Is oxidative stress evaluated in viable human spermatozoa a marker of good semen quality?

Abstract

BackgroundOxidative stress is defined as the unbalance between reactive oxygen species (ROS) production and antioxidant defences. Whereas low levels of ROS are necessary for physiological sperm functions, high levels impair fertility damaging membranes, proteins and DNA. In this study, we used two probes, CellROX® Orange and Dihydroethidium (DHE), which reveal different intracellular ROS species, to evaluate the association between the percentage of oxidized viable spermatozoa and sperm functions.MethodsThe percentage of oxidized spermatozoa was evaluated by flow cytometry with the two probes concomitantly with standard semen parameters and sperm DNA fragmentation (sDF, by TUNEL/PI). Phosphatidylserine membrane exposure, caspase 3,7 activity, sperm kinematic parameters and hyperactivated motility were evaluated by Annexin V, FLICA™ and CASA system respectively.ResultsOxidized viable spermatozoa, evaluated with both probes, were positively associated with sperm basal parameters and negatively with sDF. Also, we found that a consistent percentage of CellROX® positive viable spermatozoa were selected from whole semen during swim up procedure. Double staining of CellROX® Orange with Annexin V and FLICA™ demonstrated that viable oxidized spermatozoa do not show apoptotic features.ConclusionOverall, our results suggest that CellROX® Orange and DHE allows identification of the viable oxidized sperm fraction related to better performances.

Effects of Benzo[a]pyrene on Human Sperm Functions: An In Vitro Study

“…No increase was observed in viable spermatozoa with both probes. In a previous study, we demonstrated that CellROX ® Orange-and DHE-positive viable spermatozoa are associated with good semen characteristics and do not show apoptotic features, reflecting a sperm fraction related to better performances [34]. We hypothesized that most ROS-positive viable spermatozoa show physiological ROS levels [34] likely supporting their physiological functions [51,52].…”

“…An important aspect concerns the mechanism(s) responsible for BaP detrimental effects on spermatozoa. We investigated the possible role of induction of oxidative stress by evaluating intracellular ROS with two probes, CellROX ® Orange, which shows specificity for H 2 O 2 only in viable spermatozoa and DHE, which detects both H 2 O 2 and O 2 − species both in viable and unviable spermatozoa [34,50]. We found that all the three tested concentrations of BaP increased the percentage of ROS-positive unviable swim-up selected spermatozoa as detected with DHE after 3 h of incubation, likely reflecting the slight decrease in viability detected in the same sample (Figure 1C).…”

“…Incubation with 10 µM for 3 h reduced sperm penetration at 1 and 2 cm both in swim-up selected and unselected spermatozoa (Figure 4C,D). To study sperm intracellular ROS, we used two different probes [34] after 3 h of incubation with BaP of swim-up selected spermatozoa. By staining the samples with CellROX ® Orange, which detects ROS only in viable spermatozoa, the percentage of positive events did not differ among the control and the BaP treated samples (Figure 7A).…”

Low human sperm motility coexists with sperm nuclear DNA damage and oxidative stress in semen

“…Our results are in agreement with data obtained by other authors, who have shown that subjects with higher sORP (> 1.34; > 1.36; > 1.47) had significantly lower sperm progressive motility [111][112][113] and higher SDF. 112 Moreover, it has been shown that ROS levels are adversely correlated with progressive motility 53,106,[111][112][113][114][115][116][117][118] and positively correlated with SDF. 53,106,112,114,116 The linear associations among sperm motility, SDF and sORP were also confirmed in our current and previous studies.…”

“…112 Moreover, it has been shown that ROS levels are adversely correlated with progressive motility 53,106,[111][112][113][114][115][116][117][118] and positively correlated with SDF. 53,106,112,114,116 The linear associations among sperm motility, SDF and sORP were also confirmed in our current and previous studies. 79,87 However, Garcia-Segura et al 119 showed that sORP negatively correlated with sperm progressive motility and positively correlated with a deficiency of sperm chromatin-specific proteins (protamines).…”