**Background:** Worldwide, 15% of couples are affected by infertility, with male factors responsible for half of cases. Oxidative stress (OS), caused by an imbalance between reactive oxygen species (ROS) and antioxidants, is a key contributor to male infertility, particularly in idiopathic cases. Spermatozoa are highly susceptible to OS due to limited antioxidant enzymes (e.g., catalase is absent, superoxide dismutase activity is low) and a high content of polyunsaturated fatty acids. The oxido-reduction potential (ORP) measured by the Mioxsys system is a novel, fast, and user-friendly method to assess OS in semen, but its reliability is debated due to the influence of sperm concentration. This study aimed to confirm correlations between ORP and sperm parameters, examine the impact of sperm concentration, and determine whether intracellular ROS formation is reflected in ORP levels using three different OS inducers: menadione (superoxide anion generator), hydrogen peroxide (direct OS source), and tert-butyl hydroperoxide (lipid peroxidation inducer).
**Methods:** The study had two parts. First, a retrospective analysis of 183 semen samples from 2022 was performed, comparing oligozoospermic (<16 M/mL sperm) and non-oligozoospermic (>16 M/mL) groups for sperm parameters (motility, progressive motility, viability, DNA fragmentation) and ORP (both normalized and non-normalized). Second, experimental treatments were conducted on semen samples from 10 volunteers per group (inclusion criteria: ≥30 M/mL sperm count, ≥70% viability, maximum ORP of 1.36 mV/10^6/mL, ≥3.0 mL volume). Samples were divided into raw semen and plasma-free (washed) groups, adjusted to 20 M/mL. Aliquots were treated with 0, 1, 2, or 5 mM of H2O2, menadione, or t-BuOOH for 2 hours at 37°C, with 5% DMSO as control. After treatment, sperm motility, progressive motility, and viability were measured using an SCA SCOPE system, and ORP was measured using the Mioxsys system (30 μL sample, results in mV, normalized to sperm concentration). Statistical analysis used Mann-Whitney test.
**Key Results:** In the retrospective analysis, oligozoospermic samples (n=69, mean concentration 8.3 ± 4.1 M/mL) had significantly lower motility (25.8 ± 14.1% vs. 37.4 ± 17.8%, p<0.1%), progressive motility (19.7 ± 13.1% vs. 28.8 ± 15.8%), and viability (60.5 ± 8.7% vs. 67.4 ± 11.6%, p<0.1%), but higher normalized ORP (8.5 ± 13.9 mV/10^6/mL vs. 1.0 ± 0.9 mV/10^6/mL, p<1%) compared to non-oligozoospermic samples (n=90, mean concentration 61.6 ± 58.8 M/mL). Non-normalized ORP did not differ significantly (38.5 ± 16.6 mV vs. 40.6 ± 25.5 mV). In treatment experiments, all inducers caused concentration-dependent decreases in viability, motility, and progressive motility. For raw semen, 5 mM H2O2 caused 64.1% decrease in viability, 84.1% in motility, and 100% in progressive motility; 5 mM menadione caused 51.9%, 100%, and 100% decreases, respectively; 5 mM t-BuOOH caused 41.2%, 86.7%, and 94.1% decreases. ORP changes: H2O2 at 1, 2, 5 mM caused 0.98-, 1.13-, and 1.42-fold increments; menadione caused 3.73-, 4.41-, and 5.02-fold increments; t-BuOOH caused 1.06-, 1.25-, and 1.57-fold increments. In plasma-free samples, effects were more severe (e.g., 1 mM H2O2 caused 53.7% viability decrease vs. 12.1% in raw), and ORP increments were smaller (e.g., menadione: 1.23-, 1.27-, 1.27-fold). Initial ORP in plasma-free samples was 6-7-fold higher than raw.
**Clinical Implications:** The study confirms that ORP correlates with sperm parameters but is heavily influenced by sperm concentration, with problematic reliability below 10 × 10^6 sperm/mL and above 50 × 10^6 sperm/mL. The Mioxsys system can detect intracellular ROS from different sources, with menadione showing the strongest ORP response, suggesting low SOD activity in sperm. Seminal plasma provides significant antioxidant protection, as its removal worsened sperm damage and altered ORP patterns. While ORP measurement is fast and user-friendly, its clinical utility is limited by concentration-dependent distortions and high consumable costs. The authors recommend further studies to clarify acceptable concentration ranges and to validate ORP as a routine diagnostic tool for male oxidative stress infertility (MOSI). Antioxidant therapy remains controversial due to conflicting evidence from trials like MOXI, and current guidelines (EAU, EAA, ESHRE) do not recommend routine ROS measurement.