Infertility is a disease of the male or female reproductive system defined by the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse [1] .

An increasing number of couples of reproductive ages seek specialized centers in the hope of resolving their infertility problems [2] [3] . Historically, the social burden of infertility has been attributed to women. With the progress in diagnostic studies and infertility treatments, it is now known that male infertility impacts in the same proportions as female infertility. Male infertility individually affects 20% - 30% of cases and contributes to approximately 50% of cases [4] [5] [6] . Therefore, the male factor is the main cause of infertility in numerous couples who seek medical assistance daily, a percentage that exceeds female infertility in many places [6] .

The main risk factors in the development of male infertility can be classified based on their origin as congenital, acquired, and idiopathic [7] [8] [9] . Among the acquired factors, the most common in daily clinical practice is varicocele, with a prevalence of 20% in healthy men and up to 40% in men with infertility [10] [11] [12] , and infections caused by bacteria, viruses, or protozoa in the genitourinary tract account for approximately 10% - 15% of infertility cases in men [13] .

In men with infertility, the most common sperm abnormalities are low sperm concentration (oligospermia, <15 × 10/mL; the total number of ejaculated sperm <39 × 10/mL), low sperm motility (asthenospermia), and anomalies in sperm morphology (teratospermia) [14] .

So far, many studies have been conducted in the hope of improving sperm quality in men [8] [15] [16] [17] . However, not all therapies are completely effective, and not everyone has access to high-tech assisted reproductive techniques that may offset possible defects in spermatogenesis.

Among the various risk factors mentioned above, inflammation and oxidative stress appear as common factors affecting sperm quality in patients with infertility [18] [19] [20] [21] . Under normal conditions, low levels of reactive oxygen species (ROS) are essential for carrying out various biochemical reactions and physiological functions [22] . However, oxidative stress (OS) has been identified as the core common mechanism by which various endogenous and exogenous factors may induce idiopathic male infertility [23] .

Recent studies have shown that increased oxidative damage plays a significant role in changes in sperm quality in male patients with infertility, especially those who also have varicocele [24] . In normal situations, cellular machinery and endogenous antioxidants restore the balance of reduction and oxidation (REDOX). In the specific case of varicocele, the abnormal dilation, elongation, and tortuosity of the veins in the pampiniform plexus of the spermatic cord can increase testicular hyperthermia, testicular hypoxia/ischemia, reflux of adrenal metabolites, and oxidative stress [25] [26] . Antioxidants could therefore play an important role in the proper development of spermiogenesis, especially in patients with associated varicocele [27] .

Different trials have studied daily supplementation with antioxidant agents and micronutrients that help control the REDOX balance, such as vitamin C, vitamin E, zinc, selenium, folic acid, or L-carnitine, with positive results in sperm parameters and ultimately on pregnancy rates [27] [28] [29] [30] .

Spermotrend^®, a natural product manufactured by Catalysis S.L., is formulated with different antioxidants and micronutrients with adequate concentrations to have the necessary antioxidant ability to help maintain the integrity of the tissues that are part of the male genitourinary tract, thereby improving sperm parameters. Among its chemical ingredients, Spermotrend^® includes zinc, selenium, and L-arginine which have shown important benefits in men suffering from infertility. Zincdeficiency has been linked to infertility cases in men. Zinc supplementation has been studied in male patients with infertility, improving sperm quality parameters [31] [32] . The selenium supplementation has shown an increase in motility, sperm survival, and reduction of spermatozoa with DNA fragmentation [33] [34] . L-arginine supplementation has been found to have a crucial role in sperm production in men with varicocele and may improve fertility outcomes [35] [36] [37] , as well as erection aid [37] [38] .

The objective of this clinical trial is to study the efficacy of daily supplementation with Spermotrend^® in improving sperm quality and quantity in men suffering from male infertility with additional oxidative conditions such as varicocele.

A total of 170 patients were enrolled in the clinical trial at Clinica Seniors (Managua, Nicaragua). There was no dropout or withdrawal among the recruited patients, and all 170 patients completed the trial protocol.

Table 2 shows that 65.3% of patients had primary infertility, while 34.7% of the recruited patients had previously achieved pregnancy and were diagnosed with secondary infertility at the time of recruitment. The age range of most of the patients recruited for the trial was 30 to 40 years old both for those with primary (84.7%) or secondary (79.7%) infertility. In the evaluation of the medical history of the patients recruited for the trial, 47.1% of the total number of patients had dyslipidemia at the time of recruitment, 24.1% had diabetes mellitus, and 3.5% had thyroid-related disease. 25.3% of the recruited patients did not have a medical history of any previous metabolic-related condition. In the determination of testosterone levels, 44.1% of the recruited patients with infertility had levels below 300 ng/dL, while 55.9% of the patients had testosterone levels between 300 ng/dL and 1000 ng/dL.

