INFANT Research Centre - Doctoral Theses
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Item SErENdipITy - study of electrophysiological biomarkers (electroencephalogram and heart rate variability) in neonatal seIzures and encephalopathy(University College Cork, 2023) Pavel, Andreea; Boylan, Geraldine B.; Murray, Deirdre M.; Dempsey, Eugene M.; Health Research Board; Wellcome Trust; Science Foundation IrelandBackground In recent years, the use of conventional electroencephalographic (EEG) monitoring in newborns has increased and it is now recognised worldwide to be the gold standard monitoring for neonatal seizure detection, as well as a reliable biomarker for brain injury and long term prognosis in this population. Similarly, heart rate variability (HRV) is a non-invasive monitoring which provides information regarding the autonomic nervous system function, and has also been explored as a biomarker for brain function and prognosis in newborns. These physiological monitoring have been proven useful, especially in newborns with encephalopathy following hypoxic ischaemic injury. Despite the increased use of therapeutic hypothermia (TH) and the decrease in adverse outcomes following hypoxic ischaemic encephalopathy (HIE), the incidence of HIE is 1.6 per 1000 live births in high-income countries. Early identification of newborns with HIE which might benefit from TH is still challenging in clinical practice. HIE is also the main cause of seizures in newborns. Due to the unique physiological properties of the neonatal brain, this is the age period with the highest risk of seizures, with an incidence of 1-5/1000 live births. Recent evidence showed that seizures themselves might add to the degree of brain injury regardless of the background pathology, and a high seizure burden was associated with worse long-term neurodevelopmental outcome. The majority of neonatal seizures are subclinical (electrographic only) or have very subtle clinical manifestations, thus the importance of early biomarkers to identify newborns which will develop seizures, as well as the importance of prolonged EEG monitoring for seizure detection. Aims The aims of this thesis were to explore the value of physiological biomarkers, such as early clinical parameters, EEG and HRV features for early prediction of neonatal HIE severity and seizures, as well as neonatal seizure detection and evolution. Using early EEG and HRV analysis I aim to develop prediction models for seizure occurrence and HIE severity. In addition, I plan to assess the clinical value of a neonatal seizure detection algorithm (ANSeR algorithm). By analysing newborns with HIE and electrographic seizures, I aim to describe the temporal evolution of seizures during TH and to evaluate if seizure burden and intensity (power) correlates with HIE severity and long-term outcome. Furthermore, I aim to assess if the time to treatment of the first electrographic seizure in newborns with HIE had an impact on subsequent seizure burden and outcome. Methods To achieve my goals, I have analysed infants recruited from two European multicentre cohort studies, across eight European tertiary neonatal intensive care units, between January 2011 and February 2017 (ClinicalTrials.gov Identifier: NCT02160171 and NCT02431780). The studies included infants born at 36 to 44 weeks corrected gestational age, requiring prolonged EEG monitoring for being at high risk of developing seizures or being suspected of having seizures. All infants had continuous EEG monitoring using disposable electrodes according to the 10:20 electrode placement system for neonates (F3, F4, C3, C4, Cz, T3, T4, O1/P3 and O2/P4), with simultaneous electrocardiography (ECG) monitoring. The clinical diagnosis of HIE was established by the teams at each recruiting site based on signs of perinatal asphyxia and encephalopathy on neurological examination (modified Sarnat score within 24 hours of age) and retrospectively corroborated with abnormalities suggestive of HIE on EEG and brain MRI. The clinical grade of HIE was based on the most severe score of modified Sarnat score. The encephalographic grade of HIE was established by visually analysing the EEG background using a system described previously by our group: 0-normal EEG background, 1-mild abnormalities, 2-moderate abnormalities, 3-major abnormalities and 4-inactive EEG background. The quantitative EEG analysis was performed using the NEURAL software package, extracting a set of features for power, discontinuity, spectral distribution and inter-hemispheric connectivity. The HRV analysis was performed using an in-house software (HRV Analysis, Beta Version 1.12, ©University College Cork 2008-2012) which automatically identified R-peaks on ECG recording. The RR interval was generated as the time difference between each R peak. HRV was expressed in time, frequency and complexity features. Electrographic seizures were defined as at least one EEG channel with sudden, repetitive and evolving waveforms for minimum 10 seconds. For all infants, electrographic seizures were identified by two neurophysiology experts in neonatal EEG. Seizure characteristics and seizure quantification were calculated based on these expert annotations. Results The two studies included 504 newborns, out