# Caffeine Exposure Modulates Trophoblast Differentiation and Estradiol Synthesis

## TL;DR

Caffeine affects how placental cells develop and produce estradiol, a hormone important for pregnancy, by changing specific cell processes.

## Contribution

This study reveals that caffeine modulates trophoblast differentiation and estradiol synthesis via PKA-dependent mechanisms in human placental cells.

## Key findings

- Caffeine increases CYP19A1 mRNA and estradiol production in BeWo cells and placental explants.
- Caffeine's effect on CYP19A1 is mediated through PKA signaling, not PKC or MAPK pathways.
- Non-cytotoxic caffeine concentrations enhance aromatase expression and estradiol synthesis.

## Abstract

Differentiation of villous cytotrophoblasts into syncytiotrophoblasts is essential for placental endocrine function and estradiol production. Caffeine consumption has been linked to altered estradiol levels, but its effects on human trophoblast differentiation remain incompletely understood. This study investigated the effects of caffeine on biochemical differentiation of human trophoblasts using BeWo cells and human placental explants. Cell viability and apoptosis were assessed using CCK-8 and in situ TUNEL assays. Differentiation-associated changes were evaluated by measuring CYP19A1 and its placenta-specific promoter transcript CYP19 I.1, at the mRNA level, while aromatase protein expression and estradiol production were assessed by Western blotting and ELISA, respectively. Exposure to 2 mM caffeine reduced BeWo cell viability, whereas 1 mM caffeine had no detectable effects on cell viability or apoptosis. At non-cytotoxic concentrations, caffeine significantly increased CYP19A1 mRNA expression under both basal and forskolin-stimulated conditions and elevated estradiol production. Similar transcriptional and endocrine responses were observed in human placental explants. Pharmacological inhibition demonstrated that caffeine-induced CYP19A1 transcriptional upregulation was dependent on PKA signaling, but not on PKC or MAPK pathways. These results indicate that caffeine can modulate trophoblast biochemical differentiation via PKA-dependent regulation of placental aromatase expression and estradiol synthesis. While these findings provide mechanistic insight into caffeine-mediated effects on trophoblast endocrine function, their relevance to physiological exposure levels and in vivo placental development warrants cautious interpretation.