Cryopreservation of single-sperm from semen and testicular samples: a 5-year monocentric experience in hundreds of patients

For patients with severe oligozoospermia, cryopreservation of single human spermatozoa highlights its advantages. Previous research has focused on the development of micro-drop single sperm freezing methods with non-biological carriers over the past 20 years. However, these methods lacked hermetically sealed carriers, potentially leading to contamination. This study utilized a prospective cohort design, involving 434 male patients diagnosed with severe oligozoospermia, cryptozoospermia, and non-obstructive azoospermia (NOA) between January 2020 and December 2024. Individual viable spermatozoa were transferred to a 1 µL droplet of freezing medium using the self-designed closed straw carrier system. This carrier was then exposed to liquid nitrogen (LN2) vapor for freezing and finally plunged into LN2 for storage. Successful sperm cryopreservation was achieved in 308 patients, representing a success rate of 71.0%. The utilization rate of frozen testicular sperm was significantly higher at 88.9% compared to that of ejaculated sperm, which was only 16.6% (P < 0.001). After thawing, the median recovery rate of ejaculated spermatozoa was 85.0%, with an average motility rate of 23.4%. In contrast, the median recovery rate of testicular spermatozoa was significantly higher at 93.8% (P = 0.019). Among 64 couples undergoing intracytoplasmic sperm injection (ICSI) treatment, no significant differences were observed in fertilization rates, clinical pregnancy rates, miscarriage rates, or delivery outcomes between patients using frozen ejaculated sperm and those using frozen testicular sperm retrieval. To date, there have been high-quality frozen embryos and the birth of 10 healthy babies.