Prolonged 3.5 GHz and 24 GHz RF-EMF Exposure Alters Testicular Immune Balance, Apoptotic Gene Expression, and Sperm Function in Rats

<b>Background/Objectives:</b> The rapid rollout of 5G has renewed interest in potential reproductive effects of mid-band (3.5 GHz) and millimeter-wave (24 GHz) radiofrequency electromagnetic fields (RF-EMF). We examined frequency- and duration-dependent changes in testicular cytokines, apoptosis-related genes, and sperm quality in rats. <b>Methods:</b> Male Sprague Dawley rats (n = 6 per group) were exposed for 60 days to 3.5 GHz or 24 GHz RF-EMF for 1 h/day or 7 h/day. The sham controls were housed identically. Testicular expressions of IL-10, IL-6, IL-1β, and TNF-α were quantified; <i>Tp53</i>, <i>Bax</i>, <i>Bcl2,</i> and <i>Casp3</i> mRNA expressions were measured; and sperm concentration, viability, and motility were evaluated. <b>Results:</b> IL-10 was significantly reduced in the 24 GHz group at both 1-h and 7-h exposure duration. At 7 h, TNF-α was also lower at 24 GHz. <i>Casp3</i> expression was higher and <i>Tp53</i> was lower at 3.5 GHz at 1-h exposure duration. Sperm concentration and viability were reduced after 24 GHz exposure at 7 h, while sperm motility was reduced after 3.5 GHz exposure at both durations. <b>Conclusions:</b> Exposure to RF-EMF 3.5 GHz primarily impacts sperm motility via extrinsic pro-apoptotic pathways, while exposure to 24 GHz impacts sperm concentration and viability potentially through immune-apoptotic mechanisms, with all negative effects amplified by 7-h daily exposure.