DIFFERENTIAL SPERM PERFORMANCE AS JUDGED BY THE ZONA-FREE HAMSTER EGG PENETRATION TEST RELATIVE TO DIFFERING SPERM PENETRATION TECHNIQUES

A prospective study on 61 unselected semen samples from infertile patients was conducted to evaluate the effects of sperm preparation techniques on the outcomes of the zona-free hamster egg penetration test (HEPT) to assess the in-vitro fertilizing capacity of spermatozoa. Each semen sample was divided into two equal portions before separation of the spermatozoa from seminal plasma either by the single-tube swim-up method, or using a two-layer discontinuous Percoll gradient. Spermatozoa were incubated overnight for initiation of capacitation after which HEPT was performed. The swim-up spermatozoa were further divided into two sub-groups before HEPT as follows: with or without (control) treatment for 20 min with 50% (v/v) pooled, human follicular fluid (hFF) which had not been heat-inactivated. It was demonstrated (P < 0.05) that the Percoll-separated spermatozoa exhibited higher penetration scores (percentage penetration rate and penetration index) than the control or the hFF-treated swim-up spermatozoa. A short exposure (20 min) to hFF significantly increased the penetration scores in HEPT for swim-up spermatozoa (P < 0.05) but the average results were still significantly lower (P < 0.05) than those of the Percoll separated spermatozoa, which had received no hFF treatment. Based on these findings, we conclude that the Percoll separation technique is better than the centrifugal pelleting and single-tube swim-up technique for reducing the false-negative results in HEPT. In addition, the use of hFF can significantly improve the performance of the swim-up sperm samples in HEPT. The centrifugal pelleting and swim-up technique might have caused some damage to the swim-up spermatozoa in comparison to the Percoll separated spermatozoa, thus resulting in a poorer performance in the HEPT. This last point might also explain the inability of hFF-treated swim-up spermatozoa to exhibit a comparable performance in HEPT to the Percoll separated spermatozoa, which had not received any hFF treatment.