Abstract The purpose of this study is to develop a novel
method for the cryopreservation and efficient post-thaw
recovery of individual or small numbers of human spermatozoa.
Spermatozoa equilibrated in cryoprotectant buffer were
injected with an intracytoplasmic sperm injection (ICSI)
needle into a droplet of cryoprotectant on a homemade
cryoleaf. The droplet was of cryoprotectant and seminal
plasma at a ratio of 1:1. The sperm-loaded cryoleafwas slowly
lowered over and stored in liquid nitrogen. Spermatozoa were
thawed in a 37°C oil bath without dilution and centrifugation.
To test the fertilizing ability of these spermatozoa, the
recovered spermatozoa were injected by ICSI into 1-d-old or
in vitro-matured human oocytes. Fresh spermatozoa from the
same semen samples served as controls. The trials were
performed in two separate experiments. In the first set of
experiments, 92 spermatozoa were thawed and carefully
investigated. The spermatozoa from percutaneous epididymal
sperm aspiration had a motility recovery of 92.9% (13/14);
ejaculated spermatozoa had a motility recovery of 61.5% (48/
78), and only 1.3% (1/78) was lost. Together in the first and
second set of experiments, the fertilization rates for the fresh
and frozen–thawed spermatozoawere 67.6% (25/37) and 60.6%
(40/66), respectively (P=0.052). The mean embryo
cleavage rates in the fresh and frozen–thawed groups were
88% (22/25) and 85% (34/40), respectively (P=0.990). This
cryopreservation method for individual or small numbers of
human spermatozoa was efficient and simple. These findings
make this method a promising technique for the clinical
application of ejaculated sperm from oligozoospermic patients.