Cystatin C 3 (CST3) drives pathological progression in recurrent spontaneous abortion.

This study aimed to investigate the role of Cystatin C (CST3) in trophoblast cell (TBC) function and its contribution in the development of recurrent spontaneous abortion (RSA). We established an inbred RSA model by crossing CBA/J and DBA/2 mice. We investigated and compared expression levels of CST3 and pathological changes in decidual tissues from these RSA and normal pregnant mice. We next isolated TBCs from RSA mice and transfected them with CST3 overexpression or silencing vectors to assess alterations in cell proliferation, invasion, apoptosis, autophagy, oxidative stress and inflammatory stress responses. Results showed that CST3 expression was significantly elevated in RSA mice compared to normal pregnant mice, accompanied by edema, degeneration of decidual cells, and structural disorganization. CST3 overexpression in TBCs led to a significant reduction in cloning and invasion abilities, increased apoptosis, shortened G0-G1 phase and enhanced autophagy. Conversely, silencing CST3 reversed these cellular activities, promoting TBC activity and reducing apoptosis. Additionally, CST3 overexpression intensified inflammatory and oxidative stress in TBCs, whereas silencing CST3 alleviated these stress responses, further supporting its role in RSA progression. In conclusion, CST3 is upregulated in RSA and contributes to its progression by inhibiting TBC activity, while accelerating apoptosis and autophagy. These findings suggest that CST3 silencing may offer a novel therapeutic strategy to improve pregnancy outcomes in RSA by restoring TBC function and reducing apoptosis and stress responses.