Layout 1 DNA fragmentation index and fertility rate after microsurgical varicocelectomy Eur J Transl Myol 35 (3) 13791, 2025 doi: 10.4081/ejtm.2025.13791 Varicocele is an important cause of male infertility, affecting approximately 10-15% of the general male population, 30-35% of those with primary infertility, and 69-81% of men experiencing secondary infertility.1 The mechanisms through which varicocele causes testicular dysfunction and infertility are complex and involve multiple factors. These include small vessel blockage and blood pooling, which raise scrotal temperature and cause testicular hypoxia, reflux of toxic substances from the adrenal or kidney disruption of the hypothalamic- pituitary-gonadal axis; oxidative stress and decreased antioxidant capacity.2-6 The evidence points out the important link between vari- cocele, oxidative stress, and sperm dysfunction in infertile men. Increased levels of ROS and reduced TAC have been associated with DNA damage in sperm, including single- and double-strand breaks. This oxidative stress is critical in impairing sperm DNA integrity, which correlates with reduced fertility potential in assisted reproduction tech- niques.7-10 The Sperm Chromatin Structure Assay (SCSA) is a clini- cally relevant technique for assessing sperm DNA frag- mentation through the DNA Fragmentation Index (DFI), which reflects the percentage of sperm with denatured nu- clei.11 High DFI levels, particularly at 20% or more, are linked to lower success rates in Assisted Reproductive Abstract Varicoceles and their impact on sperm quality are considered one of the most common causes of male infertility. This study aims to examine the outcomes of unilateral and bilateral microsurgical varicocelectomy concerning DNA Fragmentation Index (DFI), different sperm parameters, and overall fertility. This is a cross-sectional study in which we analyzed 100 male patients who were diagnosed with varicocele by ultrasound and had a history of infertility of at least one year. Fifty patients had unilateral varicocele and 50 had bilateral varicocele (left side clinical plus right side subclinical) who underwent microsurgical varicocelectomy from March 21, 2022, to March 21, 2023. We compared the DFI before and after surgery, and fertility rates between the two groups were assessed. The median age of participants was 32 years, with a body mass index (BMI) of 25 kg/m² and an average infertility duration of 2 years. A comparative analysis revealed a statistically significant difference in the median years of infertility between the unilateral and bilateral groups before surgery (p=0.03). Both unilateral (p<0.001) and bilateral (p<0.001) microsurgical vari- cocelectomy significantly reduced DFI. However, the bilateral approach was better at lowering DFI than the unilateral method in microsurgical varicocelectomy (p<0.05). The pregnancy rates after surgery were 36.0% for the unilateral and 58.0% for the bilateral group; this difference was statistically significant, favoring bilateral microsurgical varicocelectomy (p=0.04). Microsurgical varicocelectomy is a viable treatment option for couples facing oligospermia, especially for those with bilateral varicoceles. Key Words: varicocele, infertility, microsurgical varicocelectomy, sperm DNA fragmentation index, oligospermia. Eur J Transl Myol 35 (3) 13791, 2025 doi: 10.4081/ejtm.2025.13791 Comparison of DNA fragmentation index and fertility rate after unilateral and bilateral microsurgical varicocelectomy Reza Mohammadi Farsani,1,2 Mahdi Azarabadi,2 Mohammad Javanbakht,2 Mahdiyar Jaberi,3 Mosa Asadi2 1Tehran University of Medical Sciences, Tehran, Iran; 2Nephrology and Urology Research Center, Clinical Science Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran; 3Dezful University of Medical Sciences, Dezful, Iran. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 221 - DNA fragmentation index and fertility rate after microsurgical varicocelectomy Eur J Transl Myol 35 (3) 13791, 2025 doi: 10.4081/ejtm.2025.13791 Technologies (ARTs) like Intracytoplasmic Sperm Injec- tion (ICSI). A DFI value of more than 25% serves as a clue to the degree of DNA fragmentation and a main pre- dictor of reproductive outcomes following ARTs. This threshold underlines the importance of DNA integrity as- sessment in the male partner because high fragmentation levels are statistically related to increased reproductive challenges.12 DFI assessment is thus critical to determin- ing male fertility and predicting reproductive outcomes, making it a significant tool in fertility investigations. Varicocelectomy is widely recognized as an effective treatment for varicocele, particularly for men with infer- tility. Whereas the initial interest was mainly focused on improvement in natural conception rates following the surgery, growing evidence highlights the benefits of var- icocelectomy on semen quality, including DNA quality, sperm DNA fragmentation, and overall sperm parame- ters.13-16 While guidelines currently recommend the treatment of varicoceles in selected cases of infertility, the effects of varicocelectomy on DFI are less clear due to limited re- search. This study aimed to elucidate the