Characterization of Trophoblast Mitochondrial Function and Responses to Testosterone Treatment in ACH-3P Cells

Introduction: Trophoblast mitochondria play important roles in placental energy metabolism, physiology and pathophysiology. Hyperandrogenism has been associated with mitochondrial abnormalities in pregnancy disorders such as pre-eclampsia, gestational diabetes, and intrauterine growth restriction, but the direct impacts of androgen exposure on placental mitochondrial function are unknown. Given the inherent limitations of studying the human placenta during pregnancy, trophoblast cell lines are routinely used to model placental biology in vitro. The aim of this study was to characterize mitochondrial respiratory function in four commonly used trophoblast cell lines to provide a basis for selecting one well-suited to investigating the impact of androgens on trophoblast mitochondrial function.

Methods: Androgen receptor expression, mitochondrial respiration (JO) and reactive oxygen species (ROS) release rates were evaluated in three human trophoblast cell lines (ACH-3P, BeWo and Swan-71) and one immortalized ovine trophoblast line (iOTR) under basal and substrate-stimulated conditions using high-resolution fluorespirometry.

Results: ACH-3P cells exhibited the greatest mitochondrial respiratory capacity and coupling efficiency of the four trophoblast lines tested, along with robust expression of androgen receptor protein that was found to co-localize with mitochondria by immunoblot and immunofluorescence. Acute testosterone administration (10 nM) tended to decrease ACH-3P mitochondrial JO and increase ROS release, while chronic (7 days) testosterone exposure increased expression of mitochondrial proteins, JO, and ROS release.

Discussion: These studies establish ACH-3P as a suitable cell line for investigating trophoblast mitochondrial function, and provide foundational evidence supporting links between hyperandrogenism and placental mitochondrial ROS production with potential relevance to several common pregnancy disorders.