Paediatrics & Child Health - Doctoral Theses

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    PiRAMiD: predicting early onset autism through maternal immune activation and proteomic discovery
    (University College Cork, 2023) Carter, Michael; Murray, Deirdre M.; Gibson, Louise; O'Keeffe, Gerard W.; English, Jane; National Children's Research Centre
    Autism spectrum disorder (ASD) is a heterogeneous developmental disorder arising early in life. ASD is composed of a wide variety of clinical characteristics, neuropsychological impairments and complex phenotypes. The classical triad of ASD symptoms includes disrupted social function, atypical verbal and non-verbal communication skills, and restricted interests with repetitive behaviours. These core symptoms often coexist with other psychiatric and neurological comorbidities. Attention Deficit Hyperactivity Disorder (ADHD), epilepsy, migraine, and anxiety are much commoner in children with ASD. Children and adults with ASD often encounter difficulties with emotional and behavioural problems (EBPs) such as emotional reactivity, aggression, and depression. Up to 50% of those affected can have intellectual disability (ID) and limited verbal communication. Social, emotional and behavioural deficits in children with ASD are also important modifiers of outcome and are linked to elevated stress, mental and physical health problems, and lower overall family and caregiver well-being. We know that early intervention can be effective, and may be parent or therapist delivered. Pharmacological treatment of ASD can be successful insofar as it is useful for symptomatic management of some ASD comorbidities such as ADHD, and depression. Although genetic susceptibilities are increasingly recognised, the mechanism of disease development in ASD remains unknown. We are aware of both common and rare genetic risk factors with more than four hundred diverse high confidence genes now linked to ASD ( Singly, these genetic factors each convey only a modest increase in ASD risk (~1%), however collectively they can contribute to a far greater risk. Both de novo and inherited genetic defects are recognised but ASD risk in progeny does not follow a clear pattern of inheritance. Estimates of heritability of ASD in twin pairs vary widely between 50 – 90%. The apparent male preponderance in ASD persists with a clear bias towards males. Rates of ASD among males exceed those of females by three or fourfold hinting at a possible sex differential genetic foundation. Up to 20% of individuals with ASD may possess copy number variants (CNV) and de novo loss of function single nucleotide variants (SNV) that are individually rare but in combination, increase an individual’s overall ASD risk. While newer methods of genetic analysis (such as whole genome sequencing) are uncovering new candidate genes with regularity, the heterogeneity of the clinical and phenotypic groups within ASD strongly suggest that in those with a genetic predisposition, environmental factors may act in concert to bring about a multisystem dysfunction leading to ASD. Despite recent advances in gene analysis, we are yet to discover a single gene determinant that can account for more than a small percent of ASD cases. The current ASD literature suggests that mutations occurring in genes involved in synapse formation, cell adhesion molecule production (Cadherin), scaffolding proteins (SHANK proteins), ion channels (sodium, calcium, and potassium channels), and signaling molecules can disrupt regulatory or coding regions and affect synapse formation, plasticity and synaptic transmission. All this suggests that we cannot explain many cases of ASD by genetic factors alone, or at least we cannot explain them using our current understanding of ASD genetics or our current techniques of genetic analysis. The flawed picture of ASD genetics has led some to investigate the role of environmental exposures in the aetiology of ASD. Researchers have identified many environmental risks in ASD. Advanced parental age, foetal environmental exposures, perinatal and obstetric events, maternal medication use, smoking and alcohol use, psychosocial hardship, nutrition and toxic exposures have all been implicated as risks in the pathogenesis of ASD. While authors attribute between 17 - 41% of ASD risk to non-genetic or environmental exposures, the exact balance between genetic and environmental determinants and their roles in aetiology remains disputed. Multiple mechanisms have been proposed through which each of these exposures may exert an influence on genetic and epigenetic risk in ASD , but there are only a handful that are likely to effect abnormal neurodevelopment. Animal models of inflammation and maternal immune activation are particularly well characterised, and have successfully modelled ASD type behaviours and social difficulties in mice, rats