GC–MS/MS analysis of seminal plasma PUFAs in distinct subgroups of infertile men: diagnostic potential and insight into mechanisms of male infertility

Infertility affects approximately 17.5% of the adult population, with the male factor contributing to nearly half of the cases. 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), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)) concentrations and ratios in seminal plasma between fertile (n = 22, aged 21-47) and infertile (n = 250, aged 24-45) 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-tandem mass spectrometry (GC-MS/MS) method were employed to determine the concentrations of PUFAs in seminal plasma. Nonparametric tests, including Mann-Whitney U test, ANOVA rank test followed by Tukey's post hoc test, Spearman's 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 μg/mL, p = 0.007; 0.029 vs. 0.035 μg/mL, p = 0.005; 0.007 vs. 0.009 μg/mL, p < 0.001; 1.519 vs. 2.174 μg/mL, p = 0.029; 0.066 vs. 0.053, p = 0.042; 0.248 vs. 0.305, p = 0.002; 0.168 vs. 0.216, p = 0.010; 0.230 vs. 0.283, p = 0.013; 0.003 vs. 0.004 p = 0.045, respectively), with LA, GLA, LA/AA, EPA/ALA, and EPA/GLA demonstrating limited (AUC < 0.650), and EPA exhibiting moderate diagnostic value (AUC = 0.709). 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 (p < 0.05). Notably, DHA, LA/DHA, ALA/DHA, GLA/DHA, AA/DHA, EPA/DHA exhibited at least one moderate correlation with semen parameters (0.4 <|R|, p < 0.05). 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-MS/MS method offers a novel approach for potential future diagnostic applications.