A simple technique to quantify human sperm-zona pellucida binding assays

A simplified method for assessing the degree of sperm-zona pellucida binding was developed. The zonae pellucidae of salt-stored, failed-fertilized human oocytes were each inseminated with between 1 x 10(5) and 1 x 10(6) motile spermatozoa/ml, prepared by a direct swim-up method from 11 individuals with normal sperm counts, as defined by the World Health Organization. Following 4 h of incubation at 37 degrees C in humidified air, the zonae pellucidae were 'washed' by vigorous pipetting to remove any loosely attached spermatozoa. The zonae were then placed individually in microwells and dissolved by exposure to acidified (pH < 2.0) medium to form a fluid monolayer. The slides were sealed and the number of spermatozoa in the monolayer counted by each of three observers within 24 h. There was good agreement in the counts between the different observers, with a mean coefficient of variation of only 7.4% and a range of 1.8-16.7%. It was notable that the highest coefficients of variation occurred at the extremes of sperm numbers and that the results were stable overnight. The method is also able to identify observer bias within this variation, indicating the potential for improvements in assay performance. The technique reported has the advantage over current sperm-zona pellucida binding assays of allowing the determination of the precise number of spermatozoa bound to a zona pellucida while producing a slide which remains stable overnight.