Human Reproduction, Vol 12, 994-1001, Copyright © 1997 by Oxford University Press
J Cohen, GJ Garrisi, TA Congedo-Ferrara, KA Kieck, TW Schimmel and RT Scott
A procedure is described that allows cryopreservation and efficient
post-thaw recovery of either a single or a small group of human
spermatozoa. This is achieved by injecting them into cell-free human, mouse
or hamster zonae pellucidae before the addition of cryoprotectant. The
method involves a combination of physical micromanipulation procedures and
glycerol-mediated cryoprotection. Zonae were tracked by positioning them in
straws between two small air bubbles prior to freezing. Spermatozoa from
poor specimens were cryopreserved and their fertilizing ability after
thawing was compared with that of fresh spermatozoa from fertile men. Human
eggs used for fertilization testing were either 1 day old or in-vitro
matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa
cryopreserved in this manner were recovered and prepared for
intracytoplasmic sperm injection. The feasibility of cryopreserving a
single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in
15 zonae, of which 14 were recovered after thawing. Ten were injected into
spare eggs, of which eight became fertilized. Spermatozoa recovered
mechanically from human zonae fertilized the same proportion of oocytes as
fresh fertile control spermatozoa. The recovery and fertilization rates
with spermatozoa frozen in animal zonae were 87 and 78% respectively. The
fertilization rate was marginally higher (P < 0.05) than that for
spermatozoa frozen in human zonae, perhaps because the latter may have
acrosome reacted more frequently. The zona pellucida appears to be an
ideally suited sterile vehicle for storage of single spermatozoa.
ARTICLES
Cryopreservation of single human spermatozoa
The Gamete and Embryo Research Laboratory, The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, NJ 07052, USA.
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