Antioxidants for male infertility: therapeutic scheme and indications. A retrospective single-center real-life study

Antioxidants for male infertility: therapeutic scheme and indications. 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A retrospective single-center real-life study Rossella Cannarella, Andrea Crafa, Raneen Sawaid Kaiyal, Shinnosuke Kuroda, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2773630/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jan, 2024 Read the published version in Minerva Endocrinology → Version 1 posted You are reading this latest preprint version Abstract This single-center real-life study was conducted to evaluate the most effective combination of nutraceuticals and the most appropriate indications for the treatment of male infertile patients. Infertile patients aged 20–55 years were treated with a combination of antioxidants (Androlen®) (Group 1), with Androlen® and a mixture of fibrinolytic molecules (Lenidase®) (Group 2), or Androlen® and other molecules different from those used for the patients of the Group 2 (Group 3). Patients were also subdivided according to the presence of varicocele, mild testicular hypotrophy, idiopathic infertility, and chronic male accessory gland infection (MAGI). Forty-three patients were enrolled. In the overall analysis, only progressive motility significantly improved after therapy. Subgroup analysis showed a significant increase in progressive motility, total motile sperm count (TMSC), and in the percentage of alive spermatozoa after treatment in the Group A. Progressive motility improved significantly in patients with varicocele, while the TMSC in patients with varicocele and those with idiopathic. The percentage of alive spermatozoa increased in patients with testicular hypotrophy. Treatment with antioxidants increases progressive sperm motility. The association of fibrinolytic or other nutraceuticals does not improve the efficacy of the treatment with antioxidants alone. The treatment was effective in patients with varicocele or idiopathic infertility. Antioxidants fibrinolytic conventional sperm parameters bio-functional sperm parameters indications male infertility varicocele idiopathic infertility Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION The correlation between male infertility and oxidative stress is well-known and demonstrated in the literature. Approximately 30% of cases of male infertility are defined as idiopathic [ 1 ]. From 30 to 80% of male sub-infertility cases are due to oxidative stress damage [ 2 ] and more than 20 to 40% of infertile patients have significantly higher semen ROS levels than fertile men [ 3 ]. A low amount of ROS production is required for sperm function [ 4 – 6 ]. However, when ROS production exceeds the natural scavenger capacity of seminal fluid, oxidative stress increases, and this results in sperm damage. Infections, autoimmune disorders, chronic disease, advanced age, alcohol consumption, smoking, high temperatures, environmental pollutants, and obesity are the most common causes of increased oxidative stress [7]. Spermatozoa are particularly susceptible to oxidative stress [ 8 ]. It negatively affects total and progressive sperm motility and normal sperm morphology. It also increases DNA damage and consequently decreases fertility [ 9 – 13 ]. Therefore, increased oxidative stress is a clinical condition that interferes with reproductive function. Seminal plasma contains natural antioxidants, such as vitamins C and E, catalase, superoxide dismutase, glutathione and glutathione peroxidase, superoxide dismutase, thioredoxin, and polyamine spermine. They act as free radical scavengers [ 14 ]. These compounds often cooperate with other substances, such as vitamin A, coenzyme Q10, carnitines, myoinositol, lycopene, and micronutrients (selenium, zinc, and copper) [ 15 ]. The Dietary Supplement Health and Education Act defines nutraceuticals as dietary supplements that contain a finely titrated combination of standard dietary biotic compounds that are often used for their anti-inflammatory, antioxidant, and anti-apoptotic properties [ 16 , 17 ]. Several studies have reported that the oral administration of antioxidants is efficacious in improving conventional sperm parameters without adverse events [ 13 , 18 ]. Antioxidants are advantageous because they are widely available and relatively inexpensive compared to other therapeutic strategies used for fertility treatment. The use of antioxidants is suggested by scientific evidence only at the end of a complete diagnostic workup [ 19 , 20 ]. Indeed, the latest Cochrane review on 6,264 subfertile patients showed, albeit with low-quality evidence, that antioxidants increase the pregnancy rate and improve the live birth rate in couples attending fertility clinics [5]. These findings support the administration of molecules with antioxidant properties in patients with abnormal sperm parameters caused by increased oxidative stress [5]. Different antioxidants are currently used, although there is no consensus on which compound to use [ 21 , 22 ]. Therefore, a combination of nutraceuticals with antioxidant properties is often used in male infertility to take advantage of their synergistic effects. Similarly, fibrinolytic compounds are used for the treatment of infertility [23], but at the moment it is not known whether they should be better prescribed in association with antioxidants or which patients could benefit more from their prescription. Furthermore, although different diseases are diagnosed as “male infertility”, they represent different clinical entities with different pathogenesis (i.e., varicocele, urogenital infection, urogenital inflammation, previous cryptorchidism, idiopathic infertility, etc.) and may benefit from antioxidants differently. On this account, this real-life single-center study aimed to evaluate the most effective combination of nutraceuticals and the most appropriate indications, in patients with male infertility. To accomplish this, we retrospectively extracted from the medical records of male patients entering the Division of Endocrinology, Metabolic Diseases and Nutrition for infertility who were prescribed antioxidants, data on the type of antioxidant prescribed, duration of therapy, clinical diagnosis, conventional and biofunctional sperm parameters before and after therapy, pregnancy outcomes, and female partner-related information. A before-after analysis of conventional and biofunctional parameters was carried out. Data were further sub-analyzed based on the type of therapy prescribed and the patient’s diagnosis. 2. MATERIALS AND METHODS 2.1. Patient selection In this single-center real-life retrospective study, we collected data from January 2014 to January 2023. Male patients between 20 and 55 years, referring to the Division of Endocrinology, Metabolic Diseases and Nutrition, University of Catania, for male infertility were evaluated for inclusion. Men with endocrinopathies, gastrointestinal, hematological, nephrological or autoimmune disorders, chemo and/or radiotherapy, and corticosteroid therapy, genetic disorders, hypogonadism, diabetes mellitus, hormone replacement therapy, smokers, drinkers, acute male accessory gland infection (MAGI) or MAGI non-respondent to first- and second-line antibiotics were not included. Meanwhile, those with varicoceles, primary testicular dysfunction leading to mild or severe testicular hypotrophy due to cryptorchidism or for unknown reasons, idiopathic infertility, and chronic or antibiotic-respondent MAGI were included. A complete medical history was reported in the database for each patient as well as a careful physical examination, laboratory workup, and ultrasound evaluation. 2.2. Experimental design Patient were treated with 1) a NAC 150 mg; α-lipoic acid 150 mg; L-carnitine 250 mg; Inositol 50 mg; Coenzyme Q10 10 mg and Acid Folic 200 mg (Androlen®) ( Group 1 ), 2) Androlen® plus a mixture of fibrinolytic molecules (Scutellaria baicalensis 400 mg, Baicalina 380 mg; Aesculus hippocastanum L. cortex 400 mg, Escin 60 mg; Bromeline) (Lenidase®) ( Group 2 ), and Androlen® plus other molecules, different from those used in Group 1 and Group 2 ( Group 3 ). Patients underwent semen analysis and evaluation of biofunctional sperm parameters before and after treatment. Treatment continued until benefits were seen (e.g. improvements in conventional and/or biofunctional sperm parameters). 