Introduction
Varicocele is defined as an abnormal dilation and/or tortuosity of the pampiniform plexus in the scrotum, accompanied by reversed blood flow and venous stasis [1]. Varicocele is the most common treatable cause of male infertility [2]. Studies report a prevalence of approximately 15% in the general male population and up to 40% in infertile men [3]. Its prevalence reaches about 35%–40% in cases of primary infertility and 80% in secondary infertility [4]. Varicoceles are unilateral on the left side in 78%–93% of cases, bilateral in 2%–20%, and unilateral on the right in 1%–7% [5]. Clinical varicoceles are detected and graded based on physical examination using the Dublin grading system: grade 0 varicocele is not visible or palpable but can be detected by ultrasonography; grade 1 is palpable only during the Valsalva maneuver; grade 2 is easily palpable with or without the Valsalva maneuver but is not visible; and grade 3 is a large varicocele that is both easily palpable and visible upon inspection [6]. A non-palpable enlargement of the venous plexus of the spermatic cord, which is diagnosed by ultrasound, is defined as a subclinical varicocele [7]. Varicocele is associated with impaired enzymatic antioxidant mechanisms and negatively impacts spermatogenesis, resulting in low sperm count, decreased motility, and a reduced percentage of morphologically normal sperm cells [2,8]. DNA damage is another potential mechanism by which varicocele contributes to infertility [9]. Patients with varicocele often exhibit high levels of sperm DNA fragmentation in their ejaculate [10]. Consequently, compared with normozoospermic individuals, those with varicocele show a higher incidence of sperm with damaged DNA [8]. Additionally, several studies have reported an association between varicocele and subnormal levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone [11]. Patients with varicocele are characterized by high levels of oxidative stress (OS), reduced total sperm count and motility, abnormal sperm morphology, and increased DNA fragmentation [12]. Under normal conditions, a balance exists between reactive oxygen species (ROS) production and antioxidant defenses. However, when antioxidant capacity decreases or ROS production increases, an imbalance occurs in favor of oxidants, leading to OS [13]. Major pathological mechanisms associated with varicocele-induced OS. Leydig cell dysfunction, scrotal hyperthermia, retrograde metabolite flow, hypoxia, and apoptosis [14]. The severity of varicocele has been directly correlated with the level of OS [15]. Several studies have demonstrated that men with varicocele exhibit higher levels of ROS in their semen [16]. Varicocelectomy can reduce OS and improve sperm parameters in oligoasthenozoospermic (OA) patients; however, it is not effective in all cases and carries risks, high costs, and the potential need for repeat interventions [17]. Oral supplementation with multiantioxidants provides a noninvasive alternative to lower OS and may benefit patients who do not improve after varicocelectomy, with lower associated risks [18,19]. Antioxidants are commonly employed in the treatment of male infertility associated with increased OS, including in patients with varicocele [20]. Excess ROS levels can harm Leydig, Sertoli, and spermatogenic cells, negatively impacting several functions of mature sperm [20]. Studies have reported that seminal ROS levels are significantly higher in men with left-sided grade 2 and 3 varicocele compared to those with grade 1 [21]. Our recent studies on infertile men with high levels of sperm DNA damage demonstrated that antioxidant therapy effectively improves sperm DNA integrity and pregnancy rates [22]. Researchers have studied the role of antioxidants in treating male factor infertility [23]. Various antioxidants, such as vitamins E and C, glutathione, coenzyme Q10, selenium, L-carnitine, and zinc, are used in treating varicocele-induced male factor infertility, either as single-agent therapy or in combination [24,25]. It has been shown that multiantioxidant treatments significantly improve semen parameters, thereby enhancing male fertility [22]. Furthermore, antioxidant supplementation can reduce OS and improve sperm parameters [26]. Antioxidant therapy may improve semen parameters, reduce the sperm DNA fragmentation index (DFI), and increase fertility potential [27]. To our knowledge, no studies have examined the impact of oral antioxidant treatment in patients with varicocele across different grades. In this prospective study, we aimed to investigate the effect of multiantioxidant supplementation on semen parameters, reproductive hormones, DNA integrity, and pregnancy rates in infertile men with varicocele associated with OA.
