Vitrification of a small number of spermatozoa in normozoospermic and severely oligozoospermic samples

Abstract
Despite broad utilization of sperm cryopreservation, little progress has been made to modify
freezing protocols or to improve rates of sperm survival. Vitrification is an alternative method
for freezing human spermatozoa without toxic permeable cryoprotectants (CPAs). The purpose
of our study was to optimize the vitrification and post thaw recovery of a small number of
spermatozoa using only nonpermeating CPAs in a closed straw system in normozoospermic
and severely oligozoospermic samples. Individual motile spermatozoa (n¼295) were selected
from semen samples of 15 normozoospermic and 10 severe oligozoospermia patients. Overall
sperm recovery after vitrification was 80% (n¼236) with 80% (n¼189) viability and 41.5%
(n¼98) retained post-warming motility. Two different loading techniques were compared to
transfer selected spermatozoa into straws in preparation for vitrification: by spontaneous
capillary action (CA) and with the aid of a polar body biopsy (PBB) pipette. There was evidence
that the PBB loading technique increases the odds of spermatozoa recovery in both subsets
(p¼0.01 and p¼0.04) in the normal and abnormal subsamples, respectively.
Abbreviations: CA: capillary action; CBS: Cryo Bio System; CPAs: cryoprotectants; DNA:
deoxyribonucleic acid; HOST: hypo-osmotic swelling test; ICSI: intracytoplasmic sperm injection;
IUI: intrauterine insemination; IVF: in vitro fertilization; OPS: open-pulled straw; PBB: polar body
biopsy; ROS: reactive oxygen species; WHO: World Health Organization