Cryopreservation of Sperm

Abstract

The basic principles for cryopreservation of sperm are not different from cryopreservation of embryos, eggs, and ovarian tissue. However freezing with sperm is much easier because there is such a minute amount of water in this small, otherwise highly specialized gamete. In fact very little advance has been made in sperm freezing research, because the results seem to be so adequate with the simplest of methods. For embryo or egg freezing, much more precise methodology is required because 70% of these cells is water. When the intracellular water freezes, it crystalizes, and occupies a large volume and is less dense. That is why ice cubes float. But it is also why cells are killed by freezing, so all methods of cryopreservation rely on preventing intra cellular ice crystal formation. However I will describe, in addition to routine sperm cryopreservation, a simple new method for freezing individual sperm for difficult cases of TESE. Slow cooing results in preferential freezing of water on the outside of the cell, where the osmolality is lower. That raises the osmolality extracellularly. This in turn draws water out of the sperm making the inside even more hyperosmolar. Thus as the temperature cools gradually and ice forms preferentially on outside of the cell, that pulls water out of the cell, and then cryoprotectants enter the cell and lower the freezing point inside the sperm cell even further. Eventually the inside of the sperm actually vitrifies when the temperature is low enough and the osmolality is high enough in this extremely low volume cell. This slow cooling need not be so precise as it is with using a controlled rate freezing machine (like for slow freeze of embryos). In fact one can simply have a vial containing sperm mixed with cryoprotectants over an open liquid nitrogen dewar, vapor freeze it and then plunge it into the tank. Before holding over liquid nitrogen vapor, it can be first put in an ordinary refrigerator at 4°C. It is amazingly simple. It is the same principle as slow freezing of embryos but it does not have to be very precise, because the water content of sperm is so low. Despite the crudeness and lack of graduality of such sperm freezing and thawing protocols, the effectiveness of this vapor slow freeze approach for sperm cryopreservation is very good. Nonetheless at the end of this chapter I shall mention a very simple, new method for single sperm isolation and cryopreservation.