Mitochondrial morphology in fertile and infertile men: image processing and morphometric analysis of the sperm midpiece

Rossana Sapiro^1,2,3*Fernanda Skowronek⁴Diego Silvera⁵Gabriel Decola⁵Mauricio Ramos⁵Santiago Pietroroia⁴Gabriela Casanova⁶Federico Lecumberry⁵Adriana Maria Cassina^3,7

• ¹Rossana Sapiro, Montevideo, Uruguay
• ²Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
• ³Centro de Investigaciones Biomédicas (CEINBIO), Montevideo, Uruguay
• ⁴Unidadd académica Histología Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
• ⁵Departamento de Procesamiento de Señales, Facultad de Ingeniería, Universidad de la República, Montevideo, Uruguay
• ⁶Unidad de Microscopía Electrónica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
• ⁷Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

The male factor is responsible for 50% of infertility cases. Numerous studies have explored the relationship between sperm morphology-assessed via optical and electron microscopy-and reproductive outcomes. In the sperm midpiece, mitochondria are arranged in a helical shape, forming a compact sheath, critical for sperm function. Disruptions in this precise mitochondrial structure, size, or organization may contribute to infertility. However, despite established links between abnormal sperm morphology and pathology, mitochondrial abnormalities in sperm remain relatively understudied. In this study, we employed computational image analysis and fluorescence labeling to quantitatively assess morphometric changes in the sperm midpiece and correlate these findings with mitochondrial ultrastructure in fertile and infertile men. Our results revealed a significant increase in midpiece area, width, and roundness in sperm from men with teratozoospermia. These findings were further validated by electron microscopy. The ultrastructural morphometric analysis demonstrated disassembled, enlarged, and irregularly shaped mitochondria in sperm from infertile men. Additionally, we applied ultrastructural morphometric analyses to apoptotic sperm samples, observing similar qualitative and quantitative mitochondrial alterations, particularly in those from infertile individuals. Taken together, our findings suggest that morphometric changes in the sperm midpiece and mitochondrial ultrastructure may be markedly associated with male infertility. Developing computational tools to detect and quantify these morphometric changes is a promising approach for enhancing diagnostic accuracy and advancing our understanding of the underlying mechanisms of male infertility.