Computer-assisted sperm motility analysis (CASA)

Computer-assisted sperm motility analysis (CASA) was performed as previously described [45]. The freshly isolated or frozen–thawed collected sperm was subjected to an assessment of sperm motility using the SMAS system (DITECT, Tokyo, Japan). The SMAS system automatically calculated several kinetic parameters of the sperm. Sperm moving at more than 25 μm/s were regarded as “progressive motile” sperm. The number of sperm analyzed per sample was at least 300, including immotile sperm. To evaluate hyperactive sperm, we further analyzed the CASA data by K-means clustering, as described in a previous report [45]. Briefly, sperm was classified into four clusters using the K-means method, based on straight-line velocity (VSL) and curvilinear velocity (VCL). Clustering analysis was performed using the JMP14 software package (SAS institute, Cary, North Carolina, USA). The cluster containing sperm with high VCL and low linearity (LIN; determined by VSL/VCL) was defined as the hyperactive sperm cluster.