Defining the valid hemizona assay: accounting for binding variability within zonae pellucidae and within semen samples from fertile males

To achieve a better understanding of the variability in sperm and oocyte binding capacities will optimize use of the hemizona assay (HZA) as a predictor of sperm function. Limitations of the HZA were more clearly delineated by current studies: (1) variability of sperm binding capacity of men over a 90-day interval; (2) variability of sperm binding using different oocytes; and (3) lower limits of the number of sperm bound from the fertile control in two laboratories. Semen was obtained from proven fertile men and one subfertile individual. The number of sperm tightly bound to the hemizona were measured and compared. In the initial study, 6 fertile control men exhibited a similar degree of variability in zona binding when studied over a 90-day interval. Average sperm binding for individuals ranged from 68 to 127. Second, 3 of the 15 simultaneous assays showed very low numbers of sperm bound, indicating that 20% of the zonae had poor binding. Third, from 18 men who had 0% fertilization in an in vitro fertilization system using mature oocytes, evaluation of their sperm by HZA was performed. The sperm bound poorly and the 95% confidence interval was 20 sperm bound. Thus, the fertile controls should bind >20 sperm to distinguish them from the infertile group in the HZA system resulting in a valid assay. With these guidelines, applications of the HZA may be made with greater reassurance of a valid bioassay of sperm fertilizing potential.