Abstract:

Objective To investigate the efficacy of discontinuous density gradient combined with swim-up method for bacterial removal in frozen semen， and to provide experimental evidence for the safe application of frozen semen in assisted reproductive technology. Methods A total of 208 frozen semen samples containing non-pathogenic bacteria， cryopreserved in our human sperm bank from January 2019 to December 2023， were selected. The thawed semen samples were processed using discontinuous density gradient combined with swim-up method. To simulate in vitro fertilization procedures， the processed sperm suspensions were subjected to 5-day in vitro culture for bacterial residue monitoring. Prior to cryopreservation， all semen samples underwent routine bacterial culture to exclude pathogenic microorganisms. Post-thaw semen samples， processed sperm suspensions， and 5-day culture media were sent to an independent third-party laboratory for comprehensive bacterial identification. Results Among the 208 frozen semen samples containing non-pathogenic bacteria， 41 bacterial species were identified， with 1 ~ 4 species per sample. After processing with discontinuous density gradient combined with swim-up method， the sterility rate of the sperm suspension reached 89.42% （186/208）， with residual bacteria including Enterococcus faecalis， Corynebacterium glucuronolyticum， Group B Streptococcus， and Corynebacterium glycinophilum. After 5-day in vitro culture， the sterility rate increased to 93.75% （195/208）， with residual bacteria limited to Enterococcus faecalis and Group B Streptococcus. Conclusions The combined method of discontinuous density gradient and swim-up technique effectively eliminates most bacteria in frozen semen. However， the clearance rate for Enterococcus faecalis and Group B Streptococcus remains below 90%. Optimization of pre-freezing bacterial screening and post-thaw processing techniques is necessary to enhance the safety of frozen semen in assisted reproductive technology.

Key words: frozen semen, microbial contamination, discontinuous density gradient method, swim-up method, assisted reproductive technology