Advanced analysis of human sperm using computer-aided sperm analysis and flagellar applications

Abstract

Semen analysis provides insight into male fertility and overall reproductive health. While such analyses were traditionally subjective, technological advances have introduced a more objective approach, such as computer-aided sperm analysis (CASA). However, conventional CASA primarily tracks sperm head centroid movement and is not designed to track the sperm component responsible for sperm motility, namely the flagellum. As a result, the correlation among sperm head kinematics, flagellar dynamics and overall sperm quality remains poorly understood. Additionally, the optimization of technical factors (such as cut-off values for sperm parameters in CASA, frame rate and chamber depth for flagellar analysis) has not yet been established, forming an integral aspect of this study. This study aimed to investigate the effects of different frame rates and chamber depths for flagellar waveform analysis. It also sought to assess how biologically relevant environments influence the functionality of sperm subpopulations. The research intended to use established mathematical modeling software based on the physical principles of movement to explain sperm motility from a physiological perspective by comparing conventional CASA with flagellar tracking software and to provide insights into the structural features of the flagellum, such as the centrioles. Human semen samples were separated into high motile (HM) and low motile (LM) sperm subpopulations by the double density gradient centrifugation technique to model fertile and subfertile samples. In the first phase of the study, sperm subpopulations in human tubal fluid (HTF) were assessed using CASA to examine the impact of technical factors such as frame rate and chamber depth on sperm kinematics, thereby optimizing and standardizing procedures to identify the technical conditions for flagellar waveform analysis. In the second phase, the subpopulations were treated with a viscous 1% methylcellulose medium and HD capacitating (HD-C) medium to evaluate sperm functionality, including sperm kinematics, flagellar waveform features and hyperactivation in biologically relevant environments.