Bisphenol A impairs developmental potential of mouse blastoids through oxidative stress

Bisphenol A (BPA) is a widely encountered environmental endocrine disruptor with detrimental effects on embryonic development and implantation. Conventional reproductive toxicity tests often rely on animal-derived gametes or embryos. However, these approaches are invasive and limited in their ability to assess early germline development. To address these limitations, we used a synthetic embryo model derived from pluripotent stem cells (PSCs) to evaluate BPA-induced embryotoxicity without using actual oocytes or embryos. Mouse PSCs were converted into chemically induced totipotent-like stem cells (ciTotiSCs) using a defined small-molecule combination. These ciTotiSCs generated blastoids (ciToti-blastoids) through three-dimensional aggregation in inverted pyramid-shaped microwell plates (AggreWell system) and cultured for 5 d. To investigate the impact of environmental toxicants on early embryogenesis, blastoids were exposed to 5  μM BPA during formation. Blastoid formation efficiency was reduced to approximately 14 % following treatment with 5 μM BPA, compared with approximately 73 % in the control. To assess implantation potential, established blastoids were further cultured on the Matrigel-coated dish. BPA-treated blastoids exhibited a reduction in inner cell mass (ICM) area and in trophectoderm (TE) area, approximately 96 % and 72 %, respectively, indicating impaired implantation potential. These detrimental effects of BPA were accompanied by elevated reactive oxygen species (ROS) levels during ciToti-blastoid formation. Co-treatment with the antioxidant glutathione (GSH; 0.5 mM), a ROS scavenger, restored blastoid formation efficiency (approximately 54 %) and resulted in a similar normalized ICM and TE areas comparable to control. Transcriptomic analysis revealed that BPA induced endoplasmic reticulum stress, DNA damage, and apoptosis, all of which were mitigated by GSH treatment. These results highlight the role of oxidative stress in BPA-induced embryotoxicity and support the utility of stem cell-derived blastoid models as egg-free platforms for assessing the reproductive toxicity of environmental chemicals.