Defined as inability to conceive after at least 12 months of unprotected sex, infertility is a huge global health burden which involves 15% of couples [1]. Varicocele is the most common detectable cause of infertility in males which is characterized by dilatation of the pampiniform venous plexus and spermatic veins [2]. Its prevalence is estimated to be approximately 15% in the general population but it should be noted that 35% of men with primary infertility and 75% of men with secondary infertility suffer from varicocele [3]. The underlying mechanism of varicocele development is not definitely known but several factors have been proposed as its cause. Elevated pressure in renal venous system, collateral venous anastomoses and incompetency in valves of the internal spermatic vein have been suggested as the underlying mechanism of varicocele formation [4]. Another suggested theory is the reflux in the collateral veins including gubernacular veins [5]. It is speculated that dilatation of cremaster and gubernacular veins in varicocele elevates the temperature, leads to reflux of the toxic metabolites and directly damages the testes by higher levels of oxidative stress in walls of varicose spermatic veins which all can be considered as the factors contributing to the infertility [6-9]. Increases in oxidative stress and decline in the antioxidant levels are correlated with DNA damages in sperm such as DNA fragmentation. DNA fragmentation leads to reduction in the ability of sperms for fertilization of oocytes in the fertility process [10,11].

The sperm DNA fragmentation index (DFI) is an appropriate parameter reflecting fertility status. It determines degrees of integrity and damage in sperm DNA [12]. Levels of DFI is correlated with success rates of assisted-reproductive techniques. A cut-off of >25% for DFI has been established for prediction of infertility problems [13]. Studies have shown that varicocelectomy can significantly decrease DFI and improve quality of DNA [14].

Many individuals with varicocele have no fertility problems while there are many patients with varicocele who remain infertile even after surgical repair of varicocele. Thus, more precise identification of infertility determinants in varicocele patients is crucial to prevent unnecessary surgeries in a cluster of patients and planning early surgeries in another group of patients for prevention of more robust damages to their fertility function. Numerous parameters have been introduced as the risk factor of infertility in varicocele but semen analysis is the most important factor reflecting the fertility function of patients, currently [15]. It should be noted that semen analysis is normal in many patients with primary infertility [16]. Hence, more precise indices are needed in these patients. DFI is one the most important and novel parameters suggested in the evaluation of these patients [17].

The impact of surgical varicocele repair on the fertility outcomes has been widely debated in the literature [18]. While several studies have reported that variocele repair improves fertility parameters regardless of the surgical technique, other authors have conducted studies which indicate superiority of one surgical technique over another [19-21]. Given the probable role of gubernacular veins in deterioration of the varicocele severity and its associated infertility, we aimed to compare the impact of two surgical methods of standard technique versus standard technique in addition to gubernacular veins closure on DFI as a parameter reflecting post-operative fertility function.

This single-blinded randomized controlled clinical trial was conducted at Imam Reza hospital of Tehran during 2020 and 2021. Inclusion criteria was infertile men from 18 to 50 years old with second and third degree varicocele who had impaired DNA fragmentation index (DFI>30%). Exclusion criteria included history of orchitis or testicular cancer, azoospermia, history of inguinal or scrotal surgery, systemic inflammatory disease and history of testicular trauma.

After taking informed consent, patients were randomly assigned to intervention and control groups. Permuted block randomization method was used to create a random sequence. We considered the block size to be 4 so that in each block 2 patients were operated by conventional method (control group) and two patients by conventional method + gubernaculectomy (intervention group). We entered information of the block size and sample size into the sealedenvelope.com website to specify the desired sequence in each block. In this way, 20 blocks of size 4 were identified to randomize the sample size of 80. With this method, we were able to divide patients into two groups in a balanced and unpredictable way. Also to hide random allocation, custom codes created by the same website were used for each person enrolled into the study. Participants were not aware of the surgical technique they underwent. Type of technique was not identifiable by the skin incision type and other features after surgery by the patients.