2.4. Semen analysis Semen analysis was performed according to WHO criteria (WHO, 2010). Semen samples were collected by masturbation into a sterile container after 2–7 days of sexual abstinence and were analyzed immediately after liquefaction. Each sample was evaluated for semen volume, pH, sperm count, progressive motility, morphology, and round cell concentration. 2.5. Flow cytometric analysis Flow cytometric analysis was performed using flow cytometer CytoFLEX (Beckman Coulter Life Science, Milan) equipped with two lasers and six total fluorescence channels (four 488 nm and two 638 nm), then the results were analyzed with the software CytExpert 1.2 and the following tests were performed. 2.5.1. Assessment of sperm DNA fragmentation Sperm DNA fragmentation was performed by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay (Apoptosis Mebstain kit, DBA s.r.l, Milan, Italy) using terminal deoxynucleotidyltransferase (TdT), an enzyme that polymerizes, at the level of DNA breaks, modified nucleotides conjugated to a fluorochrome. To obtain a negative control, TdT was omitted from the reaction mixture. Reading was performed by flow cytometry using the FL1 detector. 2.5.2. Evaluation of the sperm mitochondrial membrane potential The mitochondrial membrane potential (MMP) was evaluated by a lipophilic probe 5,5',6,6'-tetrachloro-1,1',3,3'tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, DBA S.r.l, Milan, Italy) able to selectively penetrate the mitochondria. Briefly, an aliquot containing 1x10 6 /ml of spermatozoa was incubated with JC-1 in the dark for 10 minutes at 37°C. At the end of the incubation period, the spermatozoa were washed in PBS and analyzed. In viable cells with normal membrane potential, JC-1 is found in the mitochondrial membrane in the form of aggregates that fluorescence orange fluorescence, while in cells with low membrane potential, it remains in the cytoplasm in monomeric form, giving a green fluorescence. 2.5.3. Evaluation of the degree of chromatin compactness The degree of chromatin compactness was evaluated after permeabilization of the cell membrane using LPR DNA-Prep Reagent to allow the fluorophore to access the nucleus. Subsequently, spermatozoa were incubated with Stain DNA-Prep Reagent (Beckman Coulter, IL). An aliquot of 1x10 6 spermatozoa was incubated with DNA-Prep Reagent containing 0.1% potassium cyanate, 0.1% NaN 3 , nonionic detergents, saline, and stabilizers (Beckman Coulter, IL, Milan, Italy), in the dark, at room temperature for 10 minutes. Flow cytometry analysis was performed after 30 minutes, using an FL3 detector. 2.5.4. Evaluation of sperm apoptosis/vitality Sperm apoptosis/viability was evaluated by annexin V-FITC Apoptosis (Beckman Coulter, IL, Milan, Italy). The simultaneous cell staining with PI and annexin V that binds selectively to phosphatidylserine (PS), an early signal of apoptosis, allows distinguishing alive spermatozoa (with intact cytoplasmic membrane) and apoptotic or necrotic spermatozoa. To accomplish this, an aliquot containing 0.5x10 6 /ml was suspended in a 0.5 ml buffer containing 10 µl of annexin V-FITC and 20 µl of PI (Annexin V-FITC Apoptosis, Beckman Coulter, IL, Milan, Italy) and incubated for 10 minutes in the dark. After incubation, the sample was analyzed by FL-1 (FITC) and FL3 (PI) detectors. The different staining patterns allowed us to identify the three different cell populations: viable cells (FITC negative and PI negative); early apoptotic cells with still intact cytoplasmic membrane (FITC-positive and PI-negative); and cells in late apoptosis (FITC positive and PI-positive). 2.6. Statistical analysis Data are reported as mean ± standard deviation (SD) for non-skewed variables, while non-normally distributed continuous variables as the median and interquartile range (IQR). The distribution of values was evaluated using the Shapiro-Wilk test. Before-after analysis was performed using the t -test for independent samples for normally distributed variables and the Mann-Whitney U test (which is used for independent samples) for not normally distributed. Two sub-analyses based on 1) the type of treatment administered and 2) the patients’ diagnosis (varicocele, testicular hypotrophy, idiopathic infertility, or chronic MAGI) were performed to identify the most effective therapeutic strategy and indications. Two-way analysis of variance (two-way ANOVA) with Shapiro-Wilk correction for multiple comparisons was used for subgroup analysis. Statistical analysis was performed using MedCalc Software Ltd. (Ostend, Belgium), version 19.6–64 bit. A p-value less than 0.05 was considered statically significant. 2.6. Ethics statement This study was conducted at the Division of Endocrinology, Metabolic Diseases and Nutrition of the University-Teaching hospital Policlinico “G. Rodolico – San Marco”, University of Catania (Catania, Italy). The protocol was approved by the internal Institutional Review Board. Written informed consent was obtained from each participant after a thorough explanation of the purpose and nature of all procedures used. The study was conducted according to the principles expressed in the Declaration of Helsinki. 3. RESULTS Forty-three patients with a mean age of 36.5 ± 5.5 years, a mean body mass index (BMI) of 26.4 ± 3.5 Kg/m 2 , coming from Eastern Sicily, were selected for this study. The presence of a female infertility factor (polycystic ovary syndrome, endometriosis, tubal occlusion, low oocyte quality, oligomenorrhea) occurred overall in 11 out of 43 cases. The mean age of the female partners was 33.8 ± 5.4 years. Twenty-four patients were prescribed Androlen® (Group 1), 12 received treatment with Androlen® and Lenidase® (Group 2), and 7 received Androlen® and other molecules (Group 3). The mean length of treatment was 4.4 ± 2.4 months. In addition, 13 patients had varicocele, 10 testicular hypotrophy, 17 idiopathic infertility, and 3 chronic MAGI. Interestingly, 11 couples achieved pregnancy, and 5 of them this so by natural intercourse, while 30 failed in reaching pregnancy. Miscarriages occurred in 4 cases. The length of the time to pregnancy ranged from 1 to 12 months. 3.1 Before-after analysis The results of the before-after analysis are summarized in Table 1 . Particularly, only progressive motility showed significant improvement after therapy. The other conventional sperm parameters did not change significantly. Similarly, no difference was found in biofunctional sperm parameters. Table 1 Results of the before-after analysis of conventional and bio-functional sperm parameters. Before After p Value n Value n Volume (ml) 3.0 (2.0–4.0) 42 3.0 (2.3–4.1) 41 0.55 Sperm concentration (mil/ml) 26.0 (10.0–34.0) 42 27.0 (11.0–45.0) 42 0.68 TSC (mil/ejaculate) 60.0 (38.3–110.3) 43 80.0 (27.5–130.0) 42 0.68 Progressive motility (%) 13.0 (7.0–20.0) 42 19.0 (13.0–23.0) 42 0.03 TMSC (million) 7.5 (3.9–14.7) 43 13.5 (4.1–23.8) 42 0.12 Total motility (%) 54.5 (50.0–62.0) 42 57.5 (45.0–65.0) 42 0.93 Morphology (%) 4.0 (2.0–7.0) 42 4.0 (3.0–6.0) 42 0.74 Leukocytes (mil/ml) 0.5 (0.2–0.7) 41 0.5 (0.2–0.8) 41 0.60 Alive (%) 55.0 (39.3–61.1) 18 63.6 (49.7–70.7) 20 0.15 Chromatin compactness (%) 20.1 ± 6.9 17 22.3 ± 9.6 20 0.96 Early apoptosis (%) 1.5 (0.6–3.5) 18 1.2 (0.6–2.1) 20 0.74 Late apoptosis (%) 5.6 (3.9–10.5) 18 4.6 (0.8–9.8) 20 0.38 H-MMP (%) 65.8 ± 22.4 18 55.2 ± 23.9 19 0.17 L-MMP (%) 34.2 ± 22.4 18 44.8 ± 23.9 19 0.17 SDF (%) 3.0 (0.9–5.0) 16 3.2 (0.6–6.1) 18 0.90 Data are shown as mean ± standard deviation (SD) for skewed variables, while the non-normally distributed continuous variables as the median and interquartile range (IQR). The distribution of values was evaluated using the Kruskal-Wallis test. The p values were calculated using ANOVA and Kruskal-Wallis for skewed variables, with Shapiro-Will correction for multiple comparisons. A p-value less than 0.05 was considered statistically significant. Legend : H-MMP, high mitochondrial membrane potential; L-MMP, low mitochondrial membrane potential; SDF, sperm DNA fragmentation; TMSC, total motile sperm count; TSC, total sperm count. 