Methods
1. Patients
This study was conducted in the Assisted Reproduction Unit of Mohammed VI University Hospital Center (Oujda, Morocco), the first such unit established in eastern Morocco. It was a prospective study conducted from May 2023 to December 2024, involving 133 OA patients with varicocele (grade 0, n=28; grade 1, n=32; grade 2, n=42; grade 3, n=31). The patients’ ages ranged from 26 to 59 years (mean 38.5±1.6). Based on the standard grading of varicocele, OA patients were classified into groups: G0 (grade 0, n=28), G1 (grade 1, n=32), G2 (grade 2, n=42), and G3 (grade 3, n=31).
All samples were collected from infertile patients with varicocele. Varicocele was diagnosed using ultrasound and physical examinations. Diagnostic criteria included the presence of two or more varicose veins in the relaxed state and retrograde flow lasting more than 2 seconds during the Valsalva maneuver. Based on the examinations, varicocele was classified into four grades: grade 0 (not visible or palpable but detectable by ultrasonography), grade 1 (palpable only during straining), grade 2 (palpable during routine physical examination while standing), and grade 3 (visible and palpable).
The inclusion criteria comprised a history of infertility for at least 12 months despite regular unprotected intercourse, and seminal fluid analysis showing abnormal sperm parameters, including oligozoospermia (<16 million/mL), asthenozoospermia (progressive motility <30%, total motility <42%), teratozoospermia (normal morphology <15%, according to the modified David classification), and a DFI below 25%, as defined by the 6th edition of the World Health Organization (WHO) manual for semen analysis (2021) [1]. Exclusion criteria were: smoking, alcohol use, testicular trauma, inguinal hernia surgery, cryptorchidism, sexually transmitted diseases, azoospermia, and endocrine disorders.
Fertile female partners were required, defined as those with regular menstrual cycles, age below 40, and couples not seeking assisted reproductive procedures such as in vitro fertilization or artificial insemination.
2. Composition of antioxidant tablets
All patients received an oral supplement of antioxidant tablets, taken twice daily. The main composition is presented in Table 1. Each sachet of antioxidants contained 200 mg L-carnitine, 40 mg vitamin C, 15 mg coenzyme Q10, 0.1 mg vitamin B9, 50 mg zinc, 25 mg selenium, and 200 mg arginine. The recommended dosage was two tablets per day for 6 months from the time of infertility diagnosis. The study’s aim and methods were explained to all patients, and they consented to receive antioxidant supplements. All patients signed an informed written consent form before entering the study and were informed that they could withdraw at any time without any consequences.
3. Semen sample analysis
Semen analysis was carried out in accordance with the 6th edition WHO recommendations [28]. Sperm volume, concentration, motility, and morphology were assessed. Each participant was provided with a sterile plastic container, confirmed to be non-toxic for spermatozoa (SPZ), for semen collection by masturbation after 3 days of sexual abstinence. To minimize temperature fluctuations and control the interval between sample collection and analysis, all samples were collected at our unit. Macroscopic analysis included evaluation of liquefaction time, viscosity, volume, color, and pH. The analysis was conducted under conditions that ensured sample integrity.
First, the semen samples were homogenized using a Pasteur pipette, and sperm concentration and motility were evaluated with a computer-assisted sperm analyzer (SCA; MICROPTIC). For each measurement, a 2.5 µL aliquot of the sample was loaded into a standard four-chamber slide (Leja; NL). Fast and slow progressive motility (categories A+B), non-progressive motility (C), and immotile SPZ (D) were counted. Sperm concentration and motility assessments were conducted using 10× magnification.
Sperm morphology was assessed based on the modified David criteria using the Diff-Quik kit (Dade Behring AG), which includes one fixative and two stains (A and B). Morphological evaluation was performed using a Nikon microscope with oil immersion (Nikon), and at least 100 sperm cells were counted. Strict scoring criteria were applied to classify sperm as normal or abnormal according to the modified David classification.
