Defining a hypoxic exposure paradigm for modelling preeclampsia-associated syncytiotrophoblast dysfunction in human blastocyst-derived placental stem cells

Introduction: Preeclampsia (PE) is a hypertensive pregnancy disorder in which placental dysfunction is thought to play a central role. Although hypoxia is widely used to model PE-associated syncytiotrophoblast (ST) dysfunction in vitro, considerable variability in oxygen control conditions and hypoxic exposure paradigms has limited standardization across studies. This study aimed to establish physiologically relevant oxygen control conditions and identify a hypoxic exposure paradigm that consistently recapitulates characteristic features of PE-associated ST dysfunction. Methods: Human blastocyst-derived trophoblast stem cells were differentiated into STs and exposed to physiological (8% O2), atmospheric (21% O2), or hypoxic (1% O2) conditions for 24 or 48 h. Angiogenic and apoptotic marker expression, cell death, and mitochondrial stress were assessed. Results: Compared with atmospheric oxygen, 8% O2 reduced cell death and more closely reflected physiological ST conditions. Acute hypoxia (24 h) induced a robust PE-like phenotype in STs characterized by induction of anti-angiogenic markers, sFlt-1 and sEng, reduced expression of the pro-angiogenic factor PlGF, and increased cell death, apoptotic signalling and mitochondrial stress. Notably, induction of several of these markers was attenuated or reversed following prolonged hypoxic exposure. Conclusions: These findings identify 8% O2 as a physiologically relevant control condition for modelling PE-associated ST dysfunction relative to 21% O2, and demonstrate that 24 h of severe hypoxia more consistently reproduces key molecular and cellular features of PE than prolonged hypoxia. This work establishes a framework for modelling hypoxia-induced ST dysfunction while highlighting the importance of oxygen control conditions and hypoxic exposure duration when modeling PE in vitro.