3.2 Before-after analysis based on the type of drug prescribed Group A showed a significant increase in progressive sperm motility (12.3 ± 7.8% vs. 16.1 ± 8.2%, p < 0.01) and TMSC [7.5 (2.8–10.3) vs. 16.0 (5.0–24.4), p < 0.01] after treatment with Androlen, while no significant improvement of these parameters was observed in Groups B and C. No significant difference in semen volume, sperm concentration, total sperm count, total motility, normal forms, and leukocyte concentration was found in any of the Groups (Fig. 1 ). We also found a trend towards an increase in total sperm count (71.1 ± 68.5 mil/ml vs. 96.1 ± 78.6 mil/ml, p = 0.06) in Group A and towards an increase in progressive motility (13.8 ± 8.3 mil/ml vs. 20.5 ± 14.1 mil/ml, p = 0.06) in Group B. Interestingly, Group A also showed an increase in the percentage of alive spermatozoa (60.9 ± 4.8% vs. 74.0 ± 4.8%, p < 0.01) after treatment, which was not observed in the other groups. After treatment, no differences were observed in the percentage of spermatozoa with compacted chromatin, early or late apoptosis, L-MMP, H-MMP, or fragmented DNA compared to baseline values in any of the other groups (Fig. 2 ). 3.3 Before-after analysis based on diagnoses Compared to pre-treatment values, we found a significant improvement in progressive motility [9.0 (6.0–16.0) vs. 15.0 (14.5–22.0), p < 0.05] in patients with varicocele, and in TMSC in patients with varicocele (9.0 ± 5.0 vs. 15.4 ± 10.0, p < 0.05) and in those with idiopathic infertility 7.2 (1.6–17.3) vs. 13.8 (1.1–24.9), p < 0.05] after treatment (Fig. 3 ). Regarding biofunctional sperm parameters, the percentage of alive spermatozoa significantly improved in patients with testicular hypotrophy (60.9 ± 8.1% vs. 76.5 ± 6.7%, p < 0.05) (Fig. 4 ). A trend toward an increase in the percentage of spermatozoa with L-MMP (33.3 ± 19.5% vs. 41.3 ± 19.4%, p = 0.06) and a reduction in the percentage of spermatozoa with H-MMP (66.7 ± 19.5% vs. 58.7 ± 19.4%, p = 0.06) was found in patients with varicocele. 4. DISCUSSION This was a single-center retrospective study, conducted to evaluate the best combination of nutraceuticals among those most prescribed by us, as well as which infertile patients could benefit most from the treatment. We have found that adding fibrinolytic or other nutraceuticals to an antioxidant mixture does not improve treatment efficacy. Indeed, contrary to the other treatments, patients with the latter therapeutic regimen alone showed a significant increase in progressive sperm motility, TMSC, and percentage of alive spermatozoa. Furthermore, the treatment was effective in infertile patients with varicocele or idiopathic infertility, who showed a significant improvement in progressive sperm motility and TMSC, while patients with mild testicular hypotrophy or chronic MAGI may not benefit from this type of treatment. Infertile patients have higher seminal fluid oxidative stress than fertile men [4, 7]. Consequently, antioxidants are often used for the treatment of idiopathic oligo-astheno-teratozoospermia due to their ability to counteract the deleterious effects of oxidative stress on sperm parameters and function [24]. Indeed, high levels of oxidative stress can damage the main sperm molecular components (lipid, protein, and DNA). The plasma membrane of spermatozoa plays a key role in successful fertilization because of its fluidity, flexibility, and functional activity. It consists of high levels of polyunsaturated fatty acids. Double bonds of membrane lipids can be oxidized by ROS. This causes a process called lipid peroxidation which affects membrane fluidity and, consequently, alters sperm motility [ 25 ]. Furthermore, mitochondria are a major source of ROS in spermatozoa [ 1 ], mainly produced by oxidative phosphorylation. Sperm mitochondrial function is closely related to the motility of these cells. Therefore, an imbalance between oxidative and antioxidant systems may reduce sperm motility by impairing sperm mitochondrial function [ 26 ]. Moreover, ROS can directly damage sperm DNA and spermatozoa cannot repair DNA due to the deficiency of the cytoplasmic enzyme systems involved in the molecular mechanisms of DNA repair [ 11 ]. For this reason, several molecules with antioxidant properties are used to counteract the negative effects of oxidative stress on sperm function. Among them, coenzyme Q10, carnitine, and myoinositol have been shown to improve sperm motility and protect DNA from oxidative damage [ 15 , 27 – 31 ]. Furthermore, coenzyme Q10 has shown beneficial effects on the pregnancy rate [ 32 , 33 ]. Treatment with CoQ10 increases mitochondrial DNA stability and counteracts the damage caused by increased ROS production to its specific RNA polymerase and mitochondrial transcription factor-A. These molecules play a fundamental role in mitochondrial transcription. These results suggest that antioxidant treatment could improve sperm motility by preserving the sperm transcription system from ROS damage [ 34 ]. In the present study, a mixture of antioxidants was investigated. Often during the treatment, the synergistic effect of the various molecules with antioxidant properties is exploited [ 35 ]. For example, carnitine associated with ubiquinol improves sperm mitochondrial function. Co-administration of vitamin C and vitamin E correlates positively with the improvement of motility and normal morphology, reduces the SDF rate, and increases the pregnancy rate [ 36 , 37 ]. A significant sperm concentration improvement has been reported after the administration of folic acid and zinc sulfate [ 38 ]. In contrast, Raigani and colleagues conducted a double-blind, randomized, placebo-controlled study on subfertile patients with oligo-astheno-teratozoospermia to evaluate the effects of oral folic acid and zinc sulfate supplementation on both sperm motility and MMP. The results of this study showed that zinc sulfate and folic acid supplementation did not improve sperm quality in infertile patients with severely impaired sperm parameters. Therefore, further studies are needed to define the effects of these molecules and to identify patients who may benefit from supplementation with these molecules [ 39 ]. While the currently available Cochrane meta-analyses [5,40] and the Society’s guidelines [ 41 – 44 ] do not identify, among all male infertile patients, those who could benefit most from therapy, the meta-analytical evidence suggests the positive impact of antioxidants in patients with varicocele. A meta-analysis of six studies, including 576 patients, reported a significant increase in sperm concentration, progressive and total motility, and normal morphology in the antioxidant-treated group, compared with controls, 3 months after varicocelectomy [ 45 ]. More recently, another meta-analysis reported a significant improvement in sperm concentration, progressive and total motility, and seminal volume in patients with varicocele, who did not undergo surgical repair, after oral antioxidant supplementation [ 46 ]. These latter findings are in line with those reported in the present study and suggest varicocele as one of the indications of treatment with antioxidants in infertile patients. Trials have been carried out to evaluate the benefits of administering antioxidants in patients with idiopathic infertility [ 47 , 48 ]. A prospective study on 50 patients with idiopathic infertility and 35 fertile control, evaluated the effects of CoQ10 on conventional sperm parameters and antioxidant indices in the seminal fluid, reporting an increased total and progressive motility and oxidative stress [ 48 ]. A randomized controlled study on 73 patients with idiopathic infertility and 70 controls found a significant improvement in sperm concentration, motility, and normal morphology in the group treated with L-carnitine. The administration of L-carnitine resulted in greater efficacy compared to CoQ10 and vitamin E [ 47 ]. However, despite the already existing evidence, most of the studies claiming to enroll patients with idiopathic infertility provide neither a careful description of the diagnostic workup nor of the inclusion and exclusion criteria, which may suggest the presence of misdiagnosis [ 49 ]. However, in line with the current literature, our findings support the efficacy of antioxidants in patients with idiopathic infertility. Interestingly, we found no efficacy in eugonadal patients with mild testicular hypotrophy (i.e., anamnesis positive for cryptorchidism or for apparently unexplained reasons). There are no studies in the literature aimed at evaluating the effects of antioxidants on previously cryptorchidic oligozoospermic patients with borderline-low testicular volume