Factors influencing human sperm kinematic measurements by the Celltrak computer-assisted sperm analysis system

Abstract

This study examines the effect of varying several factors, both extrinsic and technology-dependent, on the reconstruction of human sperm trajectories and the derived kinematic measurements using videotapes and the Motion Analysis Celltrak/S instrument. In semen samples from normal healthy men, curvilinear (VCL) and straight line velocities (VSL) were found to increase 1.5-fold, and linearity (LIN) of trajectories and amplitude of lateral head displacement (ALH) increased 1.25-fold when the temperature of analysis was raised from 24 to 37°C. Only VCL and VSL were found to increase significantly between 24 and 37°C for sperm samples selected by Percoll gradient and incubated in a capacitating medium. An analysis chamber of 20 μm depth was found to be adequate for seminal sperm samples while for Percoll-selected sperm samples the analysis in a 50 μm depth provided the highest proportions of spermatozoa with the highest VCL and the largest ALH. The grey level detection threshold required careful adjustment: using a threshold lower than the optimal threshold produced spurious sperm trajectories for seminal sperm samples and rejected some trajectories for Percoll-selected sperm samples. Definition of the appropriate frame rate and maximum burst speed was critical for valid trajectory reconstruction and therefore adequate derived kinematic measurements. Optimal values of these parameters were found to be 30 Hz and 400 μm/s for seminal spermatozoa and 60 Hz and 700 μm/s for selected spermatozoa. The optimal values of 'ALH path-smoothing factor' used to calculate average path and ALH were 5-10 points for seminal spermatozoa analysed at 30 Hz and 15-20 points for selected spermatozoa analysed at 60 Hz. We propose a set of standard conditions for reliable kinematic analysis of human spermatozoa using the Celltrak/S system.