In the control group, patients underwent conventional varicocele surgery by closing the spermatic vessels of the cord in the inguinal canal without using a microscope. In the intervention group, testicular delivery and gubernacular veins closure without microscope was conducted in addition to the conventional varicocelectomy. BMI, age, infertility duration, distribution of varicocele, varicocele grade (assessed by clinical examination), baseline FSH and baseline testosterone were documented. Primary endpoints were semen analysis parameter and DFI improvement after surgery. Measurements were done at baseline and three months after surgery. To collect sample for semen analysis, patients were instructed to self-collect their semen sample after 2-3 days of sexual abstinence. Samples were evaluated within one hour of collection. Semen analysis was performed manually by expert technicians based on WHO reference guidelines for semen volume, sperm count, sperm progressive motility and sperm normal morphology (22). To detect DFI, patients were instructed to abstain for at least 3 days. TNE buffer (0.15M NaCl, 0.01M HCL and 1 mM EDTA) was used for adjustment of semen density. For staining the sample, Acridine orange solution was used. Finally, sperm DFI was measured by a flow cytometer with fluorescence signals. To complete the calculation, at least 5000 sperms were evaluated with flow cytometer.

IBM SPSS software version 22 was used for statistical analysis. For descriptive analysis, frequency and percentage were used for categorical variables and mean with standard deviation were used for continuous variables. T-test and chi-square tests were used for comparison of categorical and continuous parameters between groups, respectively. Paired t-test was also used for comparing pre- and post-operative variables in each group. Statistical significance threshold was considered as p-value of 0.05.

The study was performed in accordance to the ethical codes of Declaration of Helsinki. The study was approved by Ethics committee of AJA University of Medical Sciences with the code of IR.AJAUMS.REC.1399.201. The study was registered at Iranian Registry of Clinical Trials (IRCT) with the registration number of IRCT20200122046220N2.

80 patients were randomly assigned to two intervention and control groups. 4 patients in the intervention group and 3 patients in the control group were lost to follow-up. Finally, 36 patients in the intervention group and 37 patients in the control group entered the analysis. The mean age of patients in the intervention and control groups were 31.7±4.1 and 32.1±3.9 years, respectively (p=0.509). The patients were infertile for a mean duration of 19.7±8.3 and 20.2±7.5 months in the intervention and control group, respectively (p=0.322). Comparison of BMI, distribution of varicocele, varicocele grade, baseline follicle-stimulating hormone (FSH) and baseline testosterone also showed no significant difference between two groups. The details are presented in Table 1.

Table 1: Demographic and baseline clinical parameters in the groups of the study.

Semen analysis showed significant improvements in several parameters at 3 months after surgery compared to the baseline. Comparison of semen analysis changes between two groups showed no significant difference except for progressive motility parameter (p=0.028). DFI was also measured which showed significant decrease compared to the baseline. DFI decreased from 40.3±6.9 to 33.3±4.6 in the intervention group (p=0.031) and from 39.6±5.1 to 35.7±5.3 in the control group (p=0.047). The reduction in DFI was significant in both groups but also this reduction was significantly more prominent in the intervention group (p=0.013). The details of semen analysis and DFI measurements are presented in Table 2 and Table 3.

Table 2: Preoperative and postoperative values of semen analysis parameters and DNA fragmentation index.

Table 3: Comparison of improvements in semen analysis and DNA fragmentation index between two groups.

Follow-up evaluation after three months showed 3 cases (8.33%) of varicocele recurrence or persistence in the intervention group while 4 cases (10.81%) revealed recurrence/persistence in the control group (p=0.319).

Analysis of correlation of study parameters with semen analysis variables and DFI was conducted. There was no correlation detected except for inverse correlation of DFI and sperm motility (r=-0.573, p=0.001).

In this study, we evaluated the impact of two surgical approaches on DFI and semen analysis parameters. We found out that sperm count, motility, morphology and DFI improve significantly with either of the surgical methods but improvements of sperm motility and DFI were significantly more pronounced in the intervention group compared to the control group.

While some international guidelines are in favor of positive role of varicocelectomy on male fertility [23], there are some inconsistencies in the literature regarding this issue. There are studies which have reported that surgical repair of varicocele produces promising changes in semen parameters but all authors do not agree on the beneficial role of varicocelectomy in promotion of male fertile function [19-21]. Studies proposed that any abnormality in semen analysis in addition to clinically overt varicocele in men with infertility can be considered as the indication for varicocelectomy [24]. While the debate is not still finished, studies are seeking the factors which can predict the need for surgery and the response to repair in varicocele patients.

