Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm

Objective: To determine whether the hypo-osmotic swelling test can assess viability of frozen spermatozoa. Design: Fresh semen specimens were divided into two aliquots: one was not processed and the other was processed by swim-up. Both aliquots were then cryopreserved by the liquid nitrogen vapor method. Setting: Andrology laboratory at a tertiary care facility. Patients: Eleven volunteers with proven fertility. Intervention(s): The hypo-osmotic swelling test and the sperm viability assessment by Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) and eosin-nigrosin stains were perfor med in each aliquot. Main Outcome Measure(s): The results of hypo-osmotic swelling test as indicated by swelling of sperm tails were compared with the viability results obtained by Hoechst 33258 stain before freezing and by Hoechst-33258 and eosin-nigrosin stains after freezing. Result(s): The hypo-osmotic swelling test and Hoechst 33258 viability results were correlated highly in unprocessed and processed fresh specimens (r = 0.95). After cryopreservation, there was no correlation between hypo-osmotic swelling test and Hoechst 33258 or between hypoosmotic swelling test and eosin-nigrosin. Prefreeze sperm processing did not influence the postthaw results. Viability assessed by Hoechst 33258 and eosin-nigrosin was well correlated (r = 0.72). Conclusion(s): The hypo-osmotic smelling test can accurately evaluate viability in fresh human spermatozoa but not in cryopreserved ones. Thus, this test cannot be used to select viable cells in cryopreserved samples for assisted reproductive procedures, such as intracytoplasmic sperm injection.