Sperm quality parameters were determined at baseline, at 4 months, and at 6 months of treatment through a spermiogram ( Table 3 ). At the time of recruitment, 38.2% of the patients had an ejaculate volume of less than 2 CC. 68.8% of the study population had abnormalities in sperm morphology, with the most common structural alteration being head abnormality, observed in 43.5% of them. The baseline sperm survival rate was equal to or less than 30 minutes in 43.5% of the patients. 40% of the recruited patients had oligospermia at the beginning of the study. Additionally, 76.5% of the patients showed alterations in the pH of the seminal fluid. Besides, the baseline testosterone levels were less than 300 ng/L in 44.1% of the recruited patients.

After 4 and 6 months of daily use of Spermotrend, 1 capsule of 536.62 mg/8 hours with meals, sperm parameters were re-assessed. At 4 months, sperm volume and motility improved significantly. However, it was not until 6 months of

daily Spermotrend^® administration that significant differences in sperm morphology, concentration, and pH were observed.

After 6 months of Spermotrend^®, the patients recruited for the trial showed the following results: a sperm ejaculate volume greater than 2 CC in 85.9% of the cases, normal sperm morphology in 72.9%, normal seminal pH in 59.4%, and no presence of blood in semen in 91.8% of cases.

On the other hand, ultrasound imaging studies were performed ( Table 4 ). The 170 patients recruited in this study additionally showed varicocele associated with infertility at grades 1, 2, and 3 (52.9%, 31.8%, and 21.2%, respectively) according to Dubin and Amelars’ criteria [39] at the time of recruitment.

Furthermore, out of the total number of patients, 35.3% had testicular hypotrophy with DV < 20% at the time of recruitment.

The evolution of the participants treated with Spermotrend^® showed a significant and progressive improvement in the grade of varicocele. After 6 months of treatment with Spermotrend^®, no varicocele was observed in 55.3%, and 90.6% of the patients presented improved testicular volume (testicles without hypotrophy).

According to the latest WHO statistics, approximately 50 - 80 million people worldwide suffer from infertility, and male factors are responsible for approximately 20% - 30% of all infertility cases [41] . Male infertility may be influenced by various factors that negatively impact spermatogenesis [20] [41] . Increased oxidative stress is a common factor that negatively affects sperm quality, especially in fertile-age men who have varicocele, metabolic disorders such as dyslipidemia, or inflammatory processes such as viral infections [11] [13] [14] [21] [24] . Therefore, in recent decades, the potential therapeutic benefit of administering mono and poly-formulations of antioxidants in male infertility has been assessed. The most studied antioxidants in infertility and those most administered so far include vitamins A, C, and E, L-carnitine, coenzyme Q10, folic acid, selenium, and zinc. However, the results of the studies conducted to date are inconclusive, and the efficacy of using antioxidants in the clinical improvement of male infertility has not been clarified. The main reasons for the significant difference in efficacy results lie in the reduced number of recruited patients, the considerable variability in the antioxidant doses, and the treatment periods.

Our study examines the efficacy of daily supplementation with Spermotrend^® in improving sperm quality and quantity in men suffering from male infertility with additional oxidative conditions such as varicocele. This clinical study shows that the administration of Spermotrend^®, a product containing antioxidants and balanced micronutrients, improved sperm parameters after 6 months of daily use. The components of Spermotrend are balanced to provide the required daily dose of antioxidants, which are associated with male infertility under oxidative stress conditions.

The initial study sample presented male infertility in fertile age associated with varicocele at stages I, II, and III (52.9%, 31.8%, and 21.2%, respectively), and 74.7% showed metabolic diseases (diabetes mellitus 24.1%; thyroid disorders 3.5%; and dyslipidemia 47.1%), both conditions being considered as baseline oxidative stress in the recruited patients. 44.1% had testosterone levels below the set normal range of 300 ng/dL. The development of varicocele may lead to testicular deficiency, resulting in a decrease in testosterone production by Leydig cells, oligospermia, and testicular hypotrophy with DV < 20%. Moreover, it has also been associated with DNA fragmentation in sperm, leading to a decrease in sperm motility, viability, count, and morphology [42] .

Based on all these findings, and taking into account the baseline conditions of the patients recruited, the study population was considered to have associated oxidative conditions that affected sperm quality. Of the total number of infertile patients who participated in the study, 38.2% had a seminal fluid volume of less than 2 ml (normal ejaculate volume is between 2 and 6 ml); 78.8% showed alterations in seminal pH; 68.8% had morphological abnormalities; 71.8% had low sperm survival; and 55.9% had alterations in sperm concentration.

After 4 months of daily administration of Spermotrend^®, no significant changes were observed in the morphological parameters or the sperm concentration. However, there were notable improvements in ejaculate volume and sperm motility. This is in alignment with a previous study showing that after 90 days of Spermotrend^® administration, sperm motility improved significantly, without significant changes in sperm morphology or concentration [43] . Regarding the varicocele condition, the percentage of patients with varicocele grade 2 decreased from 31.8% to 19.4%, and the percentage of patients with varicocele grade 3 decreased from 21.2% to 4.7%. 13.5% showed normal venous dilation. The rate of testicular hypotrophy with DV < 20% normalized to 11.8%.

