Optimizing the protocol for vitrification of individual spermatozoa by adjusting equilibration time

Area of Science:

• Reproductive Biology
• Cryobiology
• Andrology

Background:

• Efficient cryopreservation of limited human spermatozoa is vital for severe male infertility, particularly after surgical sperm retrieval.
• Vitrification on SpermVD® devices offers optimal cell retrieval but can reduce motility compared to bulk freezing.
• Optimal cryoprotectant exposure time for individual sperm vitrification remains undetermined.

Purpose of the Study:

• To evaluate the impact of varying cryoprotectant equilibration times on human sperm motility after vitrification.
• To compare post-warming motility rates between different equilibration times and cryoprotectants.

Main Methods:

• Vitrification of 2,925 human spermatozoa from 20 patients on SpermVD® devices using 1 µl droplets.
• Exposure of spermatozoa to cryoprotectant/washing medium mixtures for equilibration times ranging from 2 to 60 minutes.
• Evaluation of post-warming motility and comparison with bulk-frozen sperm.

Main Results:

• Post-thaw motility showed a negative correlation with cryoprotectant exposure time.
• The highest motility rate (32.1%) was achieved with an 8-minute equilibration time.
• At 10 minutes, vitrified sperm motility (31.7%) was significantly lower than bulk-frozen sperm (37.0%, p < 0.0001).

Conclusions:

• A maximum cryoprotectant equilibration time of 8 minutes is recommended for vitrifying small numbers of spermatozoa to maximize post-warming motility.
• Cryoprotectant type did not significantly influence the results.
• This finding is critical for improving fertility outcomes in patients with limited sperm samples.