Tip‐microVapour Fast Freezing: A novel easy method for cryopreserving severe oligozoospermic samples

Background: Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in nonobstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number.Objectives: In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n = 39) after cryopreservation with a tipmicroVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n = 16). Materials and methods: We used a Vapour Fast Freezing procedure using 10 mu L tips as carrier, and Test Yolk Buffer as freezing medium (tip-microVapour Fast Freezing). In a subset of samples (n = 22), we compared recovery of motility and viability as obtained with tip-microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500 mu L straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test.Results: We found a recovery rate (median [interquartile range]) of 0.29 (0.13-0.41) for progressive motility, 0.30 (0.21-0.52) for total motility and 0.48 (0.29-0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n = 22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip-microVapour Fast Freezing and Vapour Fast Freezing conducted with 500 mu L straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip-microVapour Fast Freezing than with Vapour Fast Freezing in 500 mu L straws: that is, progressive motility: 7.00 (3.00-8.50)% versus 2.00 (0.00-4.25)%, p < 0.001; total motility: 12.00 (8.00-16.25)% versus 6.50 (1.00-9.25)%, p < 0.001; viability: 29.75 (23.75-45.25) versus 22.50 (13.75-28.13), p < 0.001, respectively. In the second group of oligozoospermic samples, we found that tip-microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75-36.00]% vs. 36.00 [22.75-41.87]%, p < 0.001).Discussion and conclusion: Tip-microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.