Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotectiveagent for cryopreservation of human spermatozoa would be a solute for whichcells have the highest plasma membrane permeability, resulting in the leastamount of volume excursion during its addition and removal. To test thishypothesis, theoretical simulations were performed using membranepermeability coefficients to predict optimal procedures for the additionand removal of a cryoprotectant. Simulations were performed using data fromfour different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide,(iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulationswere applied to determine approaches for the addition and removal of 1 Mand 2 M final concentrations of cryoprotectant, allowing the spermatozoa tomaintain a cell volume within their osmotic tolerance limits. Based onthese data, ethylene glycol was predicted to be optimal for minimizingvolume excursions among the solutes evaluated. These predictions were thenexperimentally tested using glycerol as the control cryoprotectant andethylene glycol as the experimental cryoprotectant. The results indicatethat there was a higher (P < 0.05) recovery of motile spermatozoa aftercryopreservation when using 1 M ethylene glycol than with 1 M glycerol,supporting the hypothesis that use of the cryoprotectant for which the cellhas the highest permeability will result in higher cell survival.