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bioRxiv · 10.64898/2026.02.04.703749

Lipidomic and metabolomic profiling on low count human spermatozoa: A robust and reproducible method for untargeted HPLC-ESI-MS/MS-based approach

Calzado, I.·Araolaza, M.·Albizuri, M.·Odriozola, A.·Muinoa-Hoyos, I.·Ajuria-Morentin, I.·Subiran, N.
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Abstract

1.BackgroundHuman infertility affects approximately 17.5% of the global population, with male factors accounting for nearly half of all cases. The identification of reliable molecular biomarkers is crucial for improving the diagnosis and assessment of male fertility. In this study, we developed and optimized an untargeted high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) workflow for comprehensive lipidomic and metabolomic profiling of human spermatozoa using only 1.25 million cells per sample. ResultsCompared to previous reports, our optimized method achieved unprecedented analytical depth, identifying 473 lipid species and 955 structurally annotated metabolites, corresponding to nearly 7.600-fold improvements in detection efficiency per cell over published approaches. Lipidomic analysis revealed cholesterol, fatty acids, phosphatidylcholines, and phosphatidylethanolamine plasmalogens as the most abundant lipid classes, consistent with the structural complexity of the sperm plasma membrane. Metabolomic profiling showed strong enrichment of lipid-related and steroidogenic pathways, including phospholipid biosynthesis, glycerolipid metabolism and androgen and estrogen metabolism. The integration of lipidomic and metabolomic data highlighted functionally interconnected pathways related to membrane dynamics, energy metabolism, and hormone biosynthesis. ConclusionsOverall, this work establishes a robust, sensitive, and scalable analytical framework enabling high-coverage molecular characterization of spermatozoa from limited sample material, laying the groundwork for future biomarker discovery and clinical applications in male infertility research. One Sentence SummaryDevelopment of a highly sensitive untargeted HPLC-ESI-MS/MS lipidomic and metabolomic workflow that achieves unprecedented molecular coverage from only 1.25 million human spermatozoa, revealing interconnected lipid and metabolic pathways and providing a robust foundation for biomarker discovery in male infertility. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/703749v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@10b3132org.highwire.dtl.DTLVardef@1caf850org.highwire.dtl.DTLVardef@746adborg.highwire.dtl.DTLVardef@1135539_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Calzado, I., Araolaza, M., Albizuri, M., Odriozola, A., Muinoa-Hoyos, I., Ajuria-Morentin, I., Subiran, N.. 2026-02-06. Lipidomic and metabolomic profiling on low count human spermatozoa: A robust and reproducible method for untargeted HPLC-ESI-MS/MS-based approach. https://doi.org/10.64898/2026.02.04.703749

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