4. DNA fragmentation assessment
Sperm DNA damage was measured by sperm chromatin dispersion (SCD) following the procedure described in our study [29]. DNA fragmentation was also assessed using the SCD test. In cases where there is no massive sperm DNA breakage, acid denaturation and removal of nuclear proteins result in dispersed DNA loops that produce a characteristic halo. In contrast, sperm with fragmented DNA either do not develop a halo or develop a small halo. The DNA fragmentation test was performed using the Spermfunc DNA kit (BRED Life Science Technology Inc.) employing the SCD method. Agarose was heated to 90–100 °C for 5 minutes, then cooled to 37 °C for 5 minutes. Semen was then mixed thoroughly with the agarose, and the suspension was poured onto agarose-coated slides and covered with a 20×20 mm cover glass. The slides were cooled at 4 °C for 5 minutes and then gently removed from the cover glass. Subsequently, the slides were incubated in a denaturation solution at 22 °C for 7 minutes, followed by incubation in a lysis solution at room temperature for 25 minutes. After washing with water for 5 minutes, the slides were dehydrated with graded ethanol (70%, 90%, and 100% for 2 minutes each). The slides were then dried and stained with Wright's solution for 25 minutes. Sperm were observed under a light microscope and categorized based on halo size (large, medium, small, absent, or degraded). Sperm exhibiting large or medium halos were classified as having unfragmented DNA, while those with small or no halos were classified as fragmented. Two observers independently assessed 500 sperm per sample, and the DFI was calculated as:
DFI (%)=100×(number of SPZ with fragmented DNA/total number of SPZ).
5. Total motile sperm count analysis
Sperm samples were prepared using a two-layered density gradient centrifugation technique (50% and 90% isolate, Irvine Scientific). Male factor infertility was assessed by analyzing the number of motile sperm in the ejaculate. The total motile sperm count (TMSC) was calculated using the formula:
TMSC=volume×concentration×(total motility/100).
Patients were further categorized into four groups based on TMSC according to WHO references: less than 0.5×106, 0.5–1×106, 1–2×106, and greater than 2×106.
6. Hormonal evaluation
A quantitative hormone profile was conducted for all patients, measuring LH, testosterone, prolactin, and FSH. Blood samples (10 mL) were collected in dry tubes and centrifuged within 1 hour using an Eppendorf 5810R centrifuge to obtain plasma. Hormone levels were determined in the biochemistry department using the Architect ci8200 analyzer (Abbott) [18].
7. Ethical approval
This study was approved by the Research Ethical Review Committee of the Faculty of Medicine and Pharmacy of Oujda, Morocco (Approval code: 02/2023). All participants were informed about the scientific nature of the study.
8. Statistical analysis
Data were analyzed using IBM SPSS Statistics 21.0 for Windows (IBM Co.). Continuous variables were expressed as mean±standard deviation, and categorical variables as numbers and percentages (%). Data distribution was assessed using the Kolmogorov–Smirnov test. Normally distributed data were analyzed with Turkey’s multiple comparison test, and an independent t-test was used to compare mean values between varicocele groups. Semen quality, reproductive hormones, sperm DNA damage, and pregnancy rates were compared among different varicocele grades (grade 0, 1, 2, and 3) using the t-test. A p-value of 0.05 was considered statistically significant.
Results
A total of 133 infertile patients with various grades of varicocele received daily multivitamin supplementation for 6 months. Semen parameters—including total sperm count, concentration, progressive motility, vitality, morphology, and reproductive hormones—were measured before and after treatment. Additionally, sperm DNA fragmentation and the proportion of highly degraded sperm cells were assessed using the SCD test. Successful pregnancies during the treatment period and for 3 months thereafter were recorded. All subjects completed the study and were evaluated both before and after treatment.
Table 2 summarizes the demographic and baseline characteristics of patients with varicocele-associated infertility across different grades. In groups G0, G1, G2, and G3, bilateral varicocele was observed in 20.6%, 35.86%, 11.99%, and 8.28% of patients, respectively, while unilateral varicocele was observed in 79.4%, 64.14%, 88.01%, and 91.72%, respectively. The mean age was 31.62±0.51 years for grade 0 varicocele, 36.01±2.82 years for grade 1, 35.93±3.53 years for grade 2, and 39.23±1.92 years for grade 3.
