ABSTRACT
Cryopreservation and low-temperature storage of different cell types and tissues, including male and female gametes and embryos, are used worldwide and have become an integral part of most human IVF programs. Since the late 1930-1940s1,2 spermatozoa of several mammalian species, especially bovine and human, have been cryopreserved effectively. The empirical methods of cryopreservation developed in the 1950s are still used today, and are very important as they allow the preservation of male fertility before radiotherapy and/or chemotherapy.3 Such treatments and some kinds of surgical procedures may lead to testicular failure or ejaculatory dysfunction.4 During conventional freezing, water precipitates as ice and thus separates from dissolved substances. Both intracellular ice crystal formation and the high concentration of dissolved substances pose problems. Slow cooling rates aim to maintain a very delicate balance between these factors, yet often lead to cell damage mostly because of ice crystallization, but also due to osmotic and chilling injury, cytoplasm fracture, or even effects on the cytoskeleton or genome-related structures. Due to damage associated with freezing, the motility of cryopreserved spermatozoa after thawing is significantly reduced in comparison with the motility before and shows a wide interindividual variability.5 To date, the problem of cryoprotectant toxicity due to osmotic stress during
the addition and removal of cryoprotectants, and the possibility of its negative influence on the genetic apparatus has, as yet, not been solved,5,6 and cellular cryodamage can arise during slow thawing.7 At present the freezing procedures for many species including human show acceptable results, but cryoprotectant solutions and freezing equipment are necessary. Most IVF laboratories prefer programmable freezing devices. The whole freezing procedure (equilibration, freezing, and dilution of cryoprotectant) takes around 30-60 minutes. Cryopreservation by direct plunging into liquid nitrogen (vitrification) could be beneficial when compared with the ‘slow’ method, because it does not need any expensive equipment and takes only a few seconds for freezing and warming. The vitrification technique arose as an alternative to slow conventional freezing, in order to avoid the crystallization.8 The successful vitrification of frog9 and fowl spermatozoa10 has supported Luyet’s proposal. However, the subsequent attempts to vitrify mammalian spermatozoa using this technique resulted in low or no survival.11,12 Based on this, the vitrification technique was initially unacceptable for routine work. While this method of vitrification using high concentrations of permeable cryoprotectants was successfully applied in 1985 for mouse embryos,13 nevertheless it was impossible to perform this technique for spermatozoa cryopreservation because of the resulting osmotic and cytotoxic effects.5,14
The beneficial effects of glycerol and nonpermeable cryoprotectants, including sucrose, on plant cryostability were described as early as 1908.15 In 1937 this was followed by the demonstration of the positive effect of 1 mol/L glycerol on rabbit, guinea pig, bull, ram, stallion, and boar sperm1 frozen to −21°C. In the late 1940s, the results of experiments based on the use of glycerol in the UK by Polge, Smith and Parkes2 as well as in the USSR by Smirnov16
and Milovanov17 were published. These empirical methods, which were subsequently developed in the 1950s for use in many species, are still applied today. The motility of cryopreserved/thawed spermatozoa normally drops to about 50% of their pre-freezing value, with considerable intersample fluctuation.5