or those with unexplained borderline-low testicular volume. Testicular volume may represent a phenotypic sign that could help in decision-making. Indeed, while idiopathic patients with normal testicular volume appear to respond to antioxidants, those with idiopathic infertility and borderline-low testicular volume may not, due to primary testicular dysfunction. This should come as no surprise, as monogenic causes of oligozoospermia have been discovered in recent times, some of which have a minor impact on testicular volume [ 50 – 53 ]. Lastly, patients with chronic MAGI did not respond to antioxidants. MAGI is a complex entity that can recognize a bacterial etiology, even when the microbiological examination is negative [54,55]. Microbes are capable of triggering a cascade of events that increase oxidative stress [56]. Therefore, the eradication of germs should be a prerequisite before the treatment with antioxidants is started. The results of the present study should be taken with caution, due to some limitations, given by the limited size of the sample (including that of the subgroups), and by the retrospective design, which does not admit any causal relationship between the therapy and the observed findings. However, one of the strengths of the present study is the evaluation of biofunctional sperm parameters. Conversely, few studies have actually evaluated the effects of antioxidant treatment on biofunctional sperm parameters. A recent review describes that resveratrol, a natural antioxidant, significantly decreased the percentage of spermatozoa with abnormal chromatin compactness and high lipoperoxidation in spermatozoa incubated in vitro [57]. Furthermore, the rigorous diagnostic workup outlined in Materials and Methods used to include or exclude patients and the robust statistical approach are other strengths of the study. 5. CONCLUSION In conclusion, the results of this single-center retrospective study, which was undertaken to evaluate the best combination of nutraceuticals among those we prescribed most frequently, as well as which infertile patients could benefit most from treatment, indicate that the addition of fibrinolytic or other nutraceuticals to a mixture of carnitine-based antioxidants does not improve the effectiveness of the treatment obtained with the use of the latter alone. In fact, patients on the latter therapeutic regimen showed a significant increase in sperm progressive motility, TMSC, percentage of alive spermatozoa, and a good pregnancy rate. Furthermore, the treatment was effective in infertile patients with varicocele or idiopathic infertility, who showed a significant improvement in sperm progressive motility and TMSC, while patients with mild testicular hypotrophy or chronic MAGI did not benefit from this type if treatment. Prospective studies are needed to further confirm the conclusions of the present study. Declarations Acknowledgements None Author Declarations Ethics approval : This study was approved by the internal Institutional Review Board of the Division of Endocrinology, Metabolic Diseases and Nutrition of the University-Teaching hospital Policlinico “G. Rodolico – San Marco”, University of Catania (Catania, Italy). Consent to participate : Written informed consent to participate the study was obtained from each participant after a thorough explanation of the purpose and nature of all procedures used. Consent for publication : Written informed consent to publish the study was obtained from each participant. Availability of data and material: Data will be made available upon request to the corresponding author. Code availability : Not applicable. Author Contributions: Conceptualization, R.C. and A.E.C.; literature search, A.A., L.M.M., F.B.; data management: A.C., S.S.; writing original draft preparation, A.A., R.C., R.A.C., A.C.; statistical analysis, R.C., A.C.; revision and editing, R.S.K., S.K., S.L.V., A.E.C.; supervision, A.E.C. All authors have read and agreed to the published version of the manuscript. Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Conflicts of Interest: The authors have nothing to disclose and declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. References Agarwal, A., Mulgund, A., Hamada, A., Chyatte, M.R. A unique view on male infertility around the globe. Reprod Biol Endocrinol 2015; 13,37. Lanzafame, F.M., La Vignera, S., Vicari, E., Calogero, A.E. Oxidative stress and medical antioxidant treatment in male infertility. 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Antioxidant treatment with carnitines is effective in infertile patients with prostate-vesiculo-epididymitis and elevated seminal leukocyte concentrations after treatment with nonsteroidal anti-inflammatory compounds. Fertil Steril 2002; 78,1203-8. Perri, A., Ilacqua, A., Valenti, M., Aversa, A. Effects of nutraceuticals on sexual satisfaction and lower urinary tract symptoms in a cohort of young-old men. Phytother Res 2018; 32, 284-289. Greco, E., Romano, S., Iacobelli, M., Ferrero, S., Baroni, E., Minasi, M.G., Ubaldi, F., Rienzi, L., Tesarik, J. ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment. Human Reproduction 2005; 20,2590–2594. Safarinejad, M.R., Safarinejad, S. Efficacy of selenium and/or N-acetyl-cysteine for improving semen parameters in infertile men: A double-blind, placebo controlled, randomized study. J Urol 2009; 181,741–51. Raigani, M., Yaghmaei, B., Amirjannti, N., Lakpour, N., Akhondi, M.M., Zeraati, H., Hajihosseinal, M.,Sadeghi, M.R. The micronutrient supplements, zinc sulphate and folic acid, did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men. Andrologia 2014; 46, 956–962. Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. Cochrane Database Syst Rev. 2014;(12):CD007411. doi: 10.1002/14651858.CD007411.pub3. Epub 2014 Dec 15. Update in: Cochrane Database Syst Rev. 2019 Mar 14;3:CD007411. PMID: 25504418 Calogero AE, Aversa A, La Vignera S, Corona G, Ferlin A. The use of nutraceuticals in male sexual and reproductive disturbances: position statement from the Italian Society of Andrology and Sexual Medicine (SIAMS). J Endocrinol Invest. 2017 Dec;40(12):1389-1397. doi: 10.1007/s40618-017-0699-6. Epub 2017 Jun 6. PMID: 28589384. Ferlin A, Calogero AE, Krausz C, Lombardo F, Paoli D, Rago R, Scarica C, Simoni M, Foresta C, Rochira V, Sbardella E, Francavilla S, Corona G. Management of male factor infertility: position statement from the Italian Society of Andrology and Sexual Medicine (SIAMS) : Endorsing Organization: Italian Society of Embryology, Reproduction, and Research (SIERR). J Endocrinol Invest. 2022 May;45(5):1085-1113. doi: 10.1007/s40618-022-01741-6. Epub 2022 Jan 24. PMID: 35075609. Colpi GM, Francavilla S, Haidl G, Link K, Behre HM, Goulis DG, Krausz C, Giwercman A. European Academy of Andrology guideline Management of oligo-astheno-teratozoospermia. Andrology. 2018 Jul;6(4):513-524. doi: 10.1111/andr.12502. PMID: 30134082. Minhas S, Bettocchi C, Boeri L, Capogrosso P, Carvalho J, Cilesiz NC, Cocci A, Corona G, Dimitropoulos K, Gül M, Hatzichristodoulou G, Jones TH, Kadioglu A, Martínez Salamanca JI, Milenkovic U, Modgil V, Russo GI, Serefoglu EC, Tharakan T, Verze P, Salonia A; EAU Working Group on Male Sexual and Reproductive Health. European Association of Urology Guidelines on Male Sexual and Reproductive Health: 2021 Update on Male Infertility. Eur Urol. 2021 Nov;80(5):603-620. doi: 10.1016/j.eururo.2021.08.014. Epub 2021 Sep 10. PMID: 34511305 Wang J, Wang T, Ding W, Wu J, Wu G, Wang Y, Zhou Z, Xu L, Cui Y. Efficacy of antioxidant therapy on sperm quality measurements after varicocelectomy: A systematic review and meta-analysis. Andrologia. 2019 Nov;51(10):e13396. doi: 10.1111/and.13396. Epub 2019 Aug 18. PMID: 31423629. Ioannidou PG, Papanikolaou DA, Bosdou JK, Goulis DG, Lambropoulos AF, Grimbizis GF, Κolibianakis EM. Improvement in sperm quality by oral antioxidant supplementation in infertile men with varicocele who have not undergone surgical repair: Systematic review and meta-analysis. Andrologia. 