Sperm kinetics and morphology before and after fractionation on discontinuous Percoll gradient for sex preselection: computerized analyses.

Abstract

The multiple-layer discontinuous Percoll density gradient centrifugation procedure is being used for gender selection and several reports suggested separation efficiencies of over 77%. The mechanism involved in the separation of X- and Y-bearing sperm using this method seems to be the difference in sperm head dimensions or motility but supporting data are inconsistent. The specific aims of the study were to evaluate the head dimensions of sperm at the upper and lower fractions after the 8-layer Percoll gradient procedure for sex preselection and to ascertain the kinematic parameters and tail lengths of sperm derived from the 2 separate Percoll fractions. Sperm cells were obtained from thawed donor specimens (N = 20) and were layered on top of the 8-layer discontinuous Percoll gradient, which ranged from 34 to 85% in increments of 7%. After centrifugation, the resuspended sperm cells derived from the upper and lower fractions of the Percoll gradient were analyzed on the Hamilton Thorn HTM-C analyzer for differences in sperm motility patterns and sperm head dimensions. Aliquots of sperm from the 2 fractions were fixed and stained using the Spermac stain, and the lengths of each sperm tail (N = 600) were measured on the HTM-C analyzer. Sperm derived from the bottom Percoll (X) fraction had a threefold higher (p < .05) percent motility when compared with sperm from the top (Y) fraction. Sperm derived from the bottom (X) fraction maintained the higher percentage motility after 24 h of incubation. The percent total progression, rapid progression, and hyperactivation were also significantly higher (p < .05) in sperm from the bottom (X) fraction. Similarly, the curvilinear (Vcl), average path (Vap) and straight line (Vsl) velocities were significantly faster in sperm (p < .05) from the bottom (X) fraction. In contrast, the percent linearity and straightness were significantly (p < .05) higher for the top (Y) fraction. Sperm from the bottom (X) fraction have shorter (p < .07) tail length (1.6% difference) when compared with sperm from the top (Y) fraction. Although the dimensions of the sperm head from the bottom (X) fraction were numerically greater than top fraction sperm, they were not significant (p > .05). The results suggest that bottom (X) fraction sperm derived from the 8-layer discontinuous Percoll gradient for sex preselection have higher motility, progression, and hyperactivation when compared with top (Y) fraction sperm. The bottom (X) fraction sperm have greater longevity in motility and have shorter tails, supporting earlier hypotheses of sex differences in sperm parameters. However, the present data do not support observations of differences in sperm head dimensions in sperm processed for sex preselection, and an inference of a larger sperm due to more chromosome material originating from the X chromosome cannot be made.