and non-human primates. Maternal immune activation (MIA) is defined as an increase in measured levels of inflammatory markers in mothers during pregnancy. Through this process, a cytokine cascade transmits to the foetus, resulting in adverse neurodevelopmental phenotypes and even remodelling of the immature foetal brain. Many studies have profiled cytokine, chemokine, immune cell and inflammatory signatures in ASD affected individuals. Only a much smaller number have characterised cytokine profiles in expectant mothers who progressed to birth children who develop ASD. The few previous studies, which have examined gestational serum, have indicated mid-gestational upregulation in specific pro-inflammatory cytokines or indeed down-regulation in anti-inflammatory cytokines. Metabolomic analysis refers to the systematic identification and quantitation of all metabolites in a given biological sample. This collection of metabolites, known as the metabolome, is thought to directly reflect the biochemical activity of the studied system at a specific point in time. The metabolome has become an area of interest, as some inborn errors of metabolism (IEM) are clearly linked to ASD phenotypes. Phenylketonuria (PKU) and Smith-Lemli-Opitz syndrome (SLOS) are disorders of amino acid and cholesterol metabolism respectively. Untreated PKU is associated with strongly autistic phenotypes, while SLOS is phenotypically heterogeneous, but autism remains a common feature in these children. Similarly, proteomics is defined as the study of the complete protein profile in a given tissue, cell or biological sample. Proteomic studies of human sera have so far noted altered levels of proteins involved in inflammation or immune system regulation, including acute phase reactants and interleukins. Abnormalities of the complement system have also been found in ASD and other psychopathologies such as schizophrenia. Recent works demonstrate that the complement pathway can affect synaptic remodelling and has roles in neurodevelopmental processes. The initial focus of ASD research on genomics has largely failed to result in the much-hoped-for silver bullet of ASD aetiology, i.e. a common genetic cause. Instead, the genetic landscape has proven to be exceedingly complex and interdependent on a multitude of factors, including environmental exposures and other modifiers of genetic risk. Research examining the aetiology of ASD has shifted focus from genetics to a multimodal approach. In recent years, funding has become available for a far wider variety of ASD aligned research topics, beyond those with a focus on genetics. Opportunities now exist to adopt a multifaceted approach to ASD aetiology, shifting the focus from a narrow genetic base, to a broader multimodal approach to examine other potential mechanistic players. While this adds further complexity to what is already a complicated picture, the strived for parsimonious answer is simply never likely to materialise. Newer fields and modalities such as proteomics, metabolomics and machine learning will help to further refine and untangle the complicated web of ASD, and this variety of granular detail is what is likely to result in a practicable biomarker or effective therapy in the future. In this thesis using a multimodal approach (ELISA, metabolome and proteome analysis) we aim to explore further the role of MIA and alterations of the proteome and metabolome in the pathophysiology of ASD. We hope that our findings may ultimately help to identify a potential gestational biomarker of ASD, which will improve access to early diagnosis and treatment. We also aim to characterise co-morbid emotional and behavioural problems, which arise early in children with ASD and are pervasive throughout all spheres of life. Early recognition and intervention with these co-morbidities can improve treatment outcomes, patient, and family wellbeing.
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    Multi-modal assessment of newborns at risk of neonatal hypoxic ischaemic encephalopathy – the MONItOr study
    (University College Cork, 2022) Garvey, Aisling A.; Dempsey, Eugene M.; Murray, Deirdre M.; Boylan, Geraldine B.; National Children’s Research Centre, Crumlin, Ireland
