A SIMPLE TECHNIQUE TO QUANTIFY HUMAN SPERM-ZONA PELLUCIDA BINDING ASSAYS

A simplified method for assessing the degree of sperm-zona pellucida b inding was developed, The zonae pellucidae of salt-stored, failed-fert ilized human oocytes were each inseminated with between 1x10(5) and 1x 10(6) motile spermatozoa/ml, prepared by a direct swim-up method from 11 individuals with normal sperm counts, as defined by the World Healt h Organization, Following 4 h of incubation at 37 degrees C in humidif ied air, the zonae pellucidae were 'washed' by vigorous pipetting to r emove any loosely attached spermatozoa, The zonae were then placed ind ividually in microwells and dissolved by exposure to acidified (pH < 2 .0) medium to form a fluid monolayer, The slides were sealed and the n umber of spermatozoa in the monolayer counted by each of three observe rs within 24 h, There was good agreement in the counts between the dif ferent observers, with a mean coefficient of variation of only 7.4% an d a range of 1.9-16.7%. It was notable that the highest coefficients o f variation occurred at the extremes of sperm numbers and that the res ults were stable overnight, The method is also able to identify observ er bias within this variation, indicating the potential for improvemen ts in assay performance. The technique reported has the advantage over current sperm-zona pellucida binding assays of allowing the determina tion of the precise number of spermatozoa bound to a zona pellucida wh ile producing a slide which remains stable overnight.