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Cryoprotectant-Free Cryopreservation of Human Spermatozoa by Vitrification and Freezing in Vapor: Effect on Motility, DNA Integrity, and Fertilization Ability

Department of Gynecological Endocrinology and Reproductive Medicine,³ University of Bonn, 53105 Bonn, Germany Department of Obstetrics and Gynecology,⁴ University of Cologne, D-50924 Cologne, Germany Cancer Center,⁵ University of California at San Diego, La Jolla, California 92093 Department of Obstetrics and Gynecology,⁶ University of Sassari, 07100 Sassari, Italy

Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 10⁵ °C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (–160°C) with speed in the range 150–250°C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.

¹ Correspondence: Vladimir Isachenko, Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. FAX: 0049 228 2875795; vovaisachenko{at}yahoo.com

² Current address: Reproductive Medicine and Gynecological Endocrinology, Endokrinologikum Hamburg, 22767 Hamburg, Germany

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