of which 266 newborns had a diagnosis of HIE (3 newborns with HIE following postnatal collapse). Machine learning models were developed for early prediction of newborns with seizures in HIE, by analysing a cohort of 164 newborns with EEG monitoring before 12 hours of age. The best predictive models included both clinical parameters and EEG features: clinical and qualitative-EEG model (MCC (95% CI) 0.470 (0.336 to 0.602), AUC (95% CI) 0.721 (0.681 to 0.813)) and clinical and quantitative-EEG model (MCC (95% CI) 0.513 (0.376 to 0.645), AUC (95% CI) 0.746 (0.700 to 0.833)). A randomised controlled trial evaluating the real-time performance of a seizure detection algorithm (ANSeR algorithm) showed a higher detection of seizure hours in the algorithm group compared to the non-algorithm group (absolute difference (95% CI): 20.8% (3.6% to 37.1%)). Seizure hours detection between the two groups was even greater at weekends (Saturday-Sunday vs Monday-Friday), difference (95% CI): 16.6% (0.1% to 32.3%). Another study assessed the impact of the time to treatment of the first electrographic seizure on subsequent seizure burden showed significantly lower seizure burden and less seizures were noted in infants treated with anti-seizure medication (ASM) within 1 hour from seizure onset (p value=0.029 and 0.035, respectively). A similar trend was noted in the subgroup of infants who had a diagnosis of HIE (n=42). Analysis of newborns with HIE requiring TH with EEG monitoring throughout the rewarming phase, showed that newborns with seizures during active cooling and rewarming had a significantly higher seizure burden compared with newborns with seizures during active cooling exclusively (median (IQR) 167(54-275) vs 69(22-104) minutes, p=0.003). Seizure burden peaked at approximately 24 hours in both study groups and had a secondary seizure peak at 85 hours of age for the group of newborns with seizures during active cooling and rewarming. Newborns with seizures during active cooling and rewarming had a significantly higher risk of abnormal outcome compared to infants without seizures (OR(95% CI):4.62(1.40 to 15.24), p=0.012). In another study all electrographic seizures from 64 newborns with HIE and 2-year neurodevelopmental outcome were analysed. Infants in the severe HIE group had a higher seizure period, with more frequent seizures but less intense (lower mean seizure power), compared to infants in the moderate HIE group. Similar characteristics were associated with abnormal outcome at two years. Early HRV analysis was assessed to predict the EEG grade in neonatal HIE within the first 12 hours of life in 120 newborns. Performance of the HRV model was AUROC 0.837 (95% CI: 0.759-0.914), however performance of the HRV and clinical model combined had an AUROC of 0.895 (95% CI: 0.832-0.958). Conclusion Early qualitative and quantitative-EEG features alone and in combination with early clinical information can reliably predict infants that will later develop seizures in HIE, hours before seizure onset. The quantitative-EEG model proved as reliable as the analysis of a neonatal neurophysiologist expert (qualitative-EEG analysis) in predicting the likelihood of seizures, which could be also used to individualise the neurophysiology review frequency of the continuous EEG monitoring. The findings of the neonatal seizure detection algorithm trial validated in real time the performance of the ANSeR seizure detection algorithm by demonstrating its’ usefulness as a support tool for clinicians, especially during weekends when a limited number of health care professionals are available on site. The study of ASM timing for the first electrographic seizure showed that inappropriate treatment remains a concern in clinical practice, and that early anti-seizure treatment was associate with lower total seizure burden. Current findings suggest that treatment of neonatal seizures might be time-critical. The study of seizure evolution in HIE showed that one third of infants with HIE undergoing TH continued to have seizures after the completion of active cooling, increasing the overall seizure burden which might have an impact on long-term outcomes. Supporting the current guidelines recommendations, there is a clear need for continuous EEG monitoring during active cooling, rewarming and beyond when seizures persist. The seizure analysis in HIE showed that seizures were more frequent and were less intense in severe HIE compared to moderate HIE, and in newborns with adverse outcome compared to newborns with normal outcome at two years. This may have implications for seizure identification as low power seizures are usually harder to detect, especially using aEEG monitoring, which might have an impact on anti-seizure treatment and subsequently on long-term outcome. The study of early HRV analysis showed that HRV and clinical model had a good prediction of encephalopathy HIE severity in the early newborn period and may be a very useful additional tool for neonatologists who are often faced with challenging decisions about TH, especially where EEG monitoring is not available or feasible.Item Sleep and developmental outcome of the moderate to late preterm infant(University College Cork, 2023) Ryan, Mary Anne; Boylan, Geraldine