Infertility affects 15–20% of couples who try to conceive [1]. According to World Health Organization (WHO) recommendations, in half of these instances, the male factors are the reason [2]. Oxidative stress (OS) and reactive oxygen species (ROS), notably superoxide anions (O2.-), induce male infertility [3]. Lower O2.- levels are physiologically required for capacitation, acrosome reaction, hyperactivation, and sperm-oocyte fusion [4,5]. High levels of O2.- (pathologically) disrupt sperm cell membranes, cause DNA damage, and induce death. In male infertility, sperm motility, vitality, morphology, and cell concentration are diminished [6]. Oxidative stress is an imbalance between oxidants and antioxidants [7]. Mitochondria are abundant in motile sperm cells, and O2.- radicals are formed by electron leakage from the oxidation-reduction pathway [8].

Trapping and detection of O2.- is very difficult due to its short half-life (1 × 106 s) [9]. There are various methods for measuring oxidative stress (direct and indirect) [10]. The effects of O2.- on macromolecules are measured indirectly (lipids, proteins and DNA). In comparison with direct methods, these methods lack high specificity and sensitivity [11]. One of the most important direct methods is luminol-based chemiluminescence, which has high sensitivity and specificity [12,13]. The disadvantage of this method is the lack of specificity for O2.-. The luminol-based chemiluminescence approach can evaluate intracellular stress quantitatively and comprehensively with great sensitivity, and the cut-off values were calculated depending on the type of semen sample [14]. This approach detects hydrogen peroxide (H2O2), not O2.-. Since H2O2 is an intermediary of O2.-, numerous variables may affect its generation and conversion [15,16]. Therefore, specialized techniques with high sensitivity are required for the detection of O2.- [17]. Several high-sensitivity and specificity fluorescence and chemiluminescence techniques have recently been developed for the direct detection of O2.- [18]. Recently, our previous bioluminescence detection of O2.- using photoprotein aequorin was developed [19]. This process is based on the intensity of bioluminescence produced by aequorin. With the oxidation of coelenterazine (CTZ) as a superoxide-sensitive chromophore, under O2.- conditions, the regeneration of apoaequorin decreases, and finally, the intensity of bioluminescence decreases. According to the findings, the bioluminescence system based on Aeq/CTZ can be used as a very sensitive, selective, and easy-to-use probe in the detection of O2.- in laboratory conditions [19]. In this method, O2.- converts coelenterazine to coelenteramide, and then the changes in the effective concentration of coelenterazine are measured through apoaequorin [20,21]. Coelenteramide, although it binds to apoaequorin, cannot produce bioluminescence [21]. Therefore, the sensitivity of the method can be increased by trapping O2.- through coelenterazine and then measuring the bioluminescence intensity by apoaequorin [19].

The purpose of our study was to 1) analyze the correlation between O2.- levels and conventional semen parameters in normal individuals and infertile patients and 2) establish cut-off values of O2.- levels in normal individuals compared with infertile patients.