unexplained infertility; a 50% pregnancy rate and 47% live birth or ongoing pregnancy rate was seen in women with endometriosis. Of women aged 40 years and older, the pregnancy rate was 13% and the live birth or ongoing pregnancy rate was 13%. The pregnancy rates included those occurring after additional interventions, such as intrauterine insemination and in-vitro fertilisation, accounting for 12 of the 30 pregnancies. There were no treatment complications. Conclusions The conclusions of this thesis are as follows. Lipiodol flushing is a simple, inexpensive, effective fertility treatment, which carries a very low chance of complications and no increased chance of multiple pregnancy. Lipiodol treatment is particularly effective in the short term for women with endometriosis who have normal patent fallopian tubes. The fertility benefit from lipiodol treatment lasts longer for women with pure unexplained infertility than for women with endometriosis. The level of benefit from lipiodol treatment for women with unexplained and endometriosis-related infertility is of sufficient magnitude to convince most fertility specialists surveyed that it is a worthwhile treatment to offer routinely in clinical practice; however complex factors govern the implementation of an innovative fertility treatment. Observational study of the first 100 women to undergo lipiodol treatment in clinical practice has provided further evidence of the efficacy and safety of this approach. Uterine dendritic cell changes following lipiodol flushing in mice suggest that the mechanism of the fertility enhancing effect might be an immunobiologic effect on the endometrium that could improve the receptivity of the endometrium (rather than a mechanical tubal flushing effect), although this hypothesis requires further exploration in women.

Perinatal hypoxic ischaemic injury is an important cause of both neonatal death and long-term disability. The sequence of resuscitation followed by a latent phase then a secondary cascade of injury is well documented. This thesis covers key steps toward the utilization of selective hypothermia as an intervention during the latent phase to ameliorate the secondary injury and improve subsequent outcome. The technique was shown to be both feasible and well tolerated. Specifically, a rectal temperature of 35 °C and 34.5 °C, in term infants with neonatal encephalopathy, was not associated with an excessive requirement for cardio-respiratory support. Although a decrease in heart rate occurred during cooling, this was expected and there was no significant change in blood pressure during either the cooling or rewarming phase. Additional reassuring findings were that neither major electrolyte disturbance; hypoglycaemia or haematological changes, including excessive haemorrhage, were observed during hypothermia. The study of neurodevelopmental outcome established that selective cerebral hypothermia was not associated with late adverse effects and, in infants with moderate to severe encephalopathy, the combined cooled groups demonstrated a trend towards better outcome. These data confirmed the potential for selective cerebral hypothermia to provide neuroprotection following perinatal asphyxia. In further chapters cerebral CT scan was confirmed as a helpful adjunct to clinical staging in predicting neurodevelopmental outcome and important clinical experience was reported including rebound seizures following rewarming; sclerema neonatorum associated with hypothermia; and abnormal flow in the superior sagittal sinus, associated with perinatal asphyxia. Lastly a review of infants assessed but not recruited to the CoolCap trial based on aEEG criteria was performed. As these aEEG criteria could be applied to future clinical use it was considered important to ensure large numbers of infants with potential to benefit were not excluded from intervention Neurodevelopmental status for those infants excluded by the aEEG criteria was largely favourable but a small number had adverse outcome and the majority manifested short term morbidity. In conclusion, the work presented in this thesis suggests that intervention with selective hypothermia offers the potential to change disease progression and improve subsequent outcome following perinatal asphyxia at term. / Whole document restricted until 11/2014, but available by request, use the feedback form to request access.