**5. Conclusions**

From past to present, the knowledge gathered over the many years of study in this field o ffers us a few lessons that need to be taken into account. Firstly, to avoid any interference when assessing the production of ROS by sperm cells, an e fficient removal of leukocytes from the samples is mandatory. Their presence will always cast doubt and potentially lead to data misinterpretation, as clearly evidenced by the work of Whittington and Ford [11]. Secondly, many of the methods used to assess ROS in sperm cells present drawbacks and limitations during application, possibly obfuscating the true nature of the involvement of free radicals in sperm physiology and male infertility. The rational use of probes and sometimes the adoption of more than one method are recommended for a better assessment of ROS in cells. An indirect assessment of oxidative stress may also be done by the analysis of the products originated from lipid (MDA, 4-HNE, HHE) [23,61,62,67] and DNA oxidation (DNA base adduct 8-hydroxy-2-deoxyguanosine) [142–144].

Finally, although sperm are susceptible to in vitro induced and exogenous sources of ROS and its by-products, the in vivo relevance of these compounds needs further clarity. Of interest, considering only the ROS produced by sperm, our laboratory has recently found that neither O2•<sup>−</sup> nor other free radicals, which lead to 4-HNE production, are responsible for motility loss during incubation [141]. In addition, a clear distinction must be made between the physiological versus the pathological roles of ROS in sperm. While a subtle increase in ROS may be necessary for sperm function such as in capacitation, the relationship between sperm abnormality and ROS may arise from a redox imbalance within the di fferent environments to which sperm are subjected, especially in testis [145]. However, before any definitive conclusions are made, more studies using refined methodologies to look at the level of spontaneous ROS generation or lipid peroxidation in fertile and infertile males are required. In addition, while measurements of both 4-HNE and MDA have been performed in spermatozoa, the levels of 4-HHE, perhaps a more important aldehyde, still need to be evaluated.

**Funding:** This research was funded by National Health and Medical Research Council gran<sup>t</sup> number 1182948.

**Conflicts of Interest:** The authors declare no conflict of interest.
