Novel Fatigue Profiling Approach Highlights Temporal Dynamics of Human Sperm Motility

  1. Athanasia Sergounioti
  2. Efstathios Alonaris
  3. Dimitrios Rigas

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Abstract

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.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/15887836.

    This review is the result of a virtual, collaborative Live Review discussion organized and hosted by PREreview and JMIR Publications on June 26, 2025. The discussion was joined by 14 people: 2 facilitators from the PREreview Team, 1 member of the JMIR Publications team, 1 author, and 10 live review participants. The authors of this review have dedicated additional asynchronous time over the course of two weeks to help compose this final report using the notes from the Live Review. We thank all participants who contributed to the discussion and made it possible for us to provide feedback on this preprint.

    Summary

    This preprint presents a well-structured study that introduces the concept of "sperm fatigue" as a novel framework for assessing motility deterioration in human sperm. The study aims to develop and validate a new profiling method to assess infra-trajectory motility decline in human spermatozoa. Using a metric termed the "Fatigue Index" and data from computer-assisted sperm analysis (CASA) systems, the authors demonstrate that infra-trajectory motility decline is both measurable and biologically plausible, with potential links to mitochondrial dysfunction and oxidative stress. This approach aids in identifying sperm with potential subclinical impairments and informs the development of predictive biomarkers for sperm functionality and male fertility evaluation. The methodology is clearly described, and the inclusion of shared code and data exemplifies strong open science practices. Below, we summarize the main points discussed during the Live Review and offer suggestions for improving the manuscript. Minor enhancements to documentation and accessibility could further support its broader application across disciplines.

    List of major concerns and feedback:

    • Clarify dataset selection and segmentation process: The manuscript lacks sufficient detail regarding the selection and segmentation of video clips from the VISEM dataset. To improve transparency and reproducibility, please provide a clear explanation of how video segments were selected for analysis, including specific criteria such as duration, quality, sample characteristics, or relevance to the study's aims. Additionally, include a brief description of the origin of the VISEM dataset, emphasizing that the clips used in this study were curated from full-length videos. Please, clarify how the original dataset was constructed by its curators and how your study further selected, filtered, or modified these data. Finally, cite the original VISEM publication and relevant documentation to support clarity and reproducibility.

    • Missing ethics statement: Although the study likely follows ethical standards, it would be best practice to include a short ethics statement. Since the VISEM dataset is based on human sperm samples, the authors could briefly mention the original ethical approval and cite the VISSEM source publication to clarify this point 

    List of minor concerns and feedback

    • Reproducibility details can be improved: While the authors share code and data, it is not clear which versions of libraries and frameworks were used, and how they were applied in the analysis. Please provide a list of all software tools and libraries used, including their versions and sources. For example, "Data analysis was conducted using Python (version number) with the following open-source packages: scikit-learn (version number) for model building, PyMC (version number) for Bayesian inference, matplotlib (version number) for visualization, and pandas (version number) for data manipulation. Doing this will help validation and reuse, especially in less-resourced settings.

    • Figures need better resolution: Some figures (e.g., 1A and 1B) appear small or low-resolution and are difficult to read without zooming in. Increasing the image resolution and adjusting layout for clarity would improve readability. Also, authors may consider removing box characters from figure legends to make their content more readable and clear.

    • Clarify limitations and generalizability: The author mentions limitations, but it would be helpful to more clearly state what the study cannot determine. Also specifying the populations from which the samples were obtained and discussing whether results might differ in other populations would improve transparency.  

    • Visual abstract for non expert audience: Given the potential interest from a broad audience, including non-experts, reviewers suggested adding a non-technical summary of the findings—potentially in the form of a visual abstract. It may also be helpful to reflect on which other disciplinary fields could find this topic relevant.

    Concluding remarks

    Overall, Live Review participants found this to be a well-constructed study. We thank the authors of the preprint for posting their work openly for feedback. We also thank all participants of the Live Review call for their time and for engaging in the lively discussion that generated this review.

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2025.04.27.650828v4 on bioRxiv
  3. Version published to 10.1101/2025.04.27.650828v3 on bioRxiv
  4. Version published to 10.1101/2025.04.27.650828v2 on bioRxiv
  5. Version published to 10.1101/2025.04.27.650828v1 on bioRxiv