Improved sperm cryopreservation using cold cryoprotectant

Abstract

It has generally been assumed that very rapid cooling above freezing point would be deleterious to human sperm because it would result in cold shock. Consequently, most routine cryopreservation protocols involve the use of warm (20–30°C) cryoprotectant and slow cooling above the freezing point in order to minimise the risk of cold shock. In order to test this assumption, we added an equal volume of cold (4°C) cryoprotectant in a single aliquot to warm (20, 30 or 37°C) semen to induce rapid cooling. The results of this procedure were compared with those obtained using warm cryoprotectant or with the routine cryopreservation protocol used in this laboratory. The use of cold cryoprotectant resulted in a significant (P = 0.016) improvement (mean 63%, range 42%–79%) in post-thaw motility recovery compared with a standard procedure(mean 47%, range 35%–67%) and a significant (P = 0.016) improvement in post-thaw sperm velocity. A cold glycerol/egg yolk/citrate (GEYC) mixture also gave significantly higher motility recovery than GEYC equilibrated to either room temperature (20°C) or body temperature (37°C). Sperm frozen using the cold cryoprotectant protocol were as efficient at binding to and penetrating the human zona pellucida as sperm frozen with a standard protocol.The modified cryopreservation procedure may lead to improved pregnancy rates in donor insemination and in vitro fertilisation. Further investigation is required to determine how the cold cryoprotectant improves the cryopreservation outcome.