Successful use of the Cryolock device for cryopreservation of scarce human ejaculate and testicular spermatozoa.

The existing methods for cryopreservation of very low count sperm samples are complex and sub-optimal for individual spermatozoa. Our purpose is to establish an effective simple method for cryoprotecting individual spermatozoa. Samples from patients with OTA were mixed with TYB or HEPES-buffered salt solution with glycerol + glucose and placed on a Cryolock that was plunged directly into liquid nitrogen or exposed to its vapors. Thawing was performed by direct immersion into a drop of warmed medium. The favorable method was tested on diluted samples (10-50 cells) and leftover TESE specimens from patients with azoospermia. Cryopreservation was considered successful if >30 spermatozoa, (>3 motile), or >5 spermatozoa (>1 motile) in diluted and TESE samples, were detected post-thawing. A significantly higher survival rate of seminal spermatozoa was obtained when using the Cryolock with TYB and freezing with liquid nitrogen vapor, compared to HEPES glycerol-glucose (95 vs. 35% respectively). Plunging the Cryolock into liquid nitrogen was detrimental. Cryolock combined with TYB cryoprotection and liquid nitrogen vapor freezing was highly effective for cryopreservation of individual spermatozoa in diluted and TESE samples. The Cryolock may serve for freezing very low-count sperm samples and individual spermatozoa. This method offers simplicity, efficacy, use of available materials, without requiring micromanipulation equipment or skills.