{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:44:45Z","timestamp":1785422685684,"version":"3.56.0"},"reference-count":48,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,4,5]],"date-time":"2026-04-05T00:00:00Z","timestamp":1775347200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science and Innovation, Spain, by Instituto de Salud Carlos III, and was co-funded by the European Union","award":["PI24\/01600"],"award-info":[{"award-number":["PI24\/01600"]}]},{"award":["PI24\/01600"],"award-info":[{"award-number":["PI24\/01600"]}],"id":[{"id":"https:\/\/ror.org\/05r0vyz12","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Basque Government, Department of Education","award":["IT1547-22"],"award-info":[{"award-number":["IT1547-22"]}]},{"name":"Basque Government, Department of Education","award":["IT1966-26"],"award-info":[{"award-number":["IT1966-26"]}]},{"award":["IT1547-22"],"award-info":[{"award-number":["IT1547-22"]}],"id":[{"id":"https:\/\/ror.org\/00pz2fp31","id-type":"ROR","asserted-by":"publisher"}]},{"award":["IT1966-26"],"award-info":[{"award-number":["IT1966-26"]}],"id":[{"id":"https:\/\/ror.org\/00pz2fp31","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Researcher Fellowships from the Basque Government"},{"id":[{"id":"https:\/\/ror.org\/00pz2fp31","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003451","name":"UPV\/EHU","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003451","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/000xsnr85","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Human infertility affects approximately 17.5% of the global population, with male factors accounting for nearly half of all cases. Identifying reliable molecular biomarkers is crucial for improving the diagnosis and assessment of male fertility. This study established and refined an untargeted high-performance liquid chromatography\u2013electrospray ionization\u2013tandem mass spectrometry (HPLC-ESI-MS\/MS) protocol for a comprehensive lipidomic and metabolomic analysis of human spermatozoa, using only 1.25 million cells per sample. Compared with previous reports, our optimized method achieved an unparalleled level of analytical depth, identifying 473 lipid species and 955 structurally annotated metabolites. This corresponds to nearly a 7600-fold improvement in detection efficiency per cell compared with previously published approaches. Lipidomic analysis revealed that the most abundant lipid classes were glycerophospholipids (39%), cholesterol (20%) and fatty acids (19%), with cholesterol representing the single most abundant compound. This observation is consistent with the structural complexity of the sperm plasma membrane. Metabolomic profiling similarly identified glycerophospholipids (44%), eicosanoids (14%) and N-acyl amino acids (12%) as the major metabolite classes. The integration of lipidomic and metabolomic data highlighted functionally interconnected pathways related to membrane dynamics, energy metabolism, and hormone biosynthesis. Overall, this work establishes a robust, sensitive, and scalable analytical framework that enables the high-coverage molecular characterization of spermatozoa from limited sample material, laying the groundwork for future biomarker discovery and clinical applications in male infertility research.<\/jats:p>","DOI":"10.3390\/cells15070649","type":"journal-article","created":{"date-parts":[[2026,4,6]],"date-time":"2026-04-06T04:10:27Z","timestamp":1775448627000},"page":"649","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Lipidomic and Metabolomic Profiling on Low-Count Human Spermatozoa: A Robust and Reproducible Method for Untargeted HPLC-ESI-MS\/MS-Based Approach"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1787-6792","authenticated-orcid":false,"given":"Irune","family":"Calzado","sequence":"first","affiliation":[{"name":"Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Bizkaia, Spain"},{"name":"Bizkaia Health Research Institute, 48903 Barakaldo, Bizkaia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1838-4594","authenticated-orcid":false,"given":"Manu","family":"Araolaza","sequence":"additional","affiliation":[{"name":"Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Bizkaia, Spain"},{"name":"Bizkaia Health Research Institute, 48903 Barakaldo, Bizkaia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-5484-7414","authenticated-orcid":false,"given":"Mikel","family":"Albizuri","sequence":"additional","affiliation":[{"name":"Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Bizkaia, Spain"},{"name":"Bizkaia Health Research Institute, 48903 Barakaldo, Bizkaia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4540-7499","authenticated-orcid":false,"given":"Ainize","family":"Odriozola","sequence":"additional","affiliation":[{"name":"Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Bizkaia, Spain"},{"name":"Bizkaia Health Research Institute, 48903 Barakaldo, Bizkaia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5107-6991","authenticated-orcid":false,"given":"Iraia","family":"Mu\u00f1oa-Hoyos","sequence":"additional","affiliation":[{"name":"Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Bizkaia, Spain"},{"name":"Bizkaia Health Research Institute, 48903 Barakaldo, Bizkaia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-7532-0749","authenticated-orcid":false,"given":"Iratxe","family":"Ajuria-Morentin","sequence":"additional","affiliation":[{"name":"Galdakao-Usansolo Hospital, 48960 Galdakao, Bizkaia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nerea","family":"Subir\u00e1n","sequence":"additional","affiliation":[{"name":"Faculty of Medicine and Nursing, University of the Basque Country, 48940 Leioa, Bizkaia, Spain"},{"name":"Bizkaia Health Research Institute, 48903 Barakaldo, Bizkaia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,5]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2023). 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