Fig. 4: Cytokine production of peripheral blood CD4⁺ and CD8⁺ T cells from infertile and fertile individuals.

The ability to produce cytokines of CD3⁺ T cells in response to phorbol 12-myristate 13-acetate/Ionomycin (PMA/IONO) stimulation was evaluated by multiparametric flow cytometry. PBMC were isolated from fertile (FER, n = 25), Oligo-Astheno-Terato spermia (OAT, n = 30), and non-obstructive azoospermia (NOA, n = 10) men and activated for 6 h. a The percentage of CD8⁺ and CD4⁺ T cells producing the indicated cytokines is shown. Each dot represents a single donor, scatter plots indicate mean ± SEM; Kruskal-Wallis test in association with Dunn’s multiple comparison test were used to determine the statistical significance of the data. b–c The frequency of T helper subsets was evaluated in PBMC stimulated with PMA/IONO. b Representative gating strategy used to identify the frequencies of Th1 (CD4⁺IL-4-IL-17^-IFNg⁺), Th9 (CD4⁺IL-4^-IL-17^-IFNg^-IL-9⁺), Th2 (CD4⁺IL-17^-IFNg-IL-4⁺), and of Th17 (CD4⁺IL-4^-IFNg^-IL-17⁺) cell subsets; numbers indicate the percentage relative to the CD4⁺CD3⁺ gate. c Frequency of the indicated Th cell subsets among the three groups. Each dot represents a single donor; scatter plots indicate mean ± SEM; the Kruskal-Wallis test, in association with Dunn’s multiple comparison tests, was used to determine the statistical significance of the data, and statistically significant P values are reported. Source data are provided as a Source Data file.