    Background: Hypoxic ischaemic encephalopathy (HIE) is the leading cause of acquired brain injury in term infants. At present, therapeutic hypothermia (TH) is the only approved therapy for infants with moderate-severe HIE. However, it must be commenced before 6 hours of age resulting in a clinical challenge to resuscitate, stabilize, identify and stratify infants in this narrow timeframe. Furthermore, a significant proportion of infants with mild HIE will have neurodevelopmental impairment. Improved, timely identification of infants at risk of brain injury is required. The aim of this study was to improve our knowledge of the early physiology of infants with HIE by describing the evolution of electroencephalography (EEG), near-infrared spectroscopy (NIRS) and non-invasive cardiac output monitoring (NICOM) in infants with all grades of HIE and to determine whether these markers may be helpful in the identification of infants at risk of brain injury. Methods: This prospective observational study was set in a tertiary neonatal unit (November 2017-March 2020). Infants with all grades of HIE had multi-modal monitoring, including EEG, NIRS and NICOM, commenced after delivery and continued for up to 84 hours. All infants had an MRI performed in the first week of life. Healthy term controls were recruited after delivery and had NICOM monitoring at 6 and 24 hours of age. In this thesis, I also included infants recruited previously as part of four historic prospective cohorts that had early EEG monitoring. These infants were combined with infants with mild HIE from the current prospective cohort to examine the difference in EEG features between infants with mild HIE and healthy term controls. Results: Eighty-two infants were recruited in the prospective cohort (30 mild HIE, 25 moderate, 6 severe, 21 controls) and 60 infants were included from the historic cohorts. This study identified significant differences between EEG features of infants with mild HIE and controls in the first 6 hours after birth. Seventy-two percent of infants with mild HIE had some abnormal features on their continuous EEG and quantitative analysis revealed significant differences in spectral shape between the groups. In our cohort, cSO2 increased and FTOE decreased over the first 24 hours in all grades of HIE regardless of TH status. Compared to the moderate group, infants with mild HIE had significantly higher cSO2 at 6 hours (p=0.003), 9 hours (p=0.009) and 12 hours (p=0.032) and lower FTOE at 6 hours (p=0.016) and 9 hours (0.029). Beyond 18 hours, no differences were seen between the groups. NICOM was assessed in infants with HIE and compared with controls. Infants with mild HIE have a significantly higher heart rate at 6 hours of age compared with controls (p=0.034). Infants with moderate HIE undergoing TH have a significantly lower cardiac output compared with mild HIE (p=0.046) and control groups (p=0.040). Heart rate is significantly reduced (p<0.001) but stroke volume is maintained and gradually increases from 6-72 hours despite TH. Finally, we assessed the ability of EEG, NIRS and NICOM to predict short-term outcome (abnormal MRI +/- death in the first week of life). At 6 hours, none of the EEG, NIRS or NICOM measures predicted short-term outcome. At 12 hours of age, both qualitative and quantitative EEG features significantly predicted abnormal short-term outcome. Conclusion: Identification of infants at risk of brain injury immediately after birth is challenging. Objective, early biomarkers are required. This is the first study to combine EEG, NIRS and NICOM in infants with all grades of HIE. Multi-modal monitoring is feasible and this thesis provides novel insights into the underlying physiology and evolution of injury in infants with HIE. Furthermore, it reaffirms the importance of early continuous EEG in HIE.
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    Inflammation driven molecular alterations in disorders of the perinatal brain
    (University College Cork, 2021-03) Casey, Sophie; Murray, Deirdre M.; O'Keeffe, Gerard W.; Boylan, Geraldine B.; Irish Research Council
    Introduction: Inflammatory insults during the perinatal period are known to disrupt normal neurodevelopmental processes. Neonatal hypoxic ischemic encephalopathy (HIE) may occur with or without infection and is characterised by a significant immunoinflammatory response. Infection sensitises the neonatal brain to further hypoxic-ischemic injury. HIE with and without infection affects approximately 3 per thousand of all live births in the developed world. Despite the advent of therapeutic hypothermia (TH), almost half of affected neonates die or are left with lifelong disabilities or disorders. Autism Spectrum Disorder (ASD) is one such disorder and affects approximately 1.5% of the population in the developed world. Epidemiological and animal studies suggest that maternal immune dysregulation may contribute to the development of ASD. Rapid identification of neonates and infants at risk of HIE and ASD is vital, but many miss the critical therapeutic windows due to subjective and suboptimal diagnostic techniques. Circulating inflammation-associated markers such as microRNA (miRNA) and cytokines may hold the key to rapid diagnosis, providing an insight into maternal, foetal and neonatal injury cascades. Blood represents a non-invasive, and rapidly accessible medium in which to examine aberrant expression of these inflammation-driven molecular markers. In addition, miRNAs and cytokines are stably expressed