Protocol: A multi-factorial, multi-centre study, for biomarker identification in healthy controls for comparison to...

Neonatal encephalopathy suspected to be hypoxic-ischaemic encephalopathy (NESHIE) remains a leading cause of neonatal mortality and long-term neurodevelopmental impairment, particularly in low- and middle-income countries. While therapeutic hypothermia reduces mortality in moderate to severe cases, a significant proportion of affected infants continue to experience adverse neurological outcomes. This multi-centre observational study aims to elucidate the clinical and biological mechanisms underlying NESHIE by conducting a comprehensive comparative analysis of neonates with moderate to severe NESHIE and healthy term controls. Participants with NESHIE were previously recruited under an existing approved protocol (University of Pretoria ethics reference: 481/2017), and healthy neonates will be newly enrolled. The study will integrate clinical and molecular data to: (1) identify clinical risk factors associated with NESHIE; (2) perform whole genome sequencing to detect relevant genetic variants; (3) analyse DNA methylation patterns via bisulfite sequencing; (4) assess gene expression using bulk and single-cell RNA sequencing (RNA-seq); (5) characterise proteomic and metabolomic profiles through liquid chromatography-mass spectrometry of dried blood spot samples; (6) examine the placental microbiome; and (7) evaluate placental histopathological differences between groups. By offering a multi-dimensional view of the molecular and microbial landscape of NESHIE in a South African cohort, this study aims to enhance understanding of the disease pathogenesis. Ultimately, the findings may support the development of biomarkers for early diagnosis, improve risk stratification, and guide novel therapeutic strategies for affected neonates. The study has received National Health Research Database (NHRD) registration under GP_202411_053 (Gauteng) and WC_202411_026 (Western Cape), with ethics approvals granted by the University of Pretoria (184/2024), University of the Witwatersrand (250406B), and Stellenbosch University (N24/12/154_RECIP_UP184/2024) as well as their respective tertiary academic hospitals.