Oxidative stress and human semen quality: new results for diagnostic process of male infertility

Abstract Study question Is oxidative stress (OS) evaluated in human spermatozoa a predictive marker of better semen quality? Summary answer CellROX®Orange is a new fluorescent probe able to detect Reactive Oxygen Species (ROS) identifying a viable oxidized sperm fraction related to a better sperm performance. What is known already OS, defined as an unbalance between ROS production and antioxidant defences, is considered one of the causes of male infertility. OS evaluation in spermatozoa represents an important goal of research in this field because this parameter could be helpful to predict sperm fertilization ability to improve Assisted Reproductive Technology outcomes. By using different probes and methods for OS evaluation in spermatozoa or in semen, several studies were performed demonstrating a negative role of ROS on sperm functions. Such studies were not conclusive for the small number of included subjects, the high variability in the cohorts and the lack of validation. Study design, size, duration An observational study was conducted on 121 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from September 2021 to March 2022. Washed and Swim-up selected spermatozoa were incubated with two fluorescent probes, CellROX® Orange and Dihydroethidium (DHE), and revealed by flow cytometry. Participants/materials, setting, methods After routine semen analysis, the percentage of oxidized spermatozoa was evaluated with CellROX® Orange and DHE, and then correlated with standard semen parameters and sperm DNA fragmentation (sDF, evaluated by TUNEL/PI). Sperm kinematic parameters and hyperactivated motility were also assessed by C.A.S.A. system. Furthermore, CellROX® Orange positivity and phosphatidylserine membrane exposure (determined by Annexin V staining) or Caspase 3 and 7 activities (measured by FLICA™) were concomitantly evaluated. Main results and the role of chance We demonstrated that CellROX® Orange is able to detect hydrogen peroxide only in viable spermatozoa and the percentage of CellROX® Orange positive spermatozoa were positively associated with semen quality and negatively with sDF. To confirm these results, we performed the same experiments by using DHE, another probe which reveals hydrogen peroxide and superoxide anion in both viable and unviable cells. Similarly to CellROX® Orange, a positive correlation with semen quality and a negative one with sDF were found in viable spermatozoa. Conversely, in unviable spermatozoa, opposite associations were observed. These results indicate that oxidative status revealed by the two probes is related to a better sperm quality. To further investigate this possibility, we double labelled spermatozoa with CellROX® Orange and Annexin V (a marker of early signs of apoptosis) as well as with CellROX® Orange and FLICA™ (that detects caspase activity, a late sign of apoptosis). We found that CellROX® Orange mostly identifies spermatozoa without apoptotic features. Furthermore, we evaluated CellROX® Orange positivity in Swim-up selected spermatozoa, finding significantly higher levels in these samples respect to unselected samples, demonstrating once again that this probe identifies spermatozoa with better quality. Limitations, reasons for caution Our results indicate that within the viable oxidized spermatozoa there are cells with physiological and non-physiological intracellular ROS levels, however, they do not explain at what levels ROS cease to be functional and become deleterious for spermatozoa. Wider implications of the findings ROS evaluation in viable spermatozoa with the commercially available probes CellROX® Orange and DHE, allows the identification of the oxidized sperm fraction related to a sperm better performance. Therefore, the two probes may be useful to determine such fraction, likely improving the diagnostic process of male infertility. Trial registration number not applicable