The mean levels of sperm parameters in patients with grade 0 and grade 1 varicocele, as assessed by WHO guidelines and Kruger’s strict criteria, are shown in Table 3. Additionally, mean sperm parameters, reproductive hormones, sperm DNA damage, and pregnancy rates were compared before and after multiantioxidant treatment in patients classified into groups G0, G1, G2, and G3 (Tables 3 and 4). Significant differences in mean semen volume, sperm count, total sperm count, sperm motility, and normal sperm morphology were observed among groups G0, G1, G2, and G3 (p<0.0001). We also evaluated changes in reproductive hormones—including FSH, LH, testosterone, and prolactin—before and after multiantioxidant treatment in each varicocele group (Tables 3 and 4). Sperm DNA fragmentation was measured using the SCA method, and the DFI results are summarized in Tables 3 and 4. The mean DFI (percentage of sperm with denatured DNA) improved significantly after treatment with multiantioxidants across all varicocele grades.
Pregnancy outcomes were recorded for each varicocele grade. Both spontaneous pregnancies and those achieved after intrauterine insemination (IUI) were evaluated following antioxidant supplementation. IUI resulted in pregnancy rates of 11%, 9%, 1%, and 0.7% in groups G0, G1, G2, and G3, respectively. Additionally, spontaneous pregnancy was achieved in 6% of couples with grade 0 varicocele and 3% with grade 1 after multiantioxidant supplementation. No significant pregnancy rates were observed in groups G2 and G3 (Figure 1). Among the varicocele-related infertility groups, the most notable improvements were observed in the grade 0 varicocele group following antioxidant therapy.
Discussion
Varicocele results from dilation of the pampiniform venous plexus in the testicle and is significantly more common on the left side than the right [1]. It is a major surgically treatable cause of male infertility and is associated with reduced sperm quality and low sperm production [3]. Varicoceles are classified by severity into grades 0 through 3 [6]. Varicoceles lead to increased OS within the testicular parenchyma, likely due to scrotal hyperthermia, testicular hypoxia, and disruption of the blood-testis barrier [12]. Under physiological conditions, there is a balance between ROS production and antioxidant protection [13]. However, when antioxidant capacity is reduced or ROS production is elevated, an imbalance occurs, resulting in OS [20]. In varicocele, ROS have been associated with impaired semen quality, particularly when combined with other conditions such as infections, environmental factors (e.g., pollution, radiation), or lifestyle factors (e.g., smoking) [21]. Thus, inadequate oxygenation induces chronic hypoxia, which directly damages germ cells and activates ROS-generating metabolic pathways [14]. OS related to varicocele may lead to apoptosis and DNA damage in sperm [12]. Numerous studies have reported that sperm from patients with varicocele exhibit high levels of DNA damage—even in men with normospermia—indicating a strong association with OS [8]. Reduced reproductive potential has been linked to factors such as increased testicular temperature, resulting from enhanced blood flow, which leads to deteriorated sperm parameters, DNA damage, and ultimately lower pregnancy rates [1]. Goktepe et al. [30] demonstrated that varicocele is associated with elevated oxidative damage and increased DNA-damaged SPZ. Antioxidants are commonly used to treat male infertility associated with increased OS, including in patients with varicocele [27]. The prominence of antioxidant treatments is primarily due to OS being the main mediator of testicular damage in varicocele [19]. Multiantioxidant supplementation, including zinc, selenium, vitamin C, and coenzyme Q10, improves spermatogenesis by reducing OS, thereby addressing significant contributors to male infertility [22]. To further explore the impact of antioxidant supplementation in patients with clinical varicocele of different grades, this study measured semen parameters, reproductive hormones, sperm DNA damage, and pregnancy rates before and after antioxidant therapy. To our knowledge, no previous studies have evaluated the efficacy of multiantioxidant treatment in infertile men, particularly across different varicocele grades. The present results indicate that, following antioxidant therapy, infertile patients with varicocele across various grades exhibited improvements in semen quality—including increases in total sperm count, sperm concentration, total and progressive motility—and a reduction in sperm DNA damage. Furthermore, the findings revealed that higher varicocele grades correlated with poorer semen parameters and increased sperm DNA fragmentation. We studied a select group of OA patients with clinical varicocele across different grades. Antioxidant therapy appears to be an effective alternative for improving and mitigating the damage caused by varicocele. These results align with previous studies that have demonstrated the beneficial effects of antioxidant therapy on sperm redox status, semen parameters, and pregnancy outcomes in