Varicocele grade does not correlate with the severity of sperm DNA fragmentation levels nor with the amount of improvement in DNA fragmentation following varicocele repair

Introduction

The definition of varicocele is an abnormal dilation of the veins of the pampiniform plexus with valves that malfunction and is the most common correctable diagnosis in the infertile male (1). Varicocele is identified in approximately 15% of men in the general population and in up to 41% of men with primary infertility and 80% of those with secondary infertility (2). Men with varicoceles have been shown to be more likely to have higher levels of sperm DNA fragmentation (SDF) than men without varicoceles, and varicocele repair is able to reverse the elevation of SDF (3). High levels of SDF have been correlated with poor semen parameters, impaired preimplantation embryonic development, and higher rates of spontaneous pregnancy loss (4-7). The grade of the varicocele is based on the physical examination with a grade 1 varicocele being only palpable during a Valsalva maneuver, a grade 2 varicocele being palpable without Valsalva, and a grade 3 being visible to the examiner. Common clinical questions that arise when counseling men regarding expected levels of SDF improvement following varicocele repair is whether the grade of varicocele impacts the severity of the baseline SDF levels and whether the grade of the varicocele is an indicator for the amount of reduction in SDF that can be expected after varicocele repair. The medical literature is lacking in data to guide this counseling and the aim of this study was to assess the difference in the severity of SDF levels at baseline in infertile men with varicoceles based on the grade of varicocele, as well as to assess the amount of improvement in SDF in correlation with the grade of varicocele. We present this article in accordance with the STROBE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-154/rc).

Methods

Between October 2016 and January 2024, 116 men presented for infertility evaluations who were diagnosed with clinically palpable varicoceles on physical examination at a couple’s fertility center, Austin Fertility & Reproductive Medicine/Westlake IVF, and underwent subinguinal microsurgical varicocele repair by a single reproductive urologist (P.K.K.) and met inclusion criteria for this study. Exclusion criteria included when with class 3 obesity with body mass index (BMI) of 40 kg/m² or greater, tobacco users, and men who revealed leukocytospermia in the baseline semen analysis. Exclusion criteria also included men who had a history of cryptorchidism, testicular torsion, chemotherapy, radiation therapy, declined to have SDF testing performed preoperatively, postoperatively or both, were azoospermic at baseline, men who elected to have surgery for varicocele for orchialgia or hypogonadism rather than for fertility, and men who were successful achieving a pregnancy expeditiously and canceled the 3-month postoperative semen analysis. The men in this study met the criteria for infertility based on the definition by the American Society for Reproductive Medicine (ASRM) for infertility in the 2023 committee opinion. These criteria include: Couples that were unable to achieve a successful pregnancy based on a patient’s medical, reproductive, and sexual history, age, physical exam diagnoses, diagnostic testing, or any combination of the above (8). Indications for varicocele repair included repair for abnormalities of semen parameters including sperm concentration, overall motility, and forward progressive motility. Some patients had normal sperm concentration and motility parameters, but low forward progressive motility and still elected to proceed with varicocele repair based on shared decision making. Isolated teratozoospermia was not considered an indication for varicocele repair in these infertile men. Men presenting for infertility with ipsilateral orchialgia with characteristics consistent with varicocele pain regardless of semen parameters underwent varicocele repair. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the institutional review board of St. David’s Healthcare (No. 2217743-1) and individual consent for this retrospective analysis was waived. Following exemption by the St. David’s Healthcare institutional review board (IRB) (No. 2217743-1) due to the data being collected being de-identified, a retrospective chart review was performed of the electronic medical record to assess the correlation of varicocele grade to baseline conventional semen analysis parameters and sperm DNA fragmentation indices (DFIs), as well as to assess the amount of improvement in SDF following repair and potential correlations to varicocele grade. None of the men in this cohort underwent any additional adjuvant to reduce SDF after varicocele repair such as antioxidant use. All conventional semen analyses and sperm DFI assays were performed at a single on-site clinical laboratory improvement amendments (CLIA) certified andrology laboratory with sperm DFI performed by the sperm chromatin dispersion assay using Spectrum Technology’s second-generation Halosperm kit (Madrid, Spain). This assay is considered the most usable in clinical andrology laboratories and its reliability has been assessed (9). In men with bilateral varicoceles (n=13) the larger clinical varicocele grade was used to classify the patient’s grade. Of the men with bilateral varicoceles, A sperm DFI of 20% or higher was considered abnormally elevated and associated with poorer clinical outcomes, based on a large meta-analysis suggesting a new guideline for defining an elevated DFI for clinicians (10).

Statistical analysis

The Student’s t-test was performed for statistical analyses, with a P value of <0.05 considered statistically significant. Results were reported as mean ± standard error of the mean (SEM).

