Study protocol for the Bio-HEAT study: Investigating the Biological pathways from HEAT exposure to preterm birth and other adverse maternal and child health outcomes in South Africa.

Introduction: Epidemiological evidence linking heat exposure to adverse maternal and child health outcomes is compelling. However, the biological and social mechanisms underlying these associations remain poorly understood. Understanding the pathways explaining these associations is important given rising global temperatures, and the urgent need for developing and testing adaptive interventions. Methods: This transdisciplinary study in Johannesburg, South Africa, will monitor a cohort of 200 women from their second trimester until one-year postpartum, alongside their infants. Heat exposure and environmental factors will be tracked using personal, community and facility-level temperature monitors and geospatial data. Data will be collected on social conditions, medical and obstetric history, heat stress and adaptation, hydration, mental wellbeing, and sleep quality. Clinical data includes physical measurements, ultrasound, cardiotocography, and biological specimens (blood, urine, saliva) analysed for inflammatory markers, RNA, metabolic indicators, renal function and hormonal levels. Placental and cord blood analyses will assess foetal stress. Infant data will include medical history, hospital visits, neurodevelopment, anthropometric measurements, vital signs, and urine analysis. Three nested sub-studies (20-50 participants) will explore specific aspects: Sub-study 1 will use wearable devices to monitor sleep, activity, and heart rate in high-risk women; Sub-study 2 will involve qualitative interviews; and Sub-study 3 will assess breastmilk composition and volume. Planned analyses: Our primary aim is to document linkages between heat exposure and inflammatory pathways that precede preterm birth. The hypothesis that heat exposure triggers maternal inflammation will be tested by analysing epigenetic changes associated with inflammatory cytokine protein and gene expression. We will investigate thermoregulation and hydration during labour. Using isotope techniques, we assess whether heat exposure alters breastmilk composition and volume. Conceptual frameworks and graphical causal models will be developed to delineate pathways of vulnerability and protective mechanisms.