Oxidative stress occurs when the levels of reactive oxygen species (ROS) overwhelm antioxidant defences and has been strongly associated with male reproductive dysfunction. Hence, determination of ROS levels in semen is an important test for the assessment of male infertility. In this study, the percentage of live spermatozoa with oxidative stress (LOS) in native semen samples was evaluated using a flow cytometric technique, coupling the detection of sperm mitochondrial ROS with the MitoSOX™ probe (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with staining of dead cells using the LIVE/DEAD Fixable Dead Cell Stain kit (Thermo Fisher Scientific). Using this technique, LOS was evaluated in 80 infertile subjects and 16 cancer patients (testicular and haematological cancers); these patients were selected during routine semen analyses and cryopreservation of semen before cytotoxic therapy, respectively.
In infertile patients, the median LOS value was 24.80% (interquartile range [IQR]: 16.29–33.20). After grouping the patients according to the presence or absence of clinical signs of oxidative stress (inflammation and infection, smoking habit, leukocytospermia, semen viscosity, and semen bacteria), we found that the value of LOS was 28.56% (IQR: 25.01–40.79) in subjects with clinical signs of semen oxidative stress (n=42) and 17.18% (IQR: 12.18–21.71) in subjects without clinical signs (n=38; p=0.0001). To verify whether the percentage of LOS was able to identify patients with semen oxidative stress, we constructed receiver operating characteristic curves. It was found that the LOS percentage predicted the presence of clinical signs of oxidative stress with a good accuracy (area under the curve: 0.799; confidence interval: 0.692–0.906) and that when using 22.74% as a threshold value, the true-positive proportion was 86%, whereas the false-positive proportion was 21%.
Oxidative stress is one of the main mechanisms through which sperm DNA fragmentation (sDF) occurs. To verify whether live spermatozoa with mitochondrial ROS also exhibited higher levels of sDF, first live spermatozoa with and without mitochondrial ROS were sorted using a BD FACSAria™ (Becton Dickinson Biosciences, Franklin Lakes, New Jersey, USA) cell sorter. Subsequently, the two sorted fractions were processed with a comet assay. As expected, a higher amount of sDF in the spermatozoa with mitochondrial ROS was found compared to those without (median percentage tail intensity: 37.80±9.20% versus 27.90±4.80%, respectively; p=0.06).
In the 16 cancer patients, it was found that the LOS value was 41.53% (IQR: 28.71–62.10) (testicular cancer: n=9; 44.14% [IQR: 34.26–80.32]; haematological cancer: n=7; 31.22% [IQR: 18.00–47.26]), a value much higher than that observed in infertile subjects (p=0.0001), even when considering only those presenting with clinical signs of semen oxidation (p=0.017). No difference was found between patients with the two types of cancer (p=0.174).
In conclusion, this study shows a new flow cytometric technique for evaluating oxidative stress in live spermatozoa. Contrary to previous similar methods, this technique, does not use selected spermatozoa but instead investigates native semen samples, which are more representative of the in vivo condition. In addition, the procedure resulted in the identification of subjects with clinical signs of semen oxidative stress with a good accuracy. With this technique, it was found that semen from cancer patients exhibited very high levels of oxidative stress, which could explain the more detrimental effects of semen cryopreservation observed in these patients.