in the blood, so may represent robust biomarker candidates. Furthermore, while many studies of miRNA expression have been performed in clinical cases of HIE, their functional role is still largely unknown. The primary hypothesis of this thesis is that examination of temporal inflammatory profiles may improve our understanding of the pathophysiology of early-life neuronal injury and subsequent development of long-term disabilities, and may provide novel biomarkers, therapeutic targets, or candidates capable of improving long-term outcomes. The current thesis aims to identify early molecular biomarkers of HIE and ASD in clinical and preclinical models and investigate the functional role of altered miRNAs in vitro. To investigate this, we aimed to examine temporal profiles of miRNA and cytokine alterations in a large animal model of HIE, with and without inflammatory sensitisation. Following this, we aimed to identify rapidly detectable miRNA and cytokine biomarkers of HIE, with and without inflammatory sensitisation. Lastly, we aimed to investigate the role of altered miRNA biomarkers through identification and examination of their functional targets, and to examine the mid-gestational cytokine profiles of mothers who give birth to offspring with ASD in a large clinical cohort. Methods: The current thesis utilised a large cohort of HIE neonates recruited to the Validation of Biomarkers in Hypoxic Ischemic Encephalopathy (BiHIVE) 1 & 2 studies (NCT02019147) and a clinically relevant large animal model of HIE with and without inflammation-sensitisation – the neonatal porcine model. To achieve a moderate-severe level of hypoxia-ischemia (HI) in piglets, inspired oxygen was reduced to 4% for approximately 30 minutes and titrated to achieve the desired level of neuronal injury. To achieve inflammation-sensitisation, piglets received an infusion of lipopolysaccharide (LPS) prior to HI. Blood was drawn from piglets at regular intervals for 48-72h following injury. Whole blood was used for analysis of miRNA content, while serum was preferable for analysis of cytokines. The multi-national Screening for Pregnancy Endpoints (SCOPE) cohort was utilised for the examination of circulating midgestational cytokine markers of ASD following diagnosis at neurodevelopmental follow-up. Cohorts from Cork, Ireland and Auckland, New Zealand were examined. Techniques employed throughout the current thesis are state-of-the-art, and were performed by both the PhD candidate and collaborating co-authors. Neurodevelopmental outcomes for neonatal study subjects were measured using Apgar/Sarnat staging, clinical signs (Chapter 3), early intervention services teams and child psychiatrists (Chapter 6). Outcomes for animal subjects were measured using clinical signs and Haemotoxylin & Eosin (H&E)/Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of porcine brain tissues (Chapters 3 and 4). Further techniques featured in Chapter 3 include ribonucleic acid (RNA) Sequencing (RNA-Seq), Haemotoxylin & Eosin (H&E) staining of porcine brain tissues, magnetic resonance spectroscopy (MRS), RNA isolation from whole blood, complementary deoxyribonucleic acid (cDNA) synthesis, quantitative real-time polymerase chain reaction (qRT-PCR), in silico analysis, cell culture of SH-SY5Y cells and primary cultures of embryonic day (E) 14 rat midbrain, in vitro transfection of miRNA inhibitors, immunocytochemistry and fluorescent microscopy. Techniques used throughout the remainder of the thesis were performed solely by the PhD candidate. Techniques employed in Chapters 4 and 6 include electrochemiluminescent Mesoscale Discovery sandwich enzyme-linked immunosorbent (ELISA) immunoassays for analysis of serum cytokines. Finally, Chapter 5 used cell cultures of SH-SY5Y cells, in vitro transfection of miRNA inhibitors, green fluorescent protein (GFP) based reporter assays, immunocytochemistry, and fluorescent microscopy. All statistical analyses were conducted using IBM SPSS Statistics 24/26 and GraphPad Prism 7/8. Graphical representations of data were generated using GraphPad Prism 8. Results: miRNA Dysfunction in the Porcine Model of HIE Eleven candidate miRNAs were determined from a combination of results generated from microarray and RNA-Seq from a clinical cohort of hypoxic-ischemic neonates. These were then examined in the porcine model over 72h. Six miRNAs - miR-128, miR-148a, miR-151a, miR-181a, miR-181b and miR-374a, were upregulated in whole blood 1h after HI. Three - miR-374a, miR-181a and miR-181b, were specifically upregulated in moderate-severely injured piglets. miRNA levels at 1h correlated with histopathological and MRS-measured Lactate/Creatine (Lac/Cr) neuropathological outcomes. Inhibition of miR-181a in vitro resulted in increased neurite growth and increased expression of one member of the target bone morphogenetic protein (BMP) signalling pathway - BMPR2. Cytokine Dysfunction in the Porcine Model of Inflammation-Sensitised HIE Eight candidate cytokines were examined in serum samples from the inflammation-sensitised porcine model over 48h. Interleukin (IL) 6, tumor necrosis factor (TNF) α and Tau displayed a sustained inflammatory response following LPS exposure with and without hypoxia. Neuron-specific enolase (NSE) increased slowly following HI. LPS + Hypoxia-ischemia (LPS-HI) piglets displayed late increases in both NSE and C-reactive protein (CRP). TNFα and IL-6 allowed discrimination between animals who were exposed to inflammation-sensitised HI and those who were exposed to HI alone 6h following a moderate-severe HIE-like insult. miRNA Function In Vitro We previously identified the BMP signalling pathway as a predicted downstream target of miR-374a and miR-181a. The effect of miR-374a and miR-181a manipulation on the BMP-small mothers against decapentaplegic (SMAD) signalling pathway was investigated in vitro. miR-374a inhibition increased expression of BMP2 and BMPR2 but did not alter transcription of SMAD. Likewise, miR-181a inhibition was previously found to increase BMPR2 expression and did not alter SMAD transcription in vitro. Midgestational Cytokine Dysfunction in ASD Eight candidate cytokines were examined in serum samples retrieved at 15 and 20 weeks’ gestation in mothers of children affected by ASD. IL-17A concentrations were downregulated at 20 weeks’ gestation in mothers of children who progressed to develop ASD. IL-16, eotaxin, monocyte chemoattractant protein (MCP) 1, interferon (IFN) γ, IL-1β, IL-6 and IL-8 were unchanged in both groups at both 15 and 20 weeks. Conclusions: The current thesis has identified dysregulated expression of inflammation-driven molecular markers of the perinatal brain disorders HIE and ASD. Circulating levels of miRNAs (miR-128 miR-148a, miR-151a, miR-181a, miR-181b and miR-374a) and cytokines (IL-6, TNFα) were rapidly raised in response to HIE with and without inflammation-sensitisation in the porcine model. IL-17A was also dysregulated at 20 weeks’ gestation in mothers of ASD-affected children. These molecular markers may aid in rapid diagnosis, prognosis, and therapeutic decision-making for these time-sensitive disorders. Moreover, they may allow for discrimination of complex inflammation-sensitised HIE from classic HIE. Furthermore, evidence is beginning to suggest that HIE-associated miRNAs may play a functional role in the regulation of essential BMP-SMAD signalling. The current thesis puts forward novel information regarding the temporal profiles of these circulating molecular markers and begins to explore the functional roles of HIE-associated miRNAs. We hope this work will aid in the development of early blood-based biomarkers of disorders with an inflammatory milieu like HIE and ASD, and pave the way for more functional analysis of these markers.
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    Masks and tubes used to support the neonatal airway – how to improve their fit, seal and correct placement
    (University College Cork, 2019-04-17) O'Shea, Joyce E.; Dempsey, Eugene M.; O'Connell, Liam; Davis, Peter; Thio, Marta; Kamlin, Omar
    Despite the many changes in perinatal medicine in the last fifty years, infants still often and unpredictably need assistance with their breathing. Positive pressure delivered through a facemask remains the almost universal initial approach. This is then generally followed by endotracheal intubation if the infant is not responding or if prolonged support is needed. Despite many years of research into mask ventilation, it is still very challenging and leak and airway obstruction remain a problem. The thesis opens with two mask studies that try to solve this problem. The first is a manikin study that compared three different mask holds. It unfortunately found that there was no difference in the mask leak measured using the different holds. It is perhaps reasonable to change holds if the baby isn’t responding as expected. The second study aimed to measure the dimensions of preterm infants’ faces and compare these with the size of the most commonly available face masks. It found that the smallest size of some brands of mask is too large for many preterm infants. Masks of 35mm diameter are suitable for infants <29 weeks PMA or 1000g. Masks of 42 mm diameter are suitable for infants 27-33 weeks PMA or 750-2500g. The thesis then changed focus to neonatal intubation. Intubation is a challenging skill for paediatric trainees to master. In recent years success rates are decreasing. The next studies look at possible ways to change this trend. The first is a Cochrane review that examined if a stylet could improve intubation success. Only one unblended RCT has been performed and found no difference. The most sizable work of the thesis follows and is a RCT that examines if