**Acknowledgements**

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, partic-

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].

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

O2

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

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

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

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

Oxygen toxicity is an inherent double-edged sword to aerobic life. Increased oxidative insults to sperm lipids, proteins and DNA are associated with alterations of signal transduction

by metmyoglobin. Hence, the antioxidants suppress oxidative processes to

●−, in the extracellular space. Inversely, luminol is relatively membrane-permeable

●−, H2 O2

and OH● intracellularly as well as

to form 2,7-dichlorofluorescein [183]. Other

ularly O2

140 Spermatozoa - Facts and Perspectives

and reacts with a variety of ROS, including O2

that de-esterifies in the presence of intracellular H<sup>2</sup>

cells exhibiting a high level of activity [182].

8-hydroxy 2-deoxyguanosine [189].

(ABTS) to ABTS+

laboratories.

**9. Conclusions**

This study was supported by the Slovak Research and Development Agency grants APVV-15-0543 and APVV-15-0544.
