Identification of Gestation-Specific Patterns of Physiological, Protein and Cell-Free RNA Injury Markers in a Sheep Model of Regulable Preterm Fetal Hypoxia.

Fetal hypoxia remains a major unresolved clinical challenge. Using artificial placenta (AP) technology, we established a standardized model of progressive hypoxia that induces brain injury in fetal sheep. To support the development of new antenatal monitoring strategies, we further profiled gestation-specific physiological, biochemical, protein, and cfRNA responses during progressive hypoxia. Seven fetuses at 123 days gestational age (dGA) and eight fetuses at 98 dGA (term = 150 dGA) were supported on the AP. Following a 12-h stabilization period, during which FiO₂ and gas flow to the AP oxygenator were adjusted to maintain fetal SO₂ at 65-75% and pCO₂ at 35-45 mmHg, both parameters were reduced by 5% every 30 min until termination criteria were met (arterial pH < 7.0 or base excess < -12). This protocol produced 240-300 min of progressive hypoxia. Physiological parameters, blood gases, biochemical markers, and cardiac and vascular ultrasound were assessed hourly. At the end of the experiment, brain histology and plasma cfRNA profiling were performed. Progressive hypoxia induced significant increases in pCO₂ and lactate and decreases in SO₂, CtO₂, pH, and BE in both groups (p < 0.05). Gestation-specific differences were observed in pO₂, glucose, and vital signs (heart rate, mean arterial pressure, and circuit flow). AST, ALT, and NSE rose significantly only at 123 dGA (p < 0.05), whereas S100B and NT-proBNP increased in both groups. Histology confirmed hypoxic brain injury at both gestational ages. In 123 dGA fetuses, iliac artery pulsatility index and heart rate predicted dysregulation of pH, BE, SO₂, and lactate (R