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Background:

Accurate characterization of human sperm motility is crucial for understanding male fertility potential. Traditional motility assessment methods primarily focus on static velocity parameters, often overlooking temporal declines in motility during the sperm trajectory.

Objective:

We aimed to develop and validate a novel fatigue-based profiling approach to assess intra-trajectory motility decline in human spermatozoa.

Methods:

Using computer-assisted sperm analysis (CASA)-derived motion tracking data from 1,118 sperm trajectories, we introduced the Fatigue Index, a log-fold metric quantifying the decline in forward progression (VSL) over time. Fatigue status was classified using complementary strategies, including fixed and percentile-based thresholds, z-score normalization, and unsupervised clustering. Descriptive and feature-level analyses were performed to characterize motility patterns associated with fatigue.

Results:

Fatigued spermatozoa exhibited significantly lower straight-line velocity (VSL: 18.4 vs 42.7 μm/s) and steeper VSL slopes (−0.34 vs −0.08 μm/s/frame) compared to non-fatigued counterparts. The Fatigue Index reliably identified subpopulations of sperm with time-dependent motility deterioration across multiple classification schemes.

Conclusions:

Fatigue-based temporal profiling offers a new dimension for understanding sperm motility, highlighting the dynamic nature of forward progression and identifying subtle impairments that may be overlooked by conventional assessment methods. While preliminary, this approach provides a biologically grounded framework for dynamic sperm quality evaluation.