2022 Nov;54(10):e14533. doi: 10.1111/and.14533. Epub 2022 Jul 12. PMID: 35819022. Ma L, Sun Y. Comparison of L-Carnitine vs. Coq10 and Vitamin E for idiopathic male infertility: a randomized controlled trial. Eur Rev Med Pharmacol Sci. 2022 Jul;26(13):4698-4704. doi: 10.26355/eurrev_202207_29194. PMID: 35856361. Alahmar AT. Coenzyme Q10 improves sperm motility and antioxidant status in infertile men with idiopathic oligoasthenospermia. Clin Exp Reprod Med. 2022 Dec;49(4):277-284. doi: 10.5653/cerm.2022.05463. Epub 2022 Nov 4. PMID: 36482502; PMCID: PMC9732077. Agarwal A, Cannarella R, Saleh R, Harraz AM, Kandil H, Salvio G, Boitrelle F, Kuroda S, Farkouh A, Rambhatla A, Zini A, Colpi G, Gül M, Kavoussi P, Hamoda TAA, Ko E, Calik G, Toprak T, Pinggera GM, Park HJ, Ghayda RA, Minhas S, Busetto GM, Bakırcıoğlu ME, Kadioglu A, Chung E, Russo GI, Calogero AE, Ambar RF, Jayasena CN, Shah R. Impact of Antioxidant Therapy on Natural Pregnancy Outcomes and Semen Parameters in Infertile Men: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. World J Mens Health. 2023 Jan;41(1):14-48. doi: 10.5534/wjmh.220067. Epub 2022 Sep 7. PMID: 36102104; PMCID: PMC9826914. Cannarella R, Condorelli RA, Duca Y, La Vignera S, Calogero AE. New insights into the genetics of spermatogenic failure: a review of the literature. Hum Genet. 2019 Feb;138(2):125-140. doi: 10.1007/s00439-019-01974-1. Epub 2019 Jan 17. PMID: 30656449. Cannarella R, Bertelli M, Condorelli RA, Vilaj M, La Vignera S, Jezek D, Calogero AE. Analysis of 29 Targeted Genes for Non-Obstructive Azoospermia: The Relationship between Genetic Testing and Testicular Histology. World J Mens Health. 2022 Jul 14. doi: 10.5534/wjmh.220009. Epub ahead of print. PMID: 36047072. Cannarella R, Condorelli RA, Paolacci S, Barbagallo F, Guerri G, Bertelli M, La Vignera S, Calogero AE. Next-generation sequencing: toward an increase in the diagnostic yield in patients with apparently idiopathic spermatogenic failure. Asian J Androl. 2021 Jan-Feb;23(1):24-29. doi: 10.4103/aja.aja_25_20. PMID: 32655042; PMCID: PMC7831827. Precone V, Cannarella R, Paolacci S, Busetto GM, Beccari T, Stuppia L, Tonini G, Zulian A, Marceddu G, Calogero AE, Bertelli M. Male Infertility Diagnosis: Improvement of Genetic Analysis Performance by the Introduction of Pre-Diagnostic Genes in a Next-Generation Sequencing Custom-Made Panel. Front Endocrinol (Lausanne). 2021 Jan 26;11:605237. doi: 10.3389/fendo.2020.605237. PMID: 33574797; PMCID: PMC7872015. La Vignera S, Vicari E, Condorelli RA, D'Agata R, Calogero AE. Male accessory gland infection and sperm parameters (review). Int J Androl. 2011 Oct;34(5 Pt 2):e330-47. doi: 10.1111/j.1365-2605.2011.01200.x. Epub 2011 Jun 22. PMID: 21696400. La Vignera S, Crafa A, Condorelli RA, Barbagallo F, Mongioì LM, Cannarella R, Compagnone M, Aversa A, Calogero AE. Ultrasound aspects of symptomatic versus asymptomatic forms of male accessory gland inflammation. Andrology. 2021 Sep;9(5):1422-1428. doi: 10.1111/andr.13014. Epub 2021 May 6. PMID: 33818914; PMCID: PMC8596874. La Vignera S, Condorelli R, D'Agata R, Vicari E, Calogero AE. Semen alterations and flow-citometry evaluation in patients with male accessory gland infections. J Endocrinol Invest. 2012 Feb;35(2):219-23. doi: 10.3275/7924. Epub 2011 Sep 23. PMID: 21946047. Mongioi’, L.M., Perelli, S., Condorelli R.A., Barbagallo, F., Crafa, A., Cannarella, R., La Vignera, S., Calogero, A.E.The Role of Resveratrol in Human Male Fertility. Molecules. 2021; 26,2495 . Cite Share Download PDF Status: Published Journal Publication published 01 Jan, 2024 Read the published version in Minerva Endocrinology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2773630","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":197508934,"identity":"bbecf446-9741-43a2-8b8a-d30d69d61d88","order_by":0,"name":"Rossella Cannarella","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABPElEQVRIie2RwUrDQBBAtyzsXjbtdcpK+wtTCmmhir+yQTCXHloKUlBqJLC9iF7bz/AiHisFveQDAhGpFHoOBIIHEbcUtMRYPArmHZaZZR8zs0NIQcGfpdf+isuclBYlj5qw5JkD8hXcumeUUPxUZgTynYzCYKMYZt/LtMbjZRwj1Amfr5a9s+cao3R12r/rEIz8iyQm7VFG2QsCuzpBaHjiuNWcPAyajDI7mgYuwad7H3IaA+gyKtB0AsSWgilHU2JHlp4TDB2dNwuAu0zeEA494KkU7+pcU54OdisKpRnf8UDY0tJKmaI23akIM8slwpEW3RNpXamGpmIgp9oV1dDxIUCoehmFmx97He4fXPPHWylSVa/w8U3S151aOXRf4uFwVMndy3od28l6KWIT4k9CBvrLdwUFBQX/gw/K6l6rKGRlHwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4599-8487","institution":"University of Catania: Universita degli Studi di Catania","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Rossella","middleName":"","lastName":"Cannarella","suffix":""},{"id":197508935,"identity":"d5626938-12fc-4b60-be9c-b97a46a1fc4a","order_by":1,"name":"Andrea Crafa","email":"","orcid":"","institution":"University of Catania: Universita degli Studi di Catania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Crafa","suffix":""},{"id":197508936,"identity":"a56ba399-44bd-4b75-8abc-2a8a144bc716","order_by":2,"name":"Raneen Sawaid Kaiyal","email":"","orcid":"","institution":"Cleveland Clinic Glickman Urological and Kidney Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Raneen","middleName":"Sawaid","lastName":"Kaiyal","suffix":""},{"id":197508937,"identity":"b1cb59bd-72c2-4548-ac35-bbd1fd8968b6","order_by":3,"name":"Shinnosuke Kuroda","email":"","orcid":"","institution":"Cleveland Clinic Glickman Urological and Kidney Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shinnosuke","middleName":"","lastName":"Kuroda","suffix":""},{"id":197508938,"identity":"cb46d72e-d28d-400b-bb48-499f6dabe33f","order_by":4,"name":"Federica Barbagallo","email":"","orcid":"","institution":"University of Catania: Universita degli Studi di Catania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Federica","middleName":"","lastName":"Barbagallo","suffix":""},{"id":197508939,"identity":"fcf28030-c59e-4b3d-a452-0c8b0ecae9b4","order_by":5,"name":"Angela Alamo","email":"","orcid":"","institution":"University of Catania: Universita degli Studi di Catania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Alamo","suffix":""},{"id":197508940,"identity":"f04c9db6-708f-4889-954a-e47785932643","order_by":6,"name":"Laura M. 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Calogero","email":"","orcid":"","institution":"University of Catania: Universita degli Studi di Catania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aldo","middleName":"E.","lastName":"Calogero","suffix":""}],"badges":[],"createdAt":"2023-04-03 23:46:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2773630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2773630/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.23736/S2724-6507.23.04080-0","type":"published","date":"2024-01-01T09:54:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":36708261,"identity":"3a47e2f7-2211-48f0-968c-2ea51f5d7a49","added_by":"auto","created_at":"2023-05-08 14:41:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":69400,"visible":true,"origin":"","legend":"\u003cp\u003eConventional sperm parameters before and after therapy in Groups A, B, and C. Panel A. Semen volume. Panel B. Sperm concentration. Panel C. Total sperm count. Panel D. Progressive motility. Panel E. Total motile sperm count. Panel F. Total motility. Panel G. Normal forms. Panel H. Leukocytes. Group A showed a significant increase in progressive motility and total motile sperm count. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2773630/v1/467e4396c45cc18f5c4bee87.png"},{"id":36709625,"identity":"d4a106eb-255e-4412-9b9b-7d25d99c669b","added_by":"auto","created_at":"2023-05-08 14:57:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62727,"visible":true,"origin":"","legend":"\u003cp\u003eBiofunctional sperm parameters before and after therapy in Groups A, B, and C. Panel A. Percentage of alive spermatozoa. Panel B. Percentage of spermatozoa with compacted chromatin. Panel C. Percentage of spermatozoa in early apoptosis. Panel D. Percentage of spermatozoa in late apoptosis. Panel E. Percentage of spermatozoa with high-mitochondrial membrane potential (H-MMP). Panel F. Percentage of spermatozoa with low-MMP (L-MMP). Panel G. Percentage of spermatozoa with fragmented DNA. Group A showed a significant improvement in the percentage of alive spermatozoa. \u003cem\u003eLegend: \u003c/em\u003eSDF, sperm DNA fragmentation. *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2773630/v1/7c1c703f7f529ede075a2e73.png"},{"id":36708259,"identity":"ab7b5347-5cba-40e7-901e-da8165076883","added_by":"auto","created_at":"2023-05-08 14:41:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122803,"visible":true,"origin":"","legend":"\u003cp\u003eConventional sperm parameters before and after therapy according to diagnoses. Panel A. Semen volume. Panel B. Sperm concentration. Panel C. Total sperm count. Panel D. Progressive