目的 探讨非连续密度梯度法联合上游法对冷冻精液的除菌效果，为冷冻精液在辅助生殖中的安全应用提供实验依据。 方法 选取2019年1月至2023年12月医院人类精子库冻存的208例新鲜精液排除致病菌感染的含菌冷冻精液，采用非连续密度梯度法联合上游法处理冻融精液，并模拟体外受精过程对处理后精子上游液行5 d体外培养，监测细菌残留情况。冷冻前精液采用常规细菌培养排除致病菌；解冻后精液和处理后精子上游液及5 d培养液送第三方检测机构检测鉴定所有菌种。 结果 208例新鲜精液排除致病菌感染的含菌冷冻精液共检出41种菌，每例含菌1 ~ 4种。经非连续密度梯度法联合上游法处理后，精子上游液无菌率为89.42%（186/208），残留菌为粪肠球菌、解葡萄糖醛酸棒状杆菌、B群链球菌、解葡萄糖苷棒杆菌；精子上游液体外培养5 d后，无菌率提升至93.75%（195/208），残留菌为粪肠球菌和B群链球菌。 结论 非连续密度梯度法联合上游法可有效清除冷冻精液中大部分细菌，但对粪肠球菌和B群链球菌的菌种清除率小于90%，需优化冷冻前精液细菌鉴定和解冻后精液处理技术以提高冷冻精液在辅助生殖中的安全性。

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.