Microinjection-based Single-Cell Toxicological Assessment Reveals How Physiological Levels of PFOS Impair Oocyte Maturation and Developmental Competence

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental contaminant widely detected in human serum and follicular fluid and has been associated with reduced implantation rates and female fertility. However, its direct effects on mammalian oocyte maturation remain poorly understood. Here, we developed a microinjection-based single-oocyte toxicological assay to directly evaluate how physiologically relevant PFOS concentrations affect mouse oocyte maturation and early embryonic development. Microinjection of PFOS at follicular-fluid level (5.6 nM) and occupational exposure level (60 nM) significantly reduced germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) rates compared with water-injected controls. Notably, all tested concentrations (2.4 nM serum level, 5.6 nM, and 60 nM) induced abnormal polar-body formation, disrupted meiotic spindle morphology, and increased the proportion of unhealthy oocytes. PFOS exposure also significantly elevated intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential at 5.6 nM, indicating oxidative stress and mitochondrial dysfunction. Cytological analyses revealed chromosome misalignment and widened metaphase I plates, suggesting chromosome missegregation and subsequent prometaphase II arrest with defective polar bodies. Single-cell RNA sequencing of PFOS-treated oocytes exhibiting abnormal small polar bodies identified distinct transcriptional signatures, including dysregulation of genes involved in mRNA processing, chromosome segregation, mitochondrial function, and cell division. Functionally, these oocytes failed to progress beyond the 2-cell stage following in vitro fertilization, indicating loss of developmental competence. Collectively, these findings demonstrate that PFOS directly disrupts meiotic progression through spindle defects, oxidative stress, and transcriptional dysregulation, ultimately compromising oocyte quality even at environmentally relevant exposure levels.