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Is genotoxicity a suitable biomarker for varicocele?

Dear Editor, I read the recent paper published in the Andrologia titled ‘Effect of varicocele on sperm DNA damage: A systematic review and metaanalysis’ by Zhang et al. (2022). In this study, the authors concluded that ‘In conclusion, patients diagnosed with clinical varicocele had higher DNA fragmentation índex than healthy controls, which means varicocele could impair sperm DNA, consequently the fertility potential of affected men’. I agree with the statement; however, some biological concepts and guidelines are herein presented for the correct understanding of the study. First, the author elected 12 studies in the systematic review and meta-analysis. However, the scientific data were not properly evaluated. For example, the study conducted by Talebi et al. (2008) did not perform single-cell gel (comet) assay, whereas the study made by Smith et al. (2006) evaluated DNA fragmentation by means of TUNEL and sperm crhomatic dispersion assays. This information was not correctly presented in the manuscript (Table 1). The second question, refers to the approach of the study. In Results, the authors stated that ‘Considering the statistically significant heterogeneity existed among all studies, we conducted subgroups analysis according to the study region (Brazil and other countries)’ (Figure 4a). What is the biological relevance of this analysis? Particularly, why the studies conducted in Brazil (five studies) and Tunisia (one study) were comparatively evaluated with the rest of the world? In my opinion, this is not clear in the manucript and the approach does not contribute for understanding the paper. The second question that merits discussion is the relationship between genoand cytotoxicity. It has been established that genotoxicity assays are not reliable on the scenario of high cytotoxicity, because this is an important bias to the genotoxicity studies. Disregard any information about cytotoxicity leads to incorrect or even misleading data (Hartmann & Speit, 1997). This is due to the fact that cytotoxicity induces DNA fragmentation as a result of cellular death. Therefore, the correct evaluation of cytotoxicity is mandatory for genotoxicity evaluation. Although TUNEL assay is a well-established method able to evaluate DNA fragmentation, this one is widely used for detecting apoptosis (an important mechanism of cell death) in mammalian cells, and not genotoxicity. In a similar manner, it has been postulated that the occurrence of fully damaged cells (‘headhogs’) in the slides when evaluating the single-cell gel (comet) assay data is indicative of cytotoxic effect (Hartmann & Speit, 1997). In Discussion, the authors showed some putative mechanisms regarding the origin of genetic damage in sperm from varicocele patients, such as oxidative stress and inflammation. Nevertheless, cytotoxicity is an important biological mechanism for clarifying the ethiopathogenesis of varicocele and was not properly discussed in the manuscript. Taking into consideration that genotoxicity is closely associated with changes in the gene expression as well as activation of DNA repair system as far as genomic instability, cytotoxicity is related to the impairment of the expression of cell cycle regulatory proteins and subsequent abnormal proliferative activity. Certainly, such information would contribute to better understand if, and to what extent, varicocele induces cytoand/or genotoxicity in sperm cells as a result of DNA fragmentation in sperm cells. I assume that such comments are very useful and important for the correct understanding of the relevant paper evaluating genetic damage induced by varicocele in sperm cells.