B.; Mathieson, Sean; Dempsey, Eugene M.Background Sleep is the primary activity during early brain development and an essential part of healthy cognitive, physical and psychosocial development. Electroencephalography (EEG) provides detailed information about brainwave activity during sleep which changes in different sleep states and advancing gestational age (GA). The moderate to late preterm (MLP) infant is defined as an infant born between 32-36 +6 weeks GA. MLP’s are under-researched in terms of developmental outcome. We hypothesise that: • The sleep architecture of healthy MLP infants at 36 weeks may differ according to birth GA, birth weight, sex, mode of feeding or location (cot/incubator) at time of monitoring. • The developmental outcome of healthy MLP infants may be different to that of a term control group at 4 months and 18 months PMA. Aims • To describe the sleep architecture of healthy MLP infants in the neonatal unit at 36 weeks and the frequency of sleep interruptions using continuous EEG monitoring with video. • To describe parameters for the main EEG feature of quiet sleep i.e. inter-burst intervals (IBI) of MLP group with a normal developmental outcome at 18 months • To compare neurodevelopmental outcome of the MLP infant group to that of a term control (TC) infant group at 4 months and 18 months. Methods MLP infants recruited in the neonatal unit had overnight continuous EEG monitoring (12 hours) with video at 36 weeks post menstrual age (PMA). Post-acquisition, sleep states and sleep interruptions were annotated and quantified based on visual analysis of EEG, behavioural observation and cardiorespiratory parameters. Using an inter-burst interval (IBI) detection algorithm five IBI features of QS were extracted from MLP infants with a normal developmental outcome at 18 months. Outcome of MLP infant group was compared to a healthy term control (TC) group based on scores achieved in the Griffiths lll mental development scales at 4 and 18 months PMA. In comparing outcome of MLP and TC infant groups, the Mann–Whitney U test was used for continuous variables and the Chi-squared test or Fisher’s exact test was used for categorical variables. A p-value <0.05 is considered to be statistically significant. Cohens d (the standardized mean difference between the MLP and TC groups) was used as the measure of effect size. Results Ninety-eight infants had overnight EEG’s included in this study. In the neonatal unit 23.3% of sleep cycles were interrupted primarily for feeding. The total overnight sleep time (TST) was 7.09(6.61-7.76) hrs including 4.58(3.69-5.09) hrs in active sleep (AS), 2.02(1.76-2.36) hrs in quiet sleep (QS) and 0.65(0.48-0.89) hrs in indeterminate sleep (IS). The total duration of AS was significantly lower in infants born at lower GA (p= 0.007) whilst the duration of individual QS periods was significantly higher (p=0.001).Sixty infants had a normal outcome at 18 months and were included in the QS analysis study using the IBI detection algorithm. Normative data for five IBI features was extracted from QS. All IBI features were significantly longer for infants cared for in incubators although these infants were chronologically younger ( p<0.001). When neurodevelopmental outcome of the MLP and TC groups were compared at 4 and 18 months PMA, the MLP infant group achieved lower scores than in overall general development. The greatest differences were in the area of gross motor development (p <0.001, with a Cohen’s d effect size of -0.665), eye-hand coordination (p<0.001, with a Cohen’s d effect size of -0.648). Using the reported Griffith’s cut off of < 90 for delayed development, 7% (5/75) of the MLP group had delayed development at 18mths compared to 2% (2/92) of the TC group. Conclusion EEG provides an objective insight into sleep organisation and may be considered a biomarker of brain development. This thesis provides detailed analysis of the sleep EEG of a cohort of healthy MLP infant at 36 weeks PMA and provides useful reference criteria for studies that may assess brain maturation in the future, particularly for those infants in neonatal intensive care units.Item 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 CentreAutism 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 (https://www.sfari.org/resource/sfari-gene/). 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.Item A trilogy of stressors in the neonatal intensive care unit: towards therapy for preterm adversity(University College Cork, 2023) Dias Casacao, Maria Luis; McDonald, Fiona; Carolyn Sifton Foundation; Science Foundation IrelandPremature infants are born with immature breathing network and an innate immune system that responds differently to that of infants born full-term. All infants born less than 28 weeks of gestation develop apnoea of prematurity-related symptoms, which decreases to about 20% of infants born at 34 weeks of gestation and to less than 10% of those infants born beyond 34 weeks of gestation. Premature infants are not only at risk of developing breathing disorders but also at increased risk of infection due to early life vulnerability (the earlier a baby is born, the more likely is to have health problems due to immature organ development). Gram-positive (GP) infections are the