Abstract

Single sperm cryopreservation is a new method to preserve small numbers of spermatozoa in small droplets. So far, several devices have been introduced for this technique, but more studies are needed for its optimization. The aim of this study was optimizing the previous device for low number of spermatozoa and low volume semen, which led to design of Cryotop Vial device. Normal semen samples from 25 patients were prepared by swim-up method and divided into four groups: Fresh (F), Rapid freezing (R) and ultra-rapid freezing with Cryotop Device (CD) and Cryotop Vial Device (CVD). In R group, the diluted sperm suspension with sperm freezing medium was cooled in the vapor phase and then immersed in liquid nitrogen. Ultra rapid freezing was performed with sucrose in small volume using the Cryotop Device (CD) or Cryotop Vial Device (CVD). Sperm viability, motility, fine morphology, mitochondrial activity and DNA fragmentation were assessed in all samples. All sperm parameters decreased significantly in all the cryo groups compared to the fresh group. The comparison between the cryo groups showed that progressive motility (69.28 & PLUSMN; 6.82 vs. 55.68 & PLUSMN; 9.04, and 54.76 & PLUSMN; 5.34, p < 0.001) and viability (77.36 & PLUSMN; 5.48 vs.68.84 & PLUSMN; 8.51, p < 0.001, and 70.04 & PLUSMN; 7.44, P = 0.002) were significantly higher in the CVD compared to the CD and R groups respectively. DNA fragmentation was also significantly lower in both ultra-rapid freezing groups (CD and CVD) compared to the R group. Fine morphology and mitochondrial activity were not different between the cryo groups. The CVD as a cryoprotectant and centrifuge-free technique preserved sperm motility, viability and DNA integrity after cryopreservation better than other groups.