The relationship between clinically evident varicocele and disturbances in the spermatogenesis process is comprehensively studied in the literature [6,7,25]. High levels of impairments in the sperm DNA have been observed in patients with varicocele [26-28]. Higher temperature and release of reactive oxygen species (ROS) have been regarded as the potential underlying causes of sperm DNA damage in varicocele [29]. Other studies have suggested that venous stasis and reflux in varicocele patients lead to hypoxia which ultimately ends up in elevation of ROS. ROS directly effects DNA of sperms which causes DNA damage and increases in DNA fragmentation [30,31]. Semen analysis is the main tool used for evaluation of male infertility. Although this analysis offers valuable information regarding the fertility status of patients, it carries several drawbacks. One of its main limitations is that in many cases with normal morphology and motility, a high level of DNA fragmentation has been detected. In addition, interpretation of semen analysis is widely confounded by biological intra- and inter-observer variations [32]. Thus, DNA fragmentation changes occur prior to appearance of abnormality in semen analysis and does not exhibit much biological variation.

Theoretically it can be assumed that varicocelectomy abolishes venous reflux and stasis which leads to decline in ROS release and subsequent sperm DNA fragmentation. Numerous studies have evaluated the impact of varicocelectomy on semen parameters and DFI. A study by Kadioglu [33] indicated that DFI had decreased from 42.6% to 20.5% with varicocelectomy. A meta-analysis has reported that varicocelectomy could reduce DNA fragmentation significantly by a mean difference of -6.86 compared to controls. Sperm concentration, progressive motility and morphology had also improved after varicocelectomy [14]. Another meta-analysis also revealed that varicocele patients had significantly higher levels of DFI compared to healthy controls (mean difference: 9.84%). They reported that varicocelectomy could reduce DFI with a mean reduction of -3.37% [34]. Thus, varicocelectomy can be beneficial for infertile patients with high DFI. We found out that ligation of gubernacular veins in conjunction with conventional varicocelectomy can yield significantly higher reductions in DFI and more improvements in sperm motility of infertile varicocele patients. A study by Allameh [35] has also reported that in patients with varicocelectomy and testicular delivery, sperm motility improvement has been more prominent compared to conventional varicocelectomy. In line with our finding on negative correlation of DFI and sperm motility, Smit [36] also reported that increases in sperm progressive motility and reduction in DFI were significantly correlated after varicocelectomy. In contrast, a meta-analysis has evaluated three methods of varicocelectomy which all significantly improved semen analysis and DFI without any difference between surgical techniques [37]. A study by Yang et al showed no benefit with ligation of gubernacular veins over conventional varicocelectomy in terms of semen parameters [38]. Jin also reported that conventional varicocelectomy with testicular delivery and ligation of gubernacular veins does not yield more favorable outcomes compared to conventional varicocelectomy alone [39]. Hou et al also reported significant improvements in semen parameters in varicocelectomy with or without testicular delivery without any difference between the groups [40].

Our study had some limitations. We evaluated a relatively small number of patients. Larger sample sizes in the upcoming studies can yield more comprehensive results which can aid clinicians in treating patients. We also had a relatively short follow-up period in which we could not assess real pregnancy rates. Evaluation of pregnancy rates after varicocelectomy in longer follow-up periods can researchers a useful insight into the efficacy of their interventions. We also did not use microscopic surgical method which can enhance the efficacy of the surgery and its outcomes. Comparison of microscopic and non-microscopic methods in terms of postoperative fertile function can be an interesting topic in this research field. Also, due to show follow-up period, evaluation of complications was not feasible. Assessment of complications in the short and long follow-up periods can yield valuable results in the further studies.

Varicocelectomy regardless of its technique can improve semen parameters and DFI. Ligation of gubernacular veins in addition to conventional varicocelectomy can decrease DFI more significantly compared to the standard protocol. DFI in addition to the semen parameters can be considered as a parameter reflecting fertility status. The impact of this reduction in DFI after varicocelectomy on pregnancy rates should be evaluated in the future studies.

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