Vapour fast freezing with low semen volumes can highly improve motility and viability or DNA quality of cryopreserved human spermatozoa

Objectives To challenge a vapour fast freezing (VFF) cryopreservation procedure (conventional VFF) with several vitrification protocols and VFF conducted with small semen volumes (10 mu l, microVFF), in order to implement a procedure for sperm banking in subjects with small sperm number. Materials and methods Conventional VFF was conducted with test yolk buffer (TYB) as freezing medium and 500 mu l straws as carriers. MicroVFF was conducted with TYB and using tips or cell sleepers as carriers. Vitrification was performed with TYB or SpermFreeze as freezing medium and with microspheres and tips as carriers. The effect of different procedures on progressive and total motility, viability, oxidative stress and DNA fragmentation of spermatozoa (sDF) was determined. Fresh and thawed samples, the latter after adequate washing/centrifuging, were evaluated. In some experiments, motility and viability recovery was determined in thawed samples, omitting the washing/centrifuging step. Results All the cryopreservation procedures blunted sperm motility and viability and induced increase of oxidative stress and sDF. However, VFF better preserved sperm motility and viability and less induced oxidative stress and sDF than vitrification, independently from the freezing medium and the carriers used in the latter. MicroVFF with cell sleepers resulted in a percentage increase of 57.58 +/- 63.63%, 48.82 +/- 74.96% and 24.55 +/- 39.20% of, respectively, progressive and total motility and viability compared to the conventional VFF. Further, when tips were used, microVFF resulted in a percentage decrease of 15.77 +/- 20.77% of sDF with respect to conventional VFF. Finally, omission of washing/centrifuging in post thawed samples, resulted in a much lower negative effect on motility and viability. Discussion and conclusion VFF, and in particular microVFF, better prevents sperm cryodamage than vitrification. Washing/centrifuging step after sample thawing seems to be responsible for a relevant fraction of damage to sperm motility and viability. Overall, our results are promising for developing a novel strategy of sperm banking in subjects with small sperm number, where low semen volumes are mandatory.