motility. Panel E. Total motile sperm count. Panel F. Total motility. Panel G. Normal forms. Panel H. Leukocytes. After treatment, patients with varicocele show a significant increase in progressive motility compared to pre-treatment values. Patients with varicocele and those with idiopathic infertility showed a significant increase in the total motile sperm count compared to pre-treatment values. \u003cem\u003eLegend: \u003c/em\u003eVar, varicocele; TH, testicular hypotrophy; Idiop, idiopathic infertility; MAGI, male accessory gland infection. *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2773630/v1/35d7b04dd2f0c920fa88b884.png"},{"id":36709213,"identity":"3dea6bd7-7711-4360-af52-e7ba70092bc6","added_by":"auto","created_at":"2023-05-08 14:49:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52689,"visible":true,"origin":"","legend":"\u003cp\u003eBiofunctional sperm parameters before and after therapy based on patient’s diagnosis. Panel A. Percentage of alive spermatozoa. Panel B. Percentage of spermatozoa with compacted chromatin. Panel C. Percentage of spermatozoa in early apoptosis. Panel D. Percentage of spermatozoa in late apoptosis. Panel E. Percentage of spermatozoa with high-mitochondrial membrane potential (H-MMP). Panel F. Percentage of spermatozoa with low-MMP (L-MMP). Panel G. Percentage of spermatozoa with fragmented DNA. Patients with testicular hypotrophy showed a significant increase in the percentage of alive spermatozoa compared to pre-treatment values. \u003cem\u003eLegend: \u003c/em\u003eVar, varicocele; TH, testicular hypotrophy; Idiop, idiopathic infertility; MAGI, male accessory gland infection. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2773630/v1/e9f3cd8a5589def6f65be26f.png"},{"id":56241297,"identity":"8cce501b-717d-4e15-b014-f235523756fd","added_by":"auto","created_at":"2024-05-10 09:54:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1194986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2773630/v1/1ffe51bc-c304-49f7-8cfc-f060ef21ff32.pdf"}],"financialInterests":"","formattedTitle":"Antioxidants for male infertility: therapeutic scheme and indications. A retrospective single-center real-life study","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe correlation between male infertility and oxidative stress is well-known and demonstrated in the literature. Approximately 30% of cases of male infertility are defined as idiopathic [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. From 30 to 80% of male sub-infertility cases are due to oxidative stress damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and more than 20 to 40% of infertile patients have significantly higher semen ROS levels than fertile men [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA low amount of ROS production is required for sperm function [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, when ROS production exceeds the natural scavenger capacity of seminal fluid, oxidative stress increases, and this results in sperm damage. Infections, autoimmune disorders, chronic disease, advanced age, alcohol consumption, smoking, high temperatures, environmental pollutants, and obesity are the most common causes of increased oxidative stress [7].\u003c/p\u003e \u003cp\u003eSpermatozoa are particularly susceptible to oxidative stress [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It negatively affects total and progressive sperm motility and normal sperm morphology. It also increases DNA damage and consequently decreases fertility [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR7\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, increased oxidative stress is a clinical condition that interferes with reproductive function.\u003c/p\u003e \u003cp\u003eSeminal plasma contains natural antioxidants, such as vitamins C and E, catalase, superoxide dismutase, glutathione and glutathione peroxidase, superoxide dismutase, thioredoxin, and polyamine spermine. They act as free radical scavengers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These compounds often cooperate with other substances, such as vitamin A, coenzyme Q10, carnitines, myoinositol, lycopene, and micronutrients (selenium, zinc, and copper) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Dietary Supplement Health and Education Act defines nutraceuticals as dietary supplements that contain a finely titrated combination of standard dietary biotic compounds that are often used for their anti-inflammatory, antioxidant, and anti-apoptotic properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Several studies have reported that the oral administration of antioxidants is efficacious in improving conventional sperm parameters without adverse events [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Antioxidants are advantageous because they are widely available and relatively inexpensive compared to other therapeutic strategies used for fertility treatment.\u003c/p\u003e \u003cp\u003eThe use of antioxidants is suggested by scientific evidence only at the end of a complete diagnostic workup [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Indeed, the latest Cochrane review on 6,264 subfertile patients showed, albeit with low-quality evidence, that antioxidants increase the pregnancy rate and improve the live birth rate in couples attending fertility clinics [5]. These findings support the administration of molecules with antioxidant properties in patients with abnormal sperm parameters caused by increased oxidative stress [5]. Different antioxidants are currently used, although there is no consensus on which compound to use [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, a combination of nutraceuticals with antioxidant properties is often used in male infertility to take advantage of their synergistic effects. Similarly, fibrinolytic compounds are used for the treatment of infertility [23], but at the moment it is not known whether they should be better prescribed in association with antioxidants or which patients could benefit more from their prescription. Furthermore, although different diseases are diagnosed as \u0026ldquo;male infertility\u0026rdquo;, they represent different clinical entities with different pathogenesis (i.e., varicocele, urogenital infection, urogenital inflammation, previous cryptorchidism, idiopathic infertility, etc.) and may benefit from antioxidants differently.\u003c/p\u003e \u003cp\u003eOn this account, this real-life single-center study aimed to evaluate the most effective combination of nutraceuticals and the most appropriate indications, in patients with male infertility. To accomplish this, we retrospectively extracted from the medical records of male patients entering the Division of Endocrinology, Metabolic Diseases and Nutrition for infertility who were prescribed antioxidants, data on the type of antioxidant prescribed, duration of therapy, clinical diagnosis, conventional and biofunctional sperm parameters before and after therapy, pregnancy outcomes, and female partner-related information. A before-after analysis of conventional and biofunctional parameters was carried out. Data were further sub-analyzed based on the type of therapy prescribed and the patient\u0026rsquo;s diagnosis.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Patient selection\u003c/h2\u003e \u003cp\u003eIn this single-center real-life retrospective study, we collected data from January 2014 to January 2023. Male patients between 20 and 55 years, referring to the Division of Endocrinology, Metabolic Diseases and Nutrition, University of Catania, for male infertility were evaluated for inclusion. Men with endocrinopathies, gastrointestinal, hematological, nephrological or autoimmune disorders, chemo and/or radiotherapy, and corticosteroid therapy, genetic disorders, hypogonadism, diabetes mellitus, hormone replacement therapy, smokers, drinkers, acute male accessory gland infection (MAGI) or MAGI non-respondent to first- and second-line antibiotics were not included. Meanwhile, those with varicoceles, primary testicular dysfunction leading to mild or severe testicular hypotrophy due to cryptorchidism or for unknown reasons, idiopathic infertility, and chronic or antibiotic-respondent MAGI were included.