In Vitro Evaluation of the Impact of Oxygen Concentrations on Thrombomodulin Expression in a First-Trimester Trophoblast Cell Line

Introduction: The placenta is a transient organ essential for fetal development. Abnormal placental development can lead to obstetric syndromes, such as preeclampsia (PE) and fetal growth restriction (FGR). Physiological hypoxia activates intracellular pathways via hypoxia-inducible transcription factors (HIFs), which are crucial for placentation. This study uses an in vitro model to investigate the role of oxygen concentration in the expression of thrombomodulin (THBD), a protein involved in placental hemostasis. Methods: Swan 71, a first-trimester trophoblast cell line, was cultured under normoxic (21 % O2) and physiological hypoxic (2 and 8 % O2) conditions. Hypoxia/reoxygenation assays were also performed. To assess potential modulation of THBD expression by HIF-1α, cobalt chloride (CoCl₂) was used to stabilize HIF-1α, while zinc chloride (ZnCl₂) inhibited its expression. RT-qPCR and Western blot analyses were performed to quantify THBD and KLF4 expression. Results: Significant increases in THBD mRNA and protein levels were observed under 2 % O2conditions (p = 0.03). CoCl2treatment further enhanced THBD and KLF4 expression (p < 0.01 in both experiments), while ZnCl2inhibited these effects (p < 0.01). This suggests a key role for HIF-1α in THBD regulation. However, in silico analysis did not identify any direct hypoxia response elements within the human THBD gene, suggesting the presence of an indirect regulatory mechanism potentially involving KLF4. Discussion: These results suggest that physiological hypoxia positively regulates THBD expression through HIF-1α stabilization and KLF4-mediation. These findings provide insight into the molecular mechanisms of placental adaptation to hypoxia and are relevant for understanding pregnancy complications like PE and intrauterine growth restriction (IUGR).