relationship be- tween varicocelectomy and sperm DFI, hence contributing to the management of male infertility. Materials and Methods Study design This cross-sectional study enrolled 100 microsurgical var- icocelectomy candidates, comprising 50 males with uni- lateral varicocele and 50 males with bilateral varicocele (including subclinical cases). Participants were referred to Baqiyatallah Hospital in Tehran, Iran, from March 21, 2022, to March 21, 2023. Inclusion criteria include eligible males who have been diagnosed with varicocele based on preoperative clinical assessments, ultrasound examinations, and a history of at least one year of infertility. The following were excluded from participating in the study: Individuals with a history of cigarette smoking, alcohol consumption, radiotherapy, cancer, the use of cytotoxic agents, chemotherapeutic agents, and acute or chronic infections. The sample size was determined using previous studies17,18 utilizing a disease prevalence of 45%, an alpha of 0.10, a power of 90%, and an attrition rate of 20%. The sample size was calculated using the following formula Socio-demographic and anthropometric measurements Participants completed a comprehensive questionnaire as- sessing socio-demographic characteristics, including age, duration of infertility, and educational attainment before surgery. Anthropometric measurements (weight and height) were obtained to calculate body mass index (BMI) using the standard formula: BMI=weight (kg) / (height [m²]). Postoperative follow-up was conducted at one year to evaluate pregnancy outcomes (dichotomized as suc- cessful or unsuccessful). Evaluation of laboratory findings Laboratory parameters were evaluated using pre- and postoperative data extracted from hospital records. For each participant, we analyzed semen volume (mL), sperm concentration (×106/mL), total sperm count (million/mL), sperm morphology (normal forms, %), and DNA fragmen- tation index (DFI, %). Varicocele diagnosis (unilateral or bilateral) was confirmed through standardized scrotal ul- trasonography. Statistical analysis All statistical analyses were performed using SPSS ver- sion 26 (SPSS Inc., Chicago, IL, USA). Significance was considered to be at P <0.05. Quantitative variables were assessed for normal distribution using the Kolmogorov- Smirnov test. Intergroup comparisons of quantitative variables were per- formed using either independent samples t-tests (for nor- mally distributed data) or Mann-Whitney U tests (for non-normally distributed data). Qualitative variables were compared using either χ² tests or Fisher’s exact tests, as appropriate. Intragroup differences between baseline and postoperative measurements were assessed using paired t-tests. Results As presented in Table 1, the participants had a median age of 32 years, a BMI of 25 kg/m², and an average in- fertility duration of 2 years. When a comparison analysis was done between the unilateral and bilateral varicocele groups before surgery, the difference in the median years of infertility between these groups was statistically sig- nificant (p=0.03). However, no significant difference was found between the groups for other socio-demographic and anthropometric characteristics before intervention (p > 0.05). Men who underwent bilateral varicocelectomy had a sig- nificantly higher pregnancy rate than those who opted for unilateral varicocele repair. Moreover, the naturally spon- taneous pregnancies were very high in the group that un- derwent bilateral varicocelectomy. Table 2 presents the comparative outcomes of DFI and pregnancy rates between unilateral and bilateral microsur- gical varicocelectomy procedures. The mean DFI was 20.3% ±7.5 for the unilateral group (n=50) and 24.1% ±7.0 in the bilateral group (n=50). After surgeries, the DFI in the unilateral group was 16.5% ±6.1, and that of the bi- lateral group was 18.5% ±6.0. Both surgical approaches demonstrated significant reduc- tions in DFI compared to controls (unilateral: p <0.001; bilateral: p <0.001). Moreover, the bilateral approach demonstrated statistically superior outcomes compared to unilateral varicocelectomy, reflecting a clinically mean- ingful improvement in success rates (mean change: -5.5 vs. -3.9, respectively; p <0.05). - 222 - DNA fragmentation index and fertility rate after microsurgical varicocelectomy Eur J Transl Myol 35 (3) 13791, 2025 doi: 10.4081/ejtm.2025.13791 - 223 - Table 1. General characteristics of the participants in unilateral and bilateral groups before surgery. Variable Total Unilateral group Bilateral group *P-value (n=100) (n=50) (n=50) Age (y) 32.0 (28.0, 35.0) 32.0 (28.0, 35.0) 32.5 (29.0, 35.0) 0.42 Body mass index (kg/m2) 25.0 (23.0, 27.0) 26.0 (23.1, 27.7) 24.7 (23.0, 27.0) 0.23 Infertility (year) 2.0 (2.0, 3.0) 2.0 (2.0, 3.0) 3.0 (2.0, 3.3) 0.03 Semen volume (ml) 2.9 (1.8, 4.0) 3.0 (1.0, 4.5) 2.7 (1.8, 3.4) 0.20 Sperm concentration (× 106/ml) 33.0 (15.0, 68.3) 32.0 (13.5, 57.3) 35.0 (16.0, 71.8) 0.28 Total sperm (million/ml) 91.3 (28.5, 229.8) 95.8 (27.7, 221.3) 90.0 (28.8, 232.8) 0.86 Sperm morphology 2.0 (1.0, 5.0) 2.0 (1.0, 3.8) 2.0 (1.0, 5.3) 0.97 DNA fragmentation index (%) 0.15 DFI ≤15 22 (22.0) 15 (30.0) 7 (14.0) 15