Results

Of 416 varicocele repairs performed by the single surgeon over that timeframe, 116 met inclusion criteria. Exclusion criteria included men who had a history of cryptorchidism, testicular torsion, chemotherapy, radiation therapy, declined to have SDF testing performed preoperatively, postoperatively or both, were azoospermic at baseline, men who had varicocele repaired to treat orchialgia or hypogonadism rather than for fertility, and men who were successful achieving a pregnancy with their partners expeditiously and therefore did not obtain a semen analysis 3-months postoperatively. The mean age of the men in the entire cohort was 34±6.4 years of age. The only significant difference in the mean age of the men stratified by varicocele grade were that men revealing grade 3 varicoceles were younger than those that had grade 2 varicoceles (P value =0.03) (Figure 1). When assessing conventional semen analysis parameters including semen volume, sperm concentration, total percent motility, percent forward progressive motility, percent with normal morphology, and total motile sperm counts in infertile men with varicoceles, there were no significant differences in semen parameters based on the clinical grade of varicoceles. Those who had grade 3 varicoceles had a trend towards a lower percentage of sperm with normal morphology in comparison to men with grade 2 varicoceles: 2.6% and 4.3% normal morphology respectively, although the difference did not meet statistical significance (P value =0.055) (Figure 2). Morphologic assessment was performed by Kruger strict morphology criteria, per the “WHO laboratory manual for the examination and processing of human semen, sixth edition” (11). Similarly, the severity of the baseline varicocele grade did not correlate with a significant difference in the amount of improvement in conventional semen analysis parameters 3 months following varicocele repair except for the percentage of sperm with normal morphology, which surprisingly revealed that men with grade 3 varicoceles had a lesser improvement in the percentage of sperm with normal morphology in comparison to men with grade 2 varicoceles (P value =0.02) (Figure 3).

Figure 1 The ages of men based on the grade of varicocele expressed as means ± SEM. SEM, standard error of the mean.

Figure 2 Baseline conventional semen analysis parameters by grade of varicocele including semen volume (mL), sperm concentration (10⁶/mL), total motility (%), forward progressive motility (%), normal morphology (%), and total motile sperm count (10⁶). Expressed as means ± SEM. SEM, standard error of the mean.

Figure 3 The amount of improvement in conventional semen analysis parameters by grade of varicocele including semen volume (mL), sperm concentration (10⁶/mL), total motility (%), forward progressive motility (%), normal morphology (%), and total motile sperm count (10⁶). Expressed as means ± SEM. SEM, standard error of the mean.

There was no significant difference in the severity of SDF elevation in infertile men based on the grade of their varicoceles who ultimately underwent varicocele repair, however; the difference in severity of SDF in those that had grade 3 varicoceles being higher than those with grade 2 varicoceles, was not quite statistically significance but revealed a P value of 0.051 (Figure 4). The overall cohort of men (n=116) regardless of grade of varicocele demonstrated a 13.8% improvement in SDF 3 months following varicocele repair in comparison to baseline levels. Men with grade 1 varicoceles (n=7) had an improvement in SDF from 32.3% at baseline to 18.4% 3 months following varicocele repair. Men with grade 2 varicoceles (n=52) demonstrated an improvement in SDF from 31.9% to 20.8% after varicocele repair. Men with grade 3 varicoceles (n=57) had an improvement in SDF from 36.2% to 21.4%. There was not a significant correlation between the clinical grade of the varicocele and the amount of improvement in SDF 3 months following varicocele repair (Figure 5).

Figure 4 Baseline sperm DFI by grade of varicocele. Expressed as means ± SEM. DFI, DNA fragmentation index; SEM, standard error of the mean.

Figure 5 Amount of improvement in sperm DFI from baseline levels to levels 3 months after varicocele repair. Expressed as means ± SEM. DFI, DNA fragmentation index; SEM, standard error of the mean.

