The effect of glycerol and a glycerol-containing cryoprotective medium upon the motility of human sperm prior to freezing, and subsequent difficulties in assessing sperm motility following dilution

Glycerol is a widely used cryoprotective agent in the cryopreservation of human semen, however it has toxic effects on sperm. The use of computer-assisted sperm analysis (CASA) software to determine sperm motility requires specific upper concentrations of sperm to allow the accurate tracking of sperm trajectories without collisions. Various diluents have been used successfully for diluting neat semen samples but the effect of dilution of semen containing glycerol for the purpose of cryopreservation is relatively unclear. The effect on sperm motility was therefore investigated for the following: the addition of a commercial, glycerol containing cryoprotective (CPM) Quinns Advantage Sperm Freezing Medium (Origio Australasia Pty Ltd, Thornleigh NSW, Australia), 5% neat glycerol (Sigma-Aldrich Pty Ltd, Sydney NSW, Australia) and 10% neat glycerol; cell-free seminal plasma, gamete handling media G-MOPSTM PLUS (Vitrolife Pty Ltd, Sydney NSW, Australia), which contains human serum albumin, and Quinn’s Advantage™ Medium with HEPES (Origio Australasia Pty Ltd, Thornleigh NSW, Australia) supplemented with 5% human serum albumin (Origio Australasia Pty Ltd, Thornleigh NSW, Australia), and finally; combinations of the above cryoprotectants and handling media. The presence of glycerol was found to have a negative impact on sperm motility in all samples; this appeared to be in a dose-dependent fashion, with the CPM suffering the least, and 10% glycerol having the most severe reduction in progressive motility. All glycerol containing samples suffered a further reduction in progressive motility when either GMOPSTM PLUS or HEPES-buffered medium were introduced. Seminal plasma suffered no further reduction in progressive motility for CPM and 10% glycerol, but a reduction in the 5% glycerol. This study has demonstrated that semen samples containing glycerol should only be diluted with seminal plasma prior to assessment by CASA, and that HEPES-buffered medium and G-MOPSTM PLUS should not be used