To investigate whether long-term low-dose exposure to polystyrene microplastics (PS-MPs) induces ferroptosis in testicular Sertoli cells and then leads to testicular injury.

Forty 8-week-old male C57BL/6 mice were randomly divided into a control group (deionized water group) and a PS-MPs group [2.5 mg/(kg·d)], with 20 mice in each group. Corresponding agents were gavaged once a day for 12 consecutive months. HE staining and Prussian blue staining were used to detect histopathological damage and accumulation of ferrous ions in the testes. Electron transmission microscopy was employed to observe the mitochondrial morphology of testicular Sertoli cells. Mouse Sertoli cell line TM4 was divided into a Con group (standard culture) and an MPs group (2.5 μg/mL PS-MPs). After both groups underwent continuous exposure and passed up to the 100th generation, morphological changes were observed under the microscope; cell viability was detected with CCK8 assay, and production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected with a ROS probe and a mitochondrial membrane potential probe (JC-1), respectively. Flow cytometry, ferrous ion (Fe²⁺) kit and Western blotting were applied to detect cell apoptosis, intracellular iron ion content, and protein levels of key molecules of ferroptosis in tissues and cells.

Long-term exposure to PS-MPs resulted in significantly reduced diameter and thickness of mouse varicocele (P<0.01), fewer testicular Sertoli cells (P<0.05), with characteristic ferroptosis alterations in the mitochondria, and increased accumulation of ferrous ions in testicular tissue. Exposure to PS-MPs down-regulated the key molecules of ferroptosis, glutathione peroxidase 4 （GPX4） and ferritin light chain （FTL） when compared with the control group (P<0.05). In the cell model, long-term PS-MPs exposure led to morphological changes and decreased cell viability (P<0.05), more production of ROS (P<0.01), and decrease in MMP (P<0.05) of TM4 cells. The exposure had no effect on cell apoptosis, but elevated the intracellular content of ferric ions (P<0.01), and down-regulated GPX4 and FTL protein levels (P<0.05).

Long-term low-dose exposure to PS-MPs induces mitochondrial damage and oxidative stress in testicular Sertoli cells, activates the ferroptosis pathway, and ultimately leads to testicular injury in mice.