Discussion

It has been established that varicoceles negatively impact testicular function and male fertility in some men, which has been demonstrated by reduced testicular volume of the ipsilateral testicle with the varicocele (12). Varicoceles have the ability to affect all semen parameters including sperm concentration, motility, and morphology (13,14). Previous studies have suggested that there is an inverse relationship between grade of the varicocele and sperm concentration, motility, and morphology, with the most significant difference found in men with grade 3 varicoceles (15,16). Those presenting grade 3 varicoceles typically demonstrated sperm concentrations less than half of that of those that did not have varicoceles (15). Varicocele repair in randomized clinical trials revealed a significant improvement of sperm concentration by 75%, motility by 5.2%, and morphology by 8% (17). One previous study suggested that following repair of varicoceles sperm concentration and motility improved in all grades of varicoceles, but most robustly in men with grade 3 varicoceles, but that it can be up to 6 months postoperatively to show this improvement rather than 3 months (18). It may be argued if men presenting for infertility evaluations diagnosed with clinical varicoceles should have SDF obtained at the time of the diagnostic conventional semen analysis. Although the American Urological Association/ASRM (AUA/ASRM) guidelines make no specific mention of SDF within the context of varicocele, it is considered acceptable clinical practice to obtain SDF testing in men with varicoceles. The European Urology Association (EUA) guidelines state varicocele repair may be considered in men with elevated SDF and unexplained male infertility, which presumes such testing is appropriate. The Italian Society of Andrology and Sexual Medicine (SIAMS) guidelines state that assessment of sperm DNA integrity may help with counseling of couples struggling with infertility, particularly when the male partner is at higher risk for SDF, including those with varicocele. Peer reviewed published expert recommendations state SDF testing can be considered as early as the initial assessment of infertile men with palpable varicoceles regardless of whether they have normal or abnormal conventional semen parameters, and especially if assisted reproductive technology (ART) is expected or after medical treatment fails (19). A recent very large, comprehensive systemic review meta-analysis recommends obtaining SDF in men presenting with infertility with clinical varicoceles (10). Although the current study did not show a significant correlation between the grade of varicocele and the severity of baseline conventional semen parameters nor significant differences in the amount of improvement in conventional semen parameters 3 months following varicocele repair based on varicocele grade, the primary endpoints of this study were to assess the possible correlation between varicocele grade and SDF and the amount of improvement of SDF 3 months postoperatively.

In men with varicoceles, oxidative stress (OS) has been directly associated with elevated SDF levels (20). Infertile men who present with clinical varicoceles and high SDF levels have demonstrated detrimental impacts on fertility in couples trying with timed intercourse, as well as those undergoing ART (6). Previous studies have reported that men with varicoceles had a 9.84% higher level of SDF in comparison to controls without varicoceles and elevated SDF can be detected in men with normal conventional semen analysis parameters (3,21,22). A previous prospective clinical trial by our group reported a 14% mean decrease in SDF 3 months after varicocele repair (23). That is consistent with the results of this current study revealing a 13.8% improvement in SDF regardless of grade of varicocele.

This current study is one of very few in the medical literature evaluating the impact of varicocele grade on baseline SDF levels in men who ultimately underwent varicocele repair as well as the amount of improvement in SDF following varicocele repair based on varicocele grade. Interestingly, there were not significant differences in these levels of SDF at baseline nor in the amount of change in SDF based on varicocele grade 3 months after varicocele repair. This suggests that infertile men with varicoceles, regardless of the grade of their varicocele, may consider varicocele repair for improvement in SDF with optimism in expected improvement regardless of varicocele grade. In fact, the only finding in these men that met statistical significance based on varicocele grade was not related to the primary endpoint of SDF but was the unexpected finding that men with men with grade 3 varicoceles had a lesser improvement in the percentage of sperm with normal morphology in comparison to men with grade 2 varicoceles following varicocele repair (P value =0.02). As far as outcomes that were not quite statistically significant but may represent a trend, were men with grade 3 varicoceles had a trend towards a lower percentage of sperm with normal morphology in comparison to men with grade 2 varicoceles: 2.6% and 4.3% normal morphology respectively (P value =0.055); and that the difference in severity of SDF in men with grade 3 varicoceles being higher than those with grade 2 varicoceles, indicated a trend, although not quite statistically significant (P=0.051). Besides the latter finding, it is questionable if the morphological outcomes based on varicocele grade should be considered impactful enough on a man’s fertility to deserve a great deal of focus when counseling these men, considering the data that questions the clinical utility of sperm morphology with the exception of total globozoospermia (24-27). Interestingly, the only group of men that reached normalized SDF levels postoperatively were those who had grade 1 varicoceles at baseline. However, the grade 2 and grade 3 patients demonstrated significant improvements and reached nearly normalized levels, just above the 20% cutoff. The improvement in the amount of SDF amongst men presenting with all 3 grades outperformed improvements reported by a previous meta-analysis reporting an improvement of 6.86% postoperatively (28). Other studies have reported improvements of SDF ranging from 5% to 5.79% following varicocele repair (29,30).

Limitations to this study include the lack of randomization, all data was from a single surgeon at a single center, semen parameters and DFI were assessed only at 3 months post varicocele repair, the lack of data following out results at 6 months postoperatively, and the lack of pregnancy and live birth data, which this study was not designed to assess. Another limitation is that all varicocele repairs were conducted by a single reproductive urologist, without validation at other centers by other surgeons.

Conclusions

This data suggests that when counseling infertile men undergoing varicocele repair who have an elevated SDF, the grade of varicocele may not impact the amount of improvement expected in SDF following repair, suggesting that varicocele patients may be counseled similarly on expectations for response regardless of varicocele grade.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-154/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-154/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-154/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-154/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the institutional review board of St. David’s Healthcare (No. 2217743-1) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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