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

  • Fecha: 24 Marzo, 2020
  • Revista: Systems Biology in Reproductive Medicine
  • Autores: Michael Belenky, Diana Itzhakov, Vita Freger, Orna Roseman, Sarah Abehsera, Netanella Miller & Arie Berkovitz


Efficient cryopreservation of small numbers of human spermatozoa is essential in cases of severe
male infertility, especially those requiring surgical sperm retrieval. Although vitrifying individual
spermatozoa on sperm vitrification devices (SpermVD®) provided optimal cell retrieval upon
warming, motility rates tended to be lower than with bulk-freezing. Post-warming motility is
directly affected by cryoprotectant exposure; however, optimal cryoprotectant equilibration time
is unknown. We evaluated several timeframes exposing individual spermatozoa to cryoprotectant
before freezing and different cryoprotectants. A total of 2,925 spermatozoa from 20 patients
ranging from normozoospermic to moderate oligoteratoasthenozoospermic were vitrified in small
groups, on 60 SpermVD®, in 1 μl droplets of 1:1 v/v cryoprotectant/washing medium mixture. Each
group was vitrified after 2–60 minutes equilibration time. Motility of each group was evaluated
after warming. Leftover pellets were frozen in cryotubes in a mixture of 1:1 v/v cryoprotectant/
washing medium after 10 minutes equilibration at room temperature and 10 minutes on liquid
nitrogen vapors. Post-thaw motility correlated negatively with cryoprotectant exposure time. The
highest post-warming motility rate (32.1%) was observed with 8-minutes equilibration. After
10 minutes, motility rate of vitrified sperm was lower than that of bulk-freezing (31.7% vs.
37.0%, p < 0.0001). Different cryoprotectants did not affect the results. Therefore, for vitrifying
small numbers of spermatozoa, we suggest maximum equilibration time of 8-minutes to achieve
maximum motility after warming.