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Seminal plasma has emerged as a potential source of biomarkers and insights into the mechanisms of male infertility; however, the significance of polyunsaturated fatty acids (PUFAs) in this body fluid remains insufficiently explored. The main objectives of the present observational study were to compare selected PUFA (linoleic acid (LA), \u03b1-linolenic acid (ALA), \u03b3-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)) concentrations and ratios in seminal plasma between fertile (n\u2009=\u200922, aged 21\u201347) and infertile (n\u2009=\u2009250, aged 24\u201345) men, as well as within infertile subgroups, including idiopathic, to investigate their clinical utility and correlations with semen parameters. An optimized extraction procedure and a validated gas chromatography\u2013tandem mass spectrometry (GC\u2013MS\/MS) method were employed to determine the concentrations of PUFAs in seminal plasma. Nonparametric tests, including Mann\u2013Whitney U test, ANOVA rank test followed by Tukey\u2019s post hoc test, Spearman\u2019s rank test, and ROC curves were used for analyses. Among the twenty-one examined parameters, nine (LA, GLA, EPA, DHA, ALA\/LA, LA\/AA, EPA\/ALA, EPA\/GLA, EPA\/AA) exhibited significant differences between fertile control group and infertile patients (0.493 vs. 0.762\u00a0\u03bcg\/mL, <jats:italic>p<\/jats:italic>\u2009=\u20090.007; 0.029 vs. 0.035\u00a0\u03bcg\/mL, <jats:italic>p<\/jats:italic>\u2009=\u20090.005; 0.007 vs. 0.009\u00a0\u03bcg\/mL, <jats:italic>p<\/jats:italic>\u2009&lt;\u20090.001; 1.519 vs. 2.174\u00a0\u03bcg\/mL, <jats:italic>p<\/jats:italic>\u2009=\u20090.029; 0.066 vs. 0.053, <jats:italic>p<\/jats:italic>\u2009=\u20090.042; 0.248 vs. 0.305, <jats:italic>p<\/jats:italic>\u2009=\u20090.002; 0.168 vs. 0.216, <jats:italic>p<\/jats:italic>\u2009=\u20090.010; 0.230 vs. 0.283, <jats:italic>p<\/jats:italic>\u2009=\u20090.013; 0.003 vs. 0.004<jats:italic> p<\/jats:italic>\u2009=\u20090.045, respectively), with LA, GLA, LA\/AA, EPA\/ALA, and EPA\/GLA demonstrating limited (AUC\u2009&lt;\u20090.650), and EPA exhibiting moderate diagnostic value (AUC\u2009=\u20090.709). Among the subgroups of infertile patients, the teratozoospermic group displayed the most pronounced alterations in both individual PUFA concentrations and PUFA ratios compared to other groups (<jats:italic>p<\/jats:italic>\u2009&lt;\u20090.05). Notably, DHA, LA\/DHA, ALA\/DHA, GLA\/DHA, AA\/DHA, EPA\/DHA exhibited at least one moderate correlation with semen parameters (0.4\u2009&lt;|R|, <jats:italic>p<\/jats:italic>\u2009&lt;\u20090.05). Despite its exploratory nature, the present study provides insights into the mechanisms underlying male infertility and their reflection in seminal plasma composition while simultaneously outlining new directions in identifying\/excluding PUFA concentrations or ratios (especially LA, GLA, EPA, DHA, ALA\/LA, LA\/AA, EPA\/ALA, EPA\/GLA, EPA\/AA) as potential biomarkers. Moreover, the use of GC\u2013MS\/MS method offers a novel approach for potential future diagnostic applications.<\/jats:p>","DOI":"10.1038\/s41598-025-18044-4","type":"journal-article","created":{"date-parts":[[2025,10,3]],"date-time":"2025-10-03T10:28:31Z","timestamp":1759487311000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["GC\u2013MS\/MS analysis of seminal plasma PUFAs in distinct subgroups of infertile men: diagnostic potential and insight into mechanisms of male infertility"],"prefix":"10.1038","volume":"15","author":[{"given":"Kamil","family":"Rodak","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdalena","family":"Grajzer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Izabela","family":"Kokot","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ricardo","family":"Faundez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iwona","family":"Gilowska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Prescha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ewa Maria","family":"Kratz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,10,3]]},"reference":[{"key":"18044_CR1","unstructured":"WHO Laboratory Manual for the Examination and Processing of Human Semen. 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