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The objective of the study was to evaluate the benefit of different sperm preparation methods by using the hemizona assay. A total of 58 men admitted to the male infertility clinic for evaluation were tested by routine semen analysis and hemizona assay. Five different techniques (swim-up, TEST-yolk buffer, Percoll, pentoxifylline and progesterone) were used for preparation of sperm suspensions. The effect of these treatments on the sperm-binding capacity using the hemizona assay was assessed. The routine swim-up preparation was used as the reference method. Of the four preparation methods, only the TEST-yolk buffer and pentoxifylline exhibited an overall statistically significant improvement in sperm-binding capacity in comparison with the swim-up preparation method ( = 0.01 and 0.001 respectively). Following preparation with Percoll and progesterone there was no change in the mean value of binding capacity, compared with swim-up. However, examination of the effect of the four treatments on each specimen individually yielded a diversity in the response, e.g. having the capability to enhance, damage or be ineffective in sperm binding capacity. The results support the conclusion that in-vitro sperm preparation methods can affect sperm binding to the zona pellucida. Since there is a diversity in the response of sperm samples to different treatments, the hemizona assay can be used in selecting the optimal sperm preparation method prior to its use for assisted reproductive techniques. This is advocated mainly for the ‘male factor’ group. [ABSTRACT FROM PUBLISHER]