junior trainees intubation success rates are superior if they intubate with a videolaryngoscope. Two hundred and six intubations were randomised to the screen being visible to the supervisor or covered. The success rate when the instructor was able to view the videolaryngoscope screen was 66% (69/104) compared to 41% (42/102) when the screen was covered, (p<0.001), OR 2.81 (95%CI 1.54-5.17). This shows that videolaryngoscopy is a promising tool to help inexperienced trainees become proficient intubators. This study has resulted in videolaryngoscopy becoming a tool commonly used in neonatal intensive cares. The next study looks at recordings of unsuccessful intubations from the RCT. If an attempt is unsuccessful, the intubator and instructor often cannot explain why making it difficult to know what to do differently in the future. The study found that lack of intubation success was most commonly due to failure to recognize midline anatomical structures. Excessive secretions are rarely a factor in elective premeditated and routine suctioning should be discouraged. Better videolaryngoscope blade design may make it easier to direct the tube through the vocal cords. The final work of the thesis is a review that examines devices used during newborn stabilization. Evidence for their use to optimize the thermal, respiratory and cardiovascular management in the delivery room is presented. After completing all this work I think that perhaps it is time to lessen our reliance on facemasks and embrace other airway devices that are showing promise, particularly the laryngeal mask. I feel that universal intubation competency is no longer feasible but universal competency on the use of laryngeal masks probably is. This urgently needs to be addressed in paediatric training programs. Videolaryngoscopy is a promising tool that improves junior intubators’ success rates. To master intubation many intubations are still necessary but the videolaryngoscope allows the slope of the learning curve to steepen. Development is necessary to design scopes of the future that are inexpensive, easily portable and user friendly.
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    Ability of early neurological assessment and continuous EEG to predict long term neurodevelopmental outcome at 5 years in infants following hypoxic-ischaemic encephalopathy
    (University College Cork, 2018) O'Connor, Catherine M.; Murray, Deirdre M.; Boylan, Geraldine B.; Health Research Board
    Hypoxic-ischaemic encephalopathy (HIE) symptoms evolve during the first days of life and their monitoring is critical for treatment decisions and long-term outcome predictions. This thesis aims to report the five-year outcome of a HIE cohort born in the pre-therapeutic hypothermia era and to evaluate the predictive value of (a) neonatal neurological and EEG markers and (b) development in the first 24 months, for outcome. Methods: Participants were recruited at age five from two birth cohorts; HIE and Comparison. Repeated neonatal neurological assessments using the Amiel-TisonNeurological-Assessment-at-Term, continuous video EEG monitoring in the first 72 hours, and Sarnat grading at 24 hours were recorded. EEG severity grades were assigned at 6, 12 and 24 hours. Development was assessed in the HIE cohort at 6, 12 and 24 months using the Griffiths Mental Development (0-2) Revised Scales. At age five, intellectual (WPPSI-IIIUK scale), neuropsychological (NEPSY-II scales), neurological and ophthalmic testing was completed. Results: 5-year outcomes were available for 81.5% (n=53) of HIE and 71.4% (n=30) of Comparison cohorts. In HIE, 47.2% (27% mild, 47% moderate, 83% severe Sarnat), had non-intact outcome vs. 3.3% of the Comparison cohort. Non-intact outcome rates by 6-hour EEG-grade were: grade0=3%, grade1=25%, grade2=54%, grade3/4=79%. In HIE, processing speed (p=0.01) and verbal short-term memory (p=0.005) were below test norms. No significant differences were found in IQ, NEPSY-II or ocular biometry scores between children following mild and moderate HIE. Median IQ scores for mild (99(94-112),p=grade 2) at 24hours had superior positive predictive value (74%; AUROC(95%CI)=0.70(0.55-0.85) for non-intact 5-year outcome than abnormal EEG at 6 hours (68%; AUROC(95%CI)=0.71(0.56-0.87). Within-child development scores were inconsistent across the first 24 months. Although all children with intact 24-month Griffiths quotient (n=30) had intact 5-year IQ, 8/30 had non-intact overall outcome. Conclusion: Predictive value of neonatal neurological assessments and an EEG grading system for outcome was confirmed. Intact early childhood outcomes post-HIE may mask subtle adverse neuropsychological sequelae into the school years. This thesis supports emerging evidence that mild-grade HIE is not a benign condition and its inclusion in studies of neuroprotective treatments for HIE is warranted.