Revision of hypo-osmotic swelling (HOS) test with a proposition of classified grading system: its comparisons with four different types of human sperm separation technique.

In this study, we arbitrarily classified the morphological changes of sperm under hypo-osmotic condition and compared the results with four different sperm separation techniques. The morphology of classification ranged from a good swelling (SG 3 > SG 2 > SG 1), to non-swelling but reactive (SG 0-R), and completely non-swelling (SG 0). Thirty fresh semen from patients were divided into 4 groups and each processed by washing, swim up, 2-layer column and real time micro-separation system. The prevalent patterns of the swollen sperm in sequence after treatment was SG 0 > SG 1 > SG 3 > SG2 > SG 0-R in fresh semen, and the proportion of SG 0 was almost half of the total % HOS test results. However, SG 3 became the most common swollen form with a significant increase in number with any method of sperm preparation after treatment. The real time technique yielded the highest % rate of SG 3 type of swollen spermatozoa and was the richest in concentration (53+/-3.9%, P < 0.05) as compared with that of swim up (26+/-4.6%), 2-layer (20+/-4.4%), sperm washing (23+/-3.9%), and fresh semen (17+/- 2.8%), respectively. By contrast, 2-layer collected more number of SG0-R (5.3+/-1.2%, P < 0.05) pattern of spermatozoa when compared to real time and swim up. A positive correlation (r = 0.81, P < 0.002) was shown between the % total HOS spermatozoa and total motility of fresh semen but not with morphology. Collectively, an abnormally functional sperm may exist in almost half of a total sperm count from men. It would appear that a better potential fertilization capacity may reside in the more swollen sperm from HOS test than the less swollen sperm and that may be more related with the motility rather than the morphology. The HOS classification and grading system appears valuable in further evaluating sperm quality.