Canonico, Luigi Fausto (2024) A microfluidic device for sperm selection based on different in-flow motion dynamics under variable viscosity conditions. [Tesi di dottorato]

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Abstract

For decades, male fertility diagnosis has relied on conventional manual sperm analysis mainly based on count, motility, and morphology. Despite exhibiting 'normal' sperm features, a significant number of men experience infertility, emphasizing the limitations of current diagnostic methods and necessitating a transition to innovative approaches. In response to this, some laboratories have adopted computer-assisted sperm analysis (CASA) alongside manual techniques. CASA provides the evaluation of different sperm features, such as motility and morphology, overcoming the subjectivity associated with manual analysis, but still providing discrepancies among different systems. However, routine semen analysis often fails in accurately assessing the fertilizing potential of sperm. Indeed, such analysis approaches do not account for the complex environment of the female reproductive tract (FRT), where sperm encounter various morpho-physical barriers that they need to overcome to achieve the fertilization of the oocyte. Nowadays, the current gold standard in assisted reproductive technology (ART) includes intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection. Despite their prevalence, these techniques yield only a modest 25-30% success rate. Indeed, they mainly rely on common sperm selection approaches comprising multiple centrifugation steps which poses damages on the cells and select them considering motility only. This gap highlights the need for the development of innovative methods. Our study introduces a novel approach that replicates a female-like environment to analyze and select sperm based on different in-flow motion dynamics while varying the viscosity of the fluid. We started from static and then moved to dynamic conditions by utilizing glass microscope observation chambers with varying depths and a microfluidic device. Cryopreserved bovine sperm were employed as a model for this study. Our objective was to comprehensively assess sperm behavior by comparing static and dynamic measurements across different fluid viscosities, emphasizing the importance of considering both conditions for a detailed diagnosis of sperm health. Cervical mucus viscosities were mimicked using biocompatible polymer solutions, while an in-house fabricated microfluidic device allowed us to assess sperm response to fluid-flow and rheology, particularly focusing on their ability to re-orient counter-flow, i.e. rheotaxis. Given the complex rototranslational motion of sperm, which results in a 3D motility pattern, our study investigated their rolling behavior and how fluid-flow and viscosity influence it. We proposed a selection mechanism based on distinct motion patterns and fluid-flow achieving a clear separation of sperm in highly and low motile classes by means of a more physiological approach. This separation allowed the extraction of sperm with higher motility and progressivity, generally associated with a positive fertilization outcome. A notable increase of sperm velocity in deeper chambers was observed, underscoring the influence of geometric confinement, and viscosity emerged as a crucial factor, affecting sperm velocity and modifying 3D motion. It induces a planarization of sperm 3D motion patterns suggesting a distinction between normal and altered motile sperm which allows an improved selection mechanism in contrast to conventional techniques. In addition to these findings, we propose the identification of additional parameters that could potentially enhance diagnostic precision and improve diagnostic outcomes. This comprehensive research provides additional insights into the intricate dynamics VII influencing sperm motility within the FRT, offering a versatile and cost-effective approach to enhance our understanding of male fertility. In summary, the suggested microfluidic selection mechanism is versatile, since within a single device, it enables the observation of various aspects of motility and their physiological interaction. This approach addresses key aspects of fertility diagnostics and treatment strategies for individuals experiencing reproductive challenges.

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