Consistent presence of two normally distributed sperm subpopulations within normozoospermic human semen: a kinematic...

Sperm motion analysis was performed using computer-aided semen analysis (CASA) on nine normozoospermic human ejaculates following six fractionation schemes that included Percoll discontinuous gradient centrifugation, swim-up, hyper and hypo-osmotic stress in which 4600-13 400 tracks were analysed (depending on the scheme). In addition, the effect of ageing for 2 h in seminal plasma or re-addition of seminal plasma, on the kinematic properties was investigated. Analysis following sorting of the swimming velocity at 5 microm/sec intervals revealed the presence of two discrete normally distributed subpopulations selected on the basis of path velocity (VAP): these subpopulations were also present in the track speed (VCL) and progressive velocity (VSL) distributions. The validity of the two subpopulations was examined using a two-component normal mixture model and found to be highly statistically significant (p < 0.00001). The basis for the subpopulations was independent from the possession of midpiece vesicles or hyperactivation. Swim-up produced subpopulations of sperm that were significantly different from those obtained by Percoll separation. Compared with sperm in seminal plasma, swim-up and Percoll separation significantly increased the level of hyperactivated sperm at 2 h. Hyperactivation was inhibited by the re-addition of seminal plasma. Swim-up enhanced the proportion of sperm in the fast VAP subpopulation (independently of the level of hyperactivation) and whilst this altered distribution was maintained following the addition of homologous seminal plasma the mean velocity of the fast fraction was significantly decreased. Both swim-up and Percoll separation significantly increased the mean velocity of the fast subpopulation but the increase was greater with swim-up. This analysis highlights a way in which subpopulations may be detected and analysed. The procedure revealed fundamental differences in the kinematic properties of sperm processed by different methods that are used in assisted reproductive technology and showed that these were independent of hyperactivation.