most common type of late onset infection in preterm infants. Activation of specific toll-like receptors (TLRs) is reported to modulate cardiorespiratory responses during infection and may play a key role in driving homeostatic instability observed during sepsis. Firstly, we sought to characterise the expression of TLRs in the brainstem, adrenal gland and in the diaphragm respiratory muscle in naïve rats during our developmental period of interest (postnatal day (PND) 3 and PND13). These studies demonstrated mRNA expression of these receptors at PND3. TLR expression fluctuated in early life depending on the subtype and tissue examined with a relative decrease in some of the mRNA expression at PND13; TLR1, 2, 4, 6, 9 and NOD2 in sternohyoid muscle, TLR1, 2, 4 and 6 in diaphragm muscle, TLR2 in adrenal glands and NOD2 in brainstem and spinal cord, but a relative increase in mRNA expression of CX3CR1 in brain and brainstem, TLR2 and 9, IL1R1, in brain and TLR2 and TLR9 in spinal cord. Sex differences were revealed in mRNA expression of TLR9 in brain and NOD2 and IL1R1 in brainstem with upregulation of expression in males. These results relating TLRs and postnatal development suggest a developmental regulation of the immune system. Given these results, we reasoned that oxygen dysregulation coupled with GP bacterial immune stimulation would modulate redox sensitive genes and TLR expression that could alter hormonal expression and impinge on respiratory function in a sex-specific manner. To test this, we developed a novel neonatal rat model in which male and female neonatal rats were exposed to intermittent hypoxia, normoxia and hyperoxia from PND3 for 10 days, followed by combined administration of GP bacterial proteins lipoteichoic acid (LTA) and peptidoglycan (PGN). This model sought to mimic physiological challenges encountered by infants born preterm. Hypoxia challenges during the intermittent hypoxia and hyperoxia (IHH) protocol, induced a significant peripheral oxygen desaturation in treated animals. LTA and PGN (3mg and 5mg, respectively) evoked a significant immune response in PND13 rat pups when measured 3 hours post administration. Serum cytokine analysis revealed LTA&PGN triggered an increase in CCL2, IL-1α, IL-1ß, IL-5, G-CSF, IL-13, CCL3, Gro/KC, CXCL10, CX3CL1, CXCL2, IL-10, IFNy, leptin, VEGF, IL-17A and TNF-α serum concentration compared to vehicle. Interestingly, IL-1ß, Gro/KC, IL-10 and leptin expression were upregulated with combined IHH and LTA&PGN exposure. Respiratory function demonstrated an overall decrease in breathing frequency that was mainly impacted by LTA&PGN administration due to an increase in expiratory time. A decrease in minute ventilation was reported with LTA&PGN however, regarding the metabolic function, the ventilatory equivalent for carbon dioxide was similar across the groups, which is consistent with normal pH levels obtained. Additionally, a mild response to the GP challenge in the late periods of hypoxia were associated with decreased number of gasps with IHH. We analysed mRNA expression of TLRs and redox modulated genes using real-time polymerase chain reaction (RT-PCR) in brainstem, diaphragm muscle and adrenal glands. Brainstem gene expression was similar across groups. In adrenal glands tissue, there was an overall upregulation of TLRs mRNA expression with IHH exposure, except for TLR6. Moreover, TLR2 mRNA expression was upregulated with IHH compared to Sham groups, in males compared to females and in LTA&PGN compared to vehicle. An inverse trend from that of adrenal glands was reported in diaphragm muscle. We also analysed redox modulated proteins in serum using bioassays to detect 8-OHdG, 8-iso-PGF2α, AOPP and SOD in plasma. No differences were observed with IHH and LTA&PGN. However, sex differences were found in 8-iso-PGF2α and AOPP redox proteins, with upregulated expression in males compared to females. Finally, we sought to characterise the postnatal development in our animal model using a battery of developmental and motor assessments. These studies demonstrated a delay in pinnae detachment in IHH pups, a decrease in time to righting with IHH and an increase in motor and locomotion faults in IHH females. Tactile stimulation was decrease with IHH suggesting a delay in brainstem reflexes. IHH treated males presented with an increased expression of stress and anxiety-related behaviours illustrated by increased time spent in the corners of the open-field test with LTA&PGN and distance travelled with IHH, and decreased time spent in open arms in the elevated plus maze experiment in males compared to females. Altogether, these results suggest that early life stress can profoundly impact the expression of TLRs and redox genes in adrenal glands, impact the expression of cytokines such as leptin and alter development, motor coordination, stress levels in this novel neonatal rat model of early life stress. In conclusion, these studies specifically target the gaps in knowledge of the pathophysiological alterations experienced in prematurely born babies who present with impaired breathing function and contract GP infection as mimicked in this novel animal model. The results presented disclose novel insights on the physiology and hormonal alterations that