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A NaCl-free cryopreservation carrier medium based on the use of histidine as the major osmolyte was designed that was capable of sustaining human sperm motility over 6\u00a0days at ambient temperature. This medium was supplemented with ethylene glycol, glycerol, and DMSO to create the basis for a novel cryopreservation medium. Dose-dependent studies with various supplements were then conducted to optimize the effectiveness of this formulation including assessments of vitamin C, EDTA, crocin, zinc, ergothioneine, and <jats:italic>myo<\/jats:italic>-inositol, as well as the potential replacement of DMSO by Cyrene\u2122. Post-thaw samples were assessed for motility, vitality, and DNA integrity and then reassessed following sperm isolation with the Felix\u2122 System.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>The completed cryopreservation formulation comprised 4.5% ethylene glycol, 4.5% glycerol, 1% DMSO in a carrier medium supplemented with 0.4\u00a0mM vitamin C, 1\u00a0mM EDTA, and 22\u00a0mM <jats:italic>myo<\/jats:italic>-inositol. Spermatozoa frozen in this medium and isolated using the Felix\u2122 System had significantly greater total motility, progressive motility, vitality, and DNA integrity than spermatozoa frozen in a commercially available product that is widely used in infertility clinics.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>A novel cryopreservation medium has been developed in this study that represents a significant improvement over existing technologies.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1007\/s10815-025-03525-2","type":"journal-article","created":{"date-parts":[[2025,5,29]],"date-time":"2025-05-29T18:42:16Z","timestamp":1748544136000},"page":"2167-2180","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Development of an improved medium for the preservation of human spermatozoa"],"prefix":"10.1007","volume":"42","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5447-9358","authenticated-orcid":false,"given":"Alena J.","family":"Hungerford","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9864-7686","authenticated-orcid":false,"given":"Natasha","family":"Harrison","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6453-1730","authenticated-orcid":false,"given":"Hassan W.","family":"Bakos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9152-156X","authenticated-orcid":false,"given":"Robert J.","family":"Aitken","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,5,30]]},"reference":[{"key":"3525_CR1","doi-asserted-by":"publisher","first-page":"1364352","DOI":"10.3389\/frph.2024.1364352","volume":"6","author":"RJ Aitken","year":"2024","unstructured":"Aitken RJ. 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