\u003c/p\u003e \u003cp\u003eA complete medical history was reported in the database for each patient as well as a careful physical examination, laboratory workup, and ultrasound evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental design\u003c/h2\u003e \u003cp\u003ePatient were treated with 1) a NAC 150 mg; α-lipoic acid 150 mg; L-carnitine 250 mg; Inositol 50 mg; Coenzyme Q10 10 mg and Acid Folic 200 mg (Androlen\u0026reg;) (\u003cb\u003eGroup 1\u003c/b\u003e), 2) Androlen\u0026reg; plus a mixture of fibrinolytic molecules (Scutellaria baicalensis 400 mg, Baicalina 380 mg; Aesculus hippocastanum L. cortex 400 mg, Escin 60 mg; Bromeline) (Lenidase\u0026reg;) (\u003cb\u003eGroup 2\u003c/b\u003e), and Androlen\u0026reg; plus other molecules, different from those used in Group 1 and Group 2 (\u003cb\u003eGroup 3\u003c/b\u003e). Patients underwent semen analysis and evaluation of biofunctional sperm parameters before and after treatment. Treatment continued until benefits were seen (e.g. improvements in conventional and/or biofunctional sperm parameters).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Semen analysis\u003c/h2\u003e \u003cp\u003eSemen analysis was performed according to WHO criteria (WHO, 2010). Semen samples were collected by masturbation into a sterile container after 2\u0026ndash;7 days of sexual abstinence and were analyzed immediately after liquefaction. Each sample was evaluated for semen volume, pH, sperm count, progressive motility, morphology, and round cell concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Flow cytometric analysis\u003c/h2\u003e \u003cp\u003eFlow cytometric analysis was performed using flow cytometer CytoFLEX (Beckman Coulter Life Science, Milan) equipped with two lasers and six total fluorescence channels (four 488 nm and two 638 nm), then the results were analyzed with the software CytExpert 1.2 and the following tests were performed.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Assessment of sperm DNA fragmentation\u003c/h2\u003e \u003cp\u003eSperm DNA fragmentation was performed by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay (Apoptosis Mebstain kit, DBA s.r.l, Milan, Italy) using terminal deoxynucleotidyltransferase (TdT), an enzyme that polymerizes, at the level of DNA breaks, modified nucleotides conjugated to a fluorochrome. To obtain a negative control, TdT was omitted from the reaction mixture. Reading was performed by flow cytometry using the FL1 detector.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Evaluation of the sperm mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eThe mitochondrial membrane potential (MMP) was evaluated by a lipophilic probe 5,5',6,6'-tetrachloro-1,1',3,3'tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, DBA S.r.l, Milan, Italy) able to selectively penetrate the mitochondria. Briefly, an aliquot containing 1x10\u003csup\u003e6\u003c/sup\u003e/ml of spermatozoa was incubated with JC-1 in the dark for 10 minutes at 37\u0026deg;C. At the end of the incubation period, the spermatozoa were washed in PBS and analyzed. In viable cells with normal membrane potential, JC-1 is found in the mitochondrial membrane in the form of aggregates that fluorescence orange fluorescence, while in cells with low membrane potential, it remains in the cytoplasm in monomeric form, giving a green fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Evaluation of the degree of chromatin compactness\u003c/h2\u003e \u003cp\u003eThe degree of chromatin compactness was evaluated after permeabilization of the cell membrane using LPR DNA-Prep Reagent to allow the fluorophore to access the nucleus. Subsequently, spermatozoa were incubated with Stain DNA-Prep Reagent (Beckman Coulter, IL). An aliquot of 1x10\u003csup\u003e6\u003c/sup\u003e spermatozoa was incubated with DNA-Prep Reagent containing 0.1% potassium cyanate, 0.1% NaN\u003csub\u003e3\u003c/sub\u003e, nonionic detergents, saline, and stabilizers (Beckman Coulter, IL, Milan, Italy), in the dark, at room temperature for 10 minutes. Flow cytometry analysis was performed after 30 minutes, using an FL3 detector.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Evaluation of sperm apoptosis/vitality\u003c/h2\u003e \u003cp\u003eSperm apoptosis/viability was evaluated by annexin V-FITC Apoptosis (Beckman Coulter, IL, Milan, Italy). The simultaneous cell staining with PI and annexin V that binds selectively to phosphatidylserine (PS), an early signal of apoptosis, allows distinguishing alive spermatozoa (with intact cytoplasmic membrane) and apoptotic or necrotic spermatozoa. To accomplish this, an aliquot containing 0.5x10\u003csup\u003e6\u003c/sup\u003e/ml was suspended in a 0.5 ml buffer containing 10 \u0026micro;l of annexin V-FITC and 20 \u0026micro;l of PI (Annexin V-FITC Apoptosis, Beckman Coulter, IL, Milan, Italy) and incubated for 10 minutes in the dark. After incubation, the sample was analyzed by FL-1 (FITC) and FL3 (PI) detectors. The different staining patterns allowed us to identify the three different cell populations: viable cells (FITC negative and PI negative); early apoptotic cells with still intact cytoplasmic membrane (FITC-positive and PI-negative); and cells in late apoptosis (FITC positive and PI-positive).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eData are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) for non-skewed variables, while non-normally distributed continuous variables as the median and interquartile range (IQR). The distribution of values was evaluated using the Shapiro-Wilk test. Before-after analysis was performed using the \u003cem\u003et\u003c/em\u003e-test for independent samples for normally distributed variables and the Mann-Whitney U test (which is used for independent samples) for not normally distributed.\u003c/p\u003e \u003cp\u003eTwo sub-analyses based on 1) the type of treatment administered and 2) the patients\u0026rsquo; diagnosis (varicocele, testicular hypotrophy, idiopathic infertility, or chronic MAGI) were performed to identify the most effective therapeutic strategy and indications. Two-way analysis of variance (two-way ANOVA) with Shapiro-Wilk correction for multiple comparisons was used for subgroup analysis. Statistical analysis was performed using MedCalc Software Ltd. (Ostend, Belgium), version 19.6\u0026ndash;64 bit. A p-value less than 0.05 was considered statically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Ethics statement\u003c/h2\u003e \u003cp\u003eThis study was conducted at the Division of Endocrinology, Metabolic Diseases and Nutrition of the University-Teaching hospital Policlinico \u0026ldquo;G. Rodolico \u0026ndash; San Marco\u0026rdquo;, University of Catania (Catania, Italy). The protocol was approved by the internal Institutional Review Board. Written informed consent was obtained from each participant after a thorough explanation of the purpose and nature of all procedures used. The study was conducted according to the principles expressed in the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003eForty-three patients with a mean age of 36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 years, a mean body mass index (BMI) of 26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 Kg/m\u003csup\u003e2\u003c/sup\u003e, coming from Eastern Sicily, were selected for this study. The presence of a female infertility factor (polycystic ovary syndrome, endometriosis, tubal occlusion, low oocyte quality, oligomenorrhea) occurred overall in 11 out of 43 cases. The mean age of the female partners was 33.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 years. Twenty-four patients were prescribed Androlen\u0026reg; (Group 1), 12 received treatment with Androlen\u0026reg; and Lenidase\u0026reg; (Group 2), and 7 received Androlen\u0026reg; and other molecules (Group 3). The mean length of treatment was 4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 months. In addition, 13 patients had varicocele, 10 testicular hypotrophy, 17 idiopathic infertility, and 3 chronic MAGI. Interestingly, 11 couples achieved pregnancy, and 5 of them this so by natural intercourse, while 30 failed in reaching pregnancy. Miscarriages occurred in 4 cases. The length of the time to pregnancy ranged from 1 to 12 months.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Before-after analysis\u003c/h2\u003e \u003cp\u003eThe results of the before-after analysis are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Particularly, only progressive motility showed significant improvement after therapy. The other conventional sperm parameters did not change significantly. Similarly, no difference was found in biofunctional sperm parameters.