patients with varicocele. Some studies have suggested improvements in semen quality and pregnancy rates following varicocele repair, while others have not demonstrated significant benefits from antioxidant treatment alone [18]. Many studies suggest that OS is the primary factor in the pathogenesis of varicocele-related infertility, and that antioxidant therapy can improve semen quality [31]. In this study, we confirmed that multiantioxidant treatment significantly improved sperm DNA integrity, increased total sperm counts, and enhanced TMSCs in infertile OA males with clinical varicocele grades 0 through 3. Our previous study demonstrated that multiantioxidant supplementation beneficially affects fertility, particularly sperm quality, and may be a promising therapy for male infertility [22]. Gual-Frau et al. [32] confirmed the beneficial effects of antioxidant compounds in patients with grade I varicocele. In their study, patients exhibited an average reduction in sperm DNA fragmentation across varicocele grades, and the total sperm count significantly increased after 6 months of treatment. The safety of the multiantioxidant formulation was ensured by its composition, and tolerability was confirmed by the near absence of adverse effects during treatment. The efficacy of the preparation appears to be due to the cumulative action of its individual antioxidant components, and it is commercially available in Morocco. The effects of varicocele on Leydig cell function and testosterone biosynthesis in infertile men remain controversial. Some reports indicate that varicocele is associated with low serum testosterone levels, which can significantly increase following successful varicocele repair [33]. Recently, Shiraishi and Naito [34] reported no significant differences in FSH, LH, and testosterone levels among varicocele grades; however, they noted that testosterone and FSH levels significantly correlated with elevated scrotal temperature in the standing position. To our knowledge, this is the first study to explore the impact of oral multiantioxidant therapy on semen parameters, reproductive hormones, and the sperm DFI in patients with different varicocele grades. Varicocele is one of the major correctable causes of male infertility [3]. Varicocelectomy has been shown to improve semen quality in men with abnormal parameters [2]. Adjuvant drug therapy, particularly with various antioxidants, appears to be associated with improved fertility outcomes compared with no medical treatment [19]. Therefore, the use of multiantioxidants may be beneficial for improving semen quality in patients with infertility associated with different varicocele grades. While surgery remains the treatment of choice, it does not always yield successful outcomes, making adjuvant therapy an acceptable alternative [35]. Adjuvant antioxidant therapy may offer additional benefits. Studies have demonstrated that antioxidant therapy has a beneficial effect on fertility, particularly in improving sperm quality, and is therefore recommended as a potentially effective treatment for male infertility—especially in patients with varicocele [36]. Both vitamin E and zinc contribute to regulating antioxidant balance and can improve sperm concentration, the percentage of progressively motile sperm, and consequently pregnancy rates [37]. Although oxidative damage is a proposed mechanism in varicocele pathogenesis [12], it remains debated whether antioxidant supplementation should be offered to patients with varicocele-associated infertility across different grades [30]. Lehtihet et al. [38] concluded that left-sided grade 3 varicocele can induce a reversible suppression of epididymal function, and that varicocele treatment results in improved semen quality and epididymal function. Sofimajidpour et al. [39] reported statistically significant decreases in total sperm motility in men with grade 3 varicocele compared to those with grade II. In light of this study, our data indicate that antioxidant treatment is a viable alternative for improving semen quality in patients with varicocele. Notably, patients with grade 0 or grade I varicocele showed significant improvements in sperm parameters, reproductive hormones, and DNA fragmentation indices compared to those with higher varicocele grades. Our study evaluated the use of a combination of metabolic substances, antioxidants, and micronutrients to improve sperm quality in patients with varicocele across different grades. Other reports suggest that grade 3 varicoceles have more detrimental effects on semen quality than grade 1 and 2, and that surgical repair may result in greater improvements in semen quality [40].
In conclusion, semen parameters, sperm DNA fragmentation levels, and pregnancy rates were evaluated at 6 months. Adjuvant antioxidant therapy improved sperm parameters and reduced sperm DNA fragmentation. Regular use of this preparation for at least 6 months may lead to significant improvements in overall semen quality and facilitate in vitro fertilization procedures in couples where male factors contribute to infertility.
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