Significant changes in the variables indicating sperm quality were observed after 6 months of treatment with Spermotrend^® at a dosage of 3 capsules per day. The ejaculate volume significantly improved, as well as sperm motility, compared to the values at 4 months of treatment. Sperm morphology and concentration, as well as pH, also showed significant improvements. After 6 months of treatment and follow-up, 72.9% of patients had normal morphology. 64.1% showed normospermia and 16.5% hyperspermia.

Furthermore, considering the grade of varicocele evaluated through imaging studies, the patients showed an improvement in the grade of varicocele after 6 months of treatment with Spermotrend^®. 55.3% of the patients showed normal venous flow and dilation. Only 5.9% of the patients had grade 2 varicocele, and 38.8% had grade 1 varicocele, with no one showing grade 3 varicocele. Regarding testicular hypotrophy, 90.6% of the patients completed the study with normalized testicular volume by recovering normal venous flow. We hypothesized that Spermotrend^® may help control oxidative stress at the endothelial level, thus promoting the recovery of venous flow and elasticity. Furthermore, it could be due to restoring the levels of L-arginine, which are reduced by the activity of the arginase enzyme.

Therefore, this study demonstrates that daily supplementation of Spermotrend for a continuous period of 6 months has a beneficial effect on sperm quality and the control of venous flow and dilation associated with varicocele, despite the heterogeneity of altered parameters associated with male infertility. The antioxidants and micronutrients of Spermotrend^® help restore proper REDOX balance and sperm metabolism, thus promoting the correct sperm morphology, motility, mobility, and maturation [44] .

Recent studies have shown that increased oxidative damage plays a significant role in changes in sperm quality in male patients with infertility, especially those who also have varicocele [24] . In normal situations, cellular machinery and endogenous antioxidants restore the balance of reduction and oxidation (REDOX). However, in situations of excessive oxidants and/or antioxidant deficiency, oxidative damage negatively affects spermatogenesis. This is the case of varicocele where structural anomalies in the pampiniform plexus of the spermatic cord can lead to increased testicular oxidative stress [25] [26] . Thus, antioxidants could therefore play an important role in the proper development of spermiogenesis, especially in patients with associated varicocele. Our study supports the evidence that 6 months of a cocktail of antioxidants in males with infertility may help to improve sperm quality and alleviate venous flow and dilation associated with varicocele that ultimately results in infertility.

Spermotrend^® contains important chemical ingredients such as zinc, selenium, and L-arginine with recognized benefits in fertility. Zinc, one of the most commonly used supplements in men suffering from infertility, is an element present in seminal plasma and in the prostate. It is essential for the production of spermatozoa, as well as for their morphology and proper motility. It is also involved in the correct testicular development and the control of steroidogenesis [31] [45] . Selenium is another well-studied and used nutritional supplement in the treatment of male infertility. This element acts as an antioxidant, forming part of the enzyme glutathione peroxidase, and helps control the production of oxidants, especially in sperm DNA. Its supplementation has shown an increase in motility, sperm survival, and reduction of spermatozoa with DNA fragmentation [34] [46] . Human studies suggest that L-Arginine supplementation is safe and effective for men with oligospermia or asthenospermia [37] . L-arginine is a key part of sperm production. It helps in producing nitric oxide and has excellent antioxidant properties. L-arginine coordinates several different functions that are required for booming sperm growth and development. Particularly, its supplementation affects several semen parameters including morphology and motility, which can ultimately enhance male reproductive performance [47] .

Combining several supplement nutrients with recognized effectiveness in male infertility may have synergistic effects on different molecular pathways such as oxidative stress, oxide nitric production, and inflammation, among others, that ultimately might explain the profound benefits seen with Spermotrend^® in sperm quality and morphology, although further studies may ascertain this hypothesis.

Finally and given the small sample size of this clinical trial, further large-scale multicenter studies should be conducted to confirm our findings.

In this study, after 6 months of therapy with Spermotrend^®, we observed significant beneficial effects on sperm morphology, mobility, and vitality. These results suggest that nutrient supplements play an essential role in maintaining male fertility. Moreover, these results support the significance of formulations that combine several nutrient supplements as a therapy to treat male infertility and recover the functional capacity of spermatozoa. Thus, Spermotrend^® could be offered as a functional and effective short-term therapy to improve sperm parameters associated with male infertility, as well as the grade of varicocele, and it helps reduce testicular hypotrophy, restoring the functionality of the affected testicles. Based on the results obtained in this study, daily use is recommended for at least 6 months, although a large randomized controlled trial is warranted to confirm our results.

The study protocol was approved by the Ethics Committee of Clinica Seniors, Managua (Nicaragua) under code 5626 and was conducted in agreement with the Declaration of Helsinki. Written consent was obtained from all patients before study enrollment.

All authors have contributed equally to the conceptualization, methodology, investigation, writing, preparation, and drafting of the clinical trial manuscript. All authors have read and agreed to the published version of the manuscript.

This research has been funded in part by Catalysis, S.L. (Madrid, Spain), which provided the product (Spermotrend^®) used for the trial.

The datasets used and/or analyzed during the current clinical trial are available from the corresponding author upon reasonable request.

The authors thank the participants involved in this study.