Supplementary Material for “Protocol: A multi-factorial, multi-centre study, for biomarker identification in healthy controls for comparison to babies with moderate-severe NESHIE”

Abstract

Neonatal encephalopathy suspected to be hypoxic–ischaemic encephalopathy (NESHIE) continues to be a significant cause of neonatal death and long-term neurodevelopmental impairment, particularly in resource-limited settings. Although therapeutic hypothermia has improved survival in moderate to severe cases, adverse neurological outcomes remain common.This multi-centre observational study seeks to investigate the underlying clinical and biological mechanisms of NESHIE through a comparative analysis of affected neonates and healthy term infants. Previously recruited NESHIE participants will be included alongside newly enrolled controls. The study combines detailed clinical data with a broad range of molecular approaches, including whole genome sequencing, DNA methylation profiling, bulk and single-cell transcriptomics, proteomic and metabolomic analyses, and placental microbiome characterisation. In addition, placental histopathology and clinical risk factors will be examined.Through this integrated, multi-dimensional approach in a South African population, the study aims to advance understanding of NESHIE pathogenesis and contribute to the development of improved diagnostic tools, risk assessment strategies, and targeted interventions.