The guidelines dictated that the indication for varicocelectomy was infertility, as proven by abnormal sperm parameters including sperm concentration, motility, and morphology. Radiological intervention, such as sclerotherapy and embolisation, can also be used for varicocele repair. These techniques are minimally invasive; however, they result in higher recurrence rates than varicocelectomy . There is also a lack of literature on the effects of sclerotherapy and embolisation on sperm DNA fragmentation.
Varicocelectomy was not recommended for infertile patients with normal semen parameters and subclinical varicocele patients . According to a meta-analysis by Kroese et al. , the value of surgical treatment in subfertile men with subclinical varicocele and normal semen analysis is disputable. Another meta-analysis by Kim et al.  showed no significant difference in the pregnancy rates. Varicocele repair also showed no benefit in other instances. For example, whether men with non-obstructive azoospermia should be offered clinical varicocele treatment remains controversial . However, another study reported that the sperm retrieval rate was significantly higher in men who had previously undergone varicocele repair .
Abnormal sperm DFI was not included as one of the indications for varicocelectomy. Furthermore, a meta-analysis by Wang et al. concluded that there was increased sperm DNA damage in varicocele patients . Thus, the meta-analysis performed in the current study aimed to evaluate the effects of varicocelectomy on the sperm DFI and other parameters, including sperm concentration, motility, and morphology, and determine whether abnormal DFI levels could be considered as one of the indicators for varicocelectomy.
A total of 7 studies (289 patients) were analysed in this study. The results showed that varicocelectomy significantly reduced DNA fragmentation by 6.86% (mean difference − 6.86; 95% CI: − 10.04, − 3.69; p < 0.00001). The studies in this analysis used SCSA and TUNEL to assess DNA fragmentation. Using SCSA, the DNA double helix needs to be opened by denaturation process using heat or low pH to expose DNA fragments or potential DNA breaks . Exposed strands then stained using acridine orange which fluoresces green when bound to native DNA and red when bound to broken DNA [32, 33]. In contrast to SCSA, TUNEL assay does not require initial denaturation step to detect DNA fragmentation . In TUNEL, the addition of template-independent DNA polymerase called terminal deoxynucleotidyl transferase (TdT) on the 3′-hydroxyl (OH) free break-ends of single-strand (ss) DNA and double-stranded (ds) DNA allows measurement of DNA fragmentation [32, 35]. Each method has its own advantages and disadvantages. For example, dsDNA may have breaks with no free 3′-OH ends [36, 37]. Additionally, the reference value of DNA fragmentation for differentiating fertile and infertile men using both SCSA and TUNEL shows high variability. However, several studies have shown high correlation between SCSA and TUNEL, indicating that both assays expressed similar values of DNA fragmentation [15, 16].
Several studies not included in this analysis also showed similar results to the present study. These non- included studies were not similar to those included, because they did not fulfil the inclusion criteria. A study by Kadioglu et al., which showed a significant decrease (22.1%) in DFI after varicocelectomy, was not included in this analysis because it did not provide enough data to calculate the mean and SD. This study also showed that the higher the pre-operative DFI, the larger is the decrease in the post-operative DFI . Another study by Telli et al. also revealed the decrease in DFI after varicocelectomy. However, DFI was evaluated using the AOT method . AOT was reported to show higher DFI levels and variability consistently than other methods including SCSA and TUNEL . These studies and our analysis showed that varicocelectomy reduced sperm DNA fragmentation. One meta-analysis by Wang et al. also proposed varicocelectomy as the potential treatment for the increase in the DNA damage in varicocele patients . The effects of varicocele on the alteration in the amount of sperm DNA damage were studied in this meta-analysis. Additionally, this study aimed to determine the efficacious effects of varicocele repair on sperm DNA damage. However, some of the studies analysed in the meta-analysis by Wang et al. were retrospective studies or of unspecified study design . This might have led to bias in the study results, as aforementioned in the limitations of the meta-analysis. We intended to strengthen the results of our meta-analysis by selecting only prospective studies or RCTs, if available. Although we could not find any RCT, all other studies included in the analysis were prospective studies.
An explanation for the reduction in DNA fragmentation after varicocelectomy lies in the proposed pathophysiology of DNA damage in varicocele patients. Sperm DNA fragmentation occurs during spermatogenesis and sperm maturation . Hypoxia caused due to venous stasis and reflux results in an increase in reactive oxygen species (ROS). ROS directly attacks spermatozoa DNA, resulting in DNA damage and increased DNA fragmentation [39, 40]. Varicocelectomy eliminates venous stasis and reflux, thereby decreasing ROS production and thus DNA damage.
This analysis also revealed improved sperm concentration (mean difference: 9.59; 95% CI: 7.80, 11.38; p < 0.00001), progressive motility (mean difference: 8.66; 95% CI: 6.96, 10.36; p < 0.00001), and morphology (mean difference: 2.73; 95% CI: 0,65, 4.80; p = 0.01) after varicocelectomy. We also evaluated the effects of varicocelectomy on these sperm parameters, in addition to sperm DFI, to strengthen the evidence for the correlation between varicocelectomy and sperm parameters. A previous meta-analysis reported the positive effects of three different varicocelectomy surgical techniques on sperm parameters, and the results of this study were in conjunction with those of our meta-analysis .
Our analysis showed a potential negative correlation between DFI and the other sperm parameters before and after varicocelectomy. Several studies have reported the correlation between DFI and other sperm parameters. Kadioglu et al. reported that higher pre-operative DFI was associated with significant negative correlation between DFI and sperm motility (r = − 0.42, p < 0.01) . Telli et al. also reported a negative correlation between DFI and sperm motility (r = − 0.267, p = 0.043) . Yang et al. reported a negative correlation between DFI and sperm motility, concentration, and morphology (r = − 0.307, − 0.552, and − 0.620, respectively; all p < 0.01) . This study also revealed a positive correlation between DFI and age, suggesting that DFI increased as the patient aged. Furthermore, a study by Smit et al. reported that low DFI values were associated with higher rates of pregnancy, both spontaneous and though ARTs . All these evidences suggested that DFI might be as important as other sperm parameters for evaluating male fertility and fertility after varicocelectomy for a successful pregnancy.
The limitations of this meta-analysis include the heterogeneity of the included studies in terms of sample size and methods of intervention and evaluation of the outcomes. All included studies were prospective studies. Even though this was ideal, inclusion of RCTs could further strengthen the study analysis. Unfortunately, no RCT was found during the literature search of the databases. However, one preliminary RCT was found on manual searching, but this RCT included only five patients who underwent varicocelectomy and showed no before-after DFI data or p-value data, making the study undetermined for significance . This difficulty in finding RCTs could be due to the ethical problems in varicocelectomy being performed in only one group of clinical varicocele patients and not in the other group, despite their need to receive varicocelectomy as a treatment for infertility.