The methods currently employed in freezing human spermatozoa are crude compared with those used for human embryos (Royere et al., 1996
). In particular, control of the cooling rate is often primitive: samples are commonly suspended in the vapour above liquid nitrogen, resulting in significant differences in cooling rate between different samples. The resulting straw-to-straw variation and loss of viability may not be important where sperm counts are normal, but in the case of oligozoospermic or asthenozoospermic samples these losses may be highly significant. With the development of intracytoplasmic sperm injection and the availability of techniques for surgical sperm retrieval, there is an increased need to store low numbers of sperm and therefore to improve freezing techniques in order to maximize survival (Cohen et al., 1997).
Improvements in cryopreservation of human spermatozoa have been attempted in the past by the use of different cryoprotectants and extenders, and in particular, by altering the cooling rate, usually a linear reduction in temperature with time (Serafini and Marrs, 1986; Ragni et al., 1990; Henry et al., 1993; Gilmore et al., 1997). With many cell types, and mammalian embryos provide a well documented example, a well defined `optimum' rate of cooling exists, with survival decreasing at both faster and slower rates. Intriguingly, similar studies demonstrate that spermatozoa are relatively insensitive to the magnitude of the linear rate of cooling during freezing. With human spermatozoa, a very broad response curve exists with little difference in survival observed following cooling at 1°C/min up to 100°C/min (Henry et al., 1993). This response is unusual for mammalian cell types, but has received surprisingly little comment. Furthermore, the recovery of viability is comparatively low, with typically less than 60% of cells retaining motility on thawing, which given that this is achieved with such a wide range of linear cooling rates would suggest that the linearity of temperature reduction may not be appropriate.
The cooling rate dependency of cell recovery of many cell types may be predicted from computer models of their osmotic behaviour during freezing. However the predicted results with spermatozoa have not been in agreement with experimental observations (Noiles et al., 1993; Curry et al., 1995). For example, although conventional models have suggested that human sperm cells should survive cooling rates up to 10 000°C/min (Noiles et al., 1993), experimentally the survival rate begins to decline beyond 100°C/min (Henry et al., 1993). It is clear that human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been amenable to conventional approaches of cryobiology.
Many of the changes in physical properties which occur in an aqueous cryoprotectant following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of ionic species, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature (e.g. Franks, 1985). In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a non-linear manner with temperature. Conventional approaches to cryopreservation thus impose a linear change of temperature with time whilst the stresses that cells are encountering are all non-linear with time. It is therefore appropriate to examine whether improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes rather than imposing a linear temperature reduction. In order to implement the required control of external conditions a new cell freezer has been specifically developed to achieve the desired protocols.
In this investigation, human spermatozoa suspended in a standard cryoprotectant were frozen using various protocols that manipulated different aspects of the physical conditions and the effects on post-thaw survival and function were assessed in comparison with conventional techniques. In order to increase our understanding of the physical behaviour of both the spermatozoa and the cryoprotectant during freezing, fractured straws were examined using the cryostage of a scanning electron microscope and freeze substitution. In addition, some simple computational modelling of the osmotic behaviour of the spermatozoa during freezing was carried out.
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