these babies could face in similar conditions, with potential to positively contribute to the field of study by enlightening future targets of research. Future directions relying on the spatial characterisation of TLRs and leptin receptors and possible long-term influences on behavioural performance would also be helpful to better characterise the model.Item Axis of placental ageing in adverse pregnancy outcomes(University College Cork, 2023) Manna, Samprikta; McCarthy, Fergus; McCarthy, Cathal; European Chiropractors' UnionBackground: Pre-eclampsia (PE), an adverse pregnancy outcome affects 2-5% pregnancies worldwide and significantly adversely impacts both maternal and fetal outcomes. Intrauterine growth restriction (IUGR) is defined as the inability of the fetus to reach normal growth potential within the uterus as a result of various genetic, environmental, or placental factors. Premature ageing of the placenta in pregnancy outcomes such as PE and IUGR is associated with the persistent presence of oxidative stress and placental insufficiency reducing its functional capacity. Placental proteomics has been instrumental in improving our understanding of molecular mechanisms involved in the pathophysiology of placental insufficiency as well as identifying biomarkers to predict and diagnose pregnancy outcomes. In this study, we investigated cellular senescence phenotypes of PE and IUGR pregnancies by simultaneously measuring several biomarkers of senescence, as well as the proteomic signature of the placenta in healthy and adverse pregnancy outcomes PE and IUGR. Method: Maternal plasma and placental samples were collected at term (>37 weeks) and preterm (<37 weeks) gestation from nulliparous women undergoing prelabour elective Caesarean section with PE without intrauterine growth restriction (PE; n=5), PE associated with intrauterine growth restriction (n=8), intrauterine growth restriction (IUGR <10th centile) (n=6) and age-matched controls (n=20) from Cork University Maternity Hospital, Cork, Ireland. To assess cellular senescence absolute telomere length (aTL) and senescence associated genes in the placentas was performed by RTqPCR. Cyclin-dependent kinase inhibitors (p21 and p16) expression were determined by Western blotting. Senescence Associated Secretory Phenotype (SASP) were evaluated in maternal plasma by multiplex ELISA assay. Proteomic analysis of placental samples dissected into 3 sub-anatomical regions (maternal, middle, fetal) taken from 3 nulliparous healthy placentas was performed by mass-spectrometry and pathway analysis was conducted. Based on the differentially expressed proteins (DEPs), a placenta specific disease map using NaviCenta focusing on functional analysis to include the placenta specific context for healthy (n=4) compared to PE affected (n=4) and IUGR affected (n=4) placentas. Results: Placental expression of senescence associated genes CHEK1, PCNA, PTEN, CDKN2A, CCNB-1 was significantly upregulated in PE, while TBX-2, PCNA, ATM and CCNB-1 expression were significantly decreased in IUGR compared to controls. Moreover, placental p16 protein expression was significantly decreased in PE only when compared to controls placentas. We also observed that IL-6 was significantly increased in maternal circulation in PE when compared to controls; while IFN-γ was significantly increased in maternal circulation in women affected with IUGR when compared to controls. Proteomic profiling of healthy placentas divided into three sub-anatomical regions identified 1081, 1086, and 1101 proteins in maternal, middle, and fetal sub-anatomical regions respectively. Depending on sample site location and sub-anatomical regions, 374 differentially expressed proteins (DEP) were identified. When we investigated the proteomic variations between PE and IUGR placentas when compared to controls we observed 314, 391, and 378 proteins in healthy control, PE, and IUGR placenta, respectively. We performed functional analysis by combining ClusterCompare and NaviCenta to analyse a placenta-centric context, and observed regulatory elements predominantly involved in the immune regulation, complement cascade and antioxidant activities in PE and IUGR compared to control placentas. Conclusion: This thesis provides evidence of premature senescence in IUGR, while in PE, evidence of activated cell cycle checkpoint regulators is suggestive of cellular repair and proliferation rather than progression to cellular senescence. The heterogeneity within senescence molecular markers of these phenotypes highlights the complexity and disparity between pathophysiological insults unique to each obstetric complication. Proteomic profiling of sub-anatomical placental regions highlighted the variabilities between regions particularly providing evidence of senescence in these regions. Placental proteomic mapping of healthy placentas compared to adverse pregnancy outcomes PE and IUGR revealed the importance of complement system, inflammatory response, and antioxidant activity in placental function in PE placentas. The identification of novel targets such as transcription factor activity and synergistic miRNAs elements within the core regulatory network, might enlighten future placental research within adverse pregnancy outcomes.