**8. Methods for detecting reactive oxygen species**

Because high levels of ROS have been associated with a decreased male infertility, measuring ROS levels in semen is an important part of the initial evaluation as well as follow-up of men with reproductive dysfunction [10–12]. Chemiluminescence and flow cytometry are currently the most common techniques in clinical andrology to assess and study seminal OS.

Chemiluminescence measures light emitted following administration of specific reagents to a semen sample. Two major probes currently used to assess ROS generation by spermatozoa are luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (10,10′-dimethyl-9,9′ biacridinium dinitrate). Lucigenin is membrane-impermeable and responsive to ROS, particularly O2 ●−, in the extracellular space. Inversely, luminol is relatively membrane-permeable and reacts with a variety of ROS, including O2 ●−, H2 O2 and OH● intracellularly as well as extracellularly. Chemiluminescent assays are sensitive, convenient for diagnostic purposes and have relatively well-established normal ranges [11, 12]. Nevertheless, significant set up costs have to be taken into consideration, and the data generated by chemiluminescence must be interpreted carefully because a variety of factors can affect the signals obtained [181].

mechanisms crucial for fertility. The origin of ROS generation and the etiologies of increased ROS in men with low sperm quality are becoming increasingly clear, offering multiple management and/or treatment options. Recent evidence suggests that spermatozoa possess an inherent ability to generate ROS essential for the fertilization process. A variety of defense mechanisms against ROS overproduction encompassing antioxidant enzymes, vitamins and other biologically active molecules are involved in biological systems. A balance of the benefits and risks from free radical production seems to be crucial for the sperm survival and function. As male infertility continues to play an increasing role in contributing to the inability to conceive in couples of reproductive age, it is pivotal for andrologists to fully comprehend the importance of thoroughly evaluating seminal oxidative profiles in order to provide a better care for male patients with reproductive dysfunction. Although the therapeutic use of antioxidants appears attractive, clinicians need to be aware of exaggerated claims of antioxidant benefits by various commercial supplements for fertility purposes until proper multicenter trials have been completed. However, initial data emphasizing on the potential of antioxidant supplementation in improving semen quality and conception rates are indeed encouraging.

Physiological and Pathological Roles of Free Radicals in Male Reproduction

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This study was supported by the Slovak Research and Development Agency grants APVV-

**Acknowledgements**

15-0543 and APVV-15-0544.

**List of abbreviations**

AA Ascorbic acid

Ca2+ Calcium

CAT Catalase

Fe2+ Ferrous ion Fe3+ Ferric ion

FR Free radical

AR Acrosome reaction

ATP Adenosine triphosphate

ABTS 2,20-azino-di-3-ethylbenzthiazoline sulfonate

ARTs Artificial reproduction techniques

cAMP Cyclic adenosine monophosphate

G6PD Glucose-6-phosphate dehydrogenase

A possible solution to the disadvantages associated with the chemiluminescence approach can be found in a variety of redox-sensitive fluorescence probes that can be loaded into spermatozoa and subsequently monitored by flow cytometry [182]. Two probes can be used. Dihydroethidium or hydroethidine is a non-fluorescent probe that is oxidized by the superoxide to become ethidium bromide, which will stain the mitochondrial and nuclear DNA [183, 184]. The other fluorescent probe is 2,7-dichlorofluorescein diacetate, a stable non-fluorescent cell-permeable probe that de-esterifies in the presence of intracellular H<sup>2</sup> O2 to form 2,7-dichlorofluorescein [183]. Other ROS such as peroxynitrite, HOCl, and OH● can also oxidize this probe [184]. Flow cytometry has a higher specificity, accuracy, sensitivity and reproducibility than fluorescent microscopy or chemiluminescence. A large number of cells can easily be analyzed, leading to high specificity and sensitivity [185]. One major disadvantage is that sophisticated and expensive hardware is needed. Also, the results do not quantify the target ROS but simply indicate the percentage of cells exhibiting a high level of activity [182].

Other methods to assess the oxidative balance in semen include indirect measurements such as the total antioxidant assay. This protocol is based on the ability of all antioxidants present in the sample to cease the oxidation of 2,20-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) to ABTS+ by metmyoglobin. Hence, the antioxidants suppress oxidative processes to a degree that is proportional to their final concentration, which may be detected at 750 nm [186]. Another option is to assess the activity of antioxidant enzymes (SOD, CAT, GPx) or the redox potential defined by the ratio of oxidized and reduced glutathione using commercially available assay kits. A popular option is the measurement of oxidative end-products, including protein carbonyls [187], lipid hydroperoxides [188], MDA [98] and oxidative DNA adduct 8-hydroxy 2-deoxyguanosine [189].

Despite a remarkable progress in the evolution and design of new techniques to evaluate seminal OS, more straight-forward and accessible assays with well-defined and clinically significant physiological ranges reflecting normal sperm functions have yet to be introduced in order for oxidative stress to become a standard sub- or infertility marker in andrology laboratories.