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of the before-after analysis of conventional and bio-functional sperm parameters.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBefore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eAfter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolume (ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0 (2.0\u0026ndash;4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.0 (2.3\u0026ndash;4.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm concentration (mil/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.0 (10.0\u0026ndash;34.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.0 (11.0\u0026ndash;45.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSC (mil/ejaculate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60.0 (38.3\u0026ndash;110.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e80.0 (27.5\u0026ndash;130.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgressive motility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.0 (7.0\u0026ndash;20.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19.0 (13.0\u0026ndash;23.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTMSC (million)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.5 (3.9\u0026ndash;14.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.5 (4.1\u0026ndash;23.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal motility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.5 (50.0\u0026ndash;62.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57.5 (45.0\u0026ndash;65.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorphology (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.0 (2.0\u0026ndash;7.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.0 (3.0\u0026ndash;6.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeukocytes (mil/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5 (0.2\u0026ndash;0.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5 (0.2\u0026ndash;0.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlive (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55.0 (39.3\u0026ndash;61.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.6 (49.7\u0026ndash;70.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChromatin compactness (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEarly apoptosis (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.5 (0.6\u0026ndash;3.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.2 (0.6\u0026ndash;2.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLate apoptosis (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.6 (3.9\u0026ndash;10.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.6 (0.8\u0026ndash;9.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH-MMP (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65.8\u0026thinsp;\u0026plusmn;\u0026thinsp;22.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.2\u0026thinsp;\u0026plusmn;\u0026thinsp;23.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-MMP (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34.2\u0026thinsp;\u0026plusmn;\u0026thinsp;22.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44.8\u0026thinsp;\u0026plusmn;\u0026thinsp;23.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSDF (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0 (0.9\u0026ndash;5.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.2 (0.6\u0026ndash;6.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eData are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) for skewed variables, while the non-normally distributed continuous variables as the median and interquartile range (IQR). The distribution of values was evaluated using the Kruskal-Wallis test. The p values were calculated using ANOVA and Kruskal-Wallis for skewed variables, with Shapiro-Will correction for multiple comparisons. A p-value less than 0.05 was considered statistically significant. \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLegend\u003c/span\u003e: H-MMP, high mitochondrial membrane potential; L-MMP, low mitochondrial membrane potential; SDF, sperm DNA fragmentation; TMSC, total motile sperm count; TSC, total sperm count.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Before-after analysis based on the type of drug prescribed\u003c/h2\u003e \u003cp\u003eGroup A showed a significant increase in progressive sperm motility (12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8% vs. 16.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and TMSC [7.5 (2.8\u0026ndash;10.3) vs. 16.0 (5.0\u0026ndash;24.4), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01] after treatment with Androlen, while no significant improvement of these parameters was observed in Groups B and C. No significant difference in semen volume, sperm concentration, total sperm count, total motility, normal forms, and leukocyte concentration was found in any of the Groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We also found a trend towards an increase in total sperm count (71.1\u0026thinsp;\u0026plusmn;\u0026thinsp;68.5 mil/ml vs. 96.1\u0026thinsp;\u0026plusmn;\u0026thinsp;78.6 mil/ml, p\u0026thinsp;=\u0026thinsp;0.06) in Group A and towards an increase in progressive motility (13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3 mil/ml vs. 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;14.1 mil/ml, p\u0026thinsp;=\u0026thinsp;0.06) in Group B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, Group A also showed an increase in the percentage of alive spermatozoa (60.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8% vs. 74.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) after treatment, which was not observed in the other groups. After treatment, no differences were observed in the percentage of spermatozoa with compacted chromatin, early or late apoptosis, L-MMP, H-MMP, or fragmented DNA compared to baseline values in any of the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Before-after analysis based on diagnoses\u003c/h2\u003e \u003cp\u003eCompared to pre-treatment values, we found a significant improvement in progressive motility [9.0 (6.0\u0026ndash;16.0) vs. 15.0 (14.5\u0026ndash;22.0), p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] in patients with varicocele, and in TMSC in patients with varicocele (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0 vs. 15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and in those with idiopathic infertility 7.2 (1.6\u0026ndash;17.3) vs. 13.8 (1.1\u0026ndash;24.9), p\u0026thinsp;\u0026lt;\u0026thinsp;0.05] after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding biofunctional sperm parameters, the percentage of alive spermatozoa significantly improved in patients with testicular hypotrophy (60.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1% vs. 76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A trend toward an increase in the percentage of spermatozoa with L-MMP (33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5% vs. 41.3\u0026thinsp;\u0026plusmn;\u0026thinsp;19.4%, p\u0026thinsp;=\u0026thinsp;0.06) and a reduction in the percentage of spermatozoa with H-MMP (66.7\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5% vs. 58.7\u0026thinsp;\u0026plusmn;\u0026thinsp;19.4%, p\u0026thinsp;=\u0026thinsp;0.06) was found in patients with varicocele.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThis was a single-center retrospective study, conducted to evaluate the best combination of nutraceuticals among those most prescribed by us, as well as which infertile patients could benefit most from the treatment. We have found that adding fibrinolytic or other nutraceuticals to an antioxidant mixture does not improve treatment efficacy. Indeed, contrary to the other treatments, patients with the latter therapeutic regimen alone showed a significant increase in progressive sperm motility, TMSC, and percentage of alive spermatozoa. Furthermore, the treatment was effective in infertile patients with varicocele or idiopathic infertility, who showed a significant improvement in progressive sperm motility and TMSC, while patients with mild testicular hypotrophy or chronic MAGI may not benefit from this type of treatment.\u003c/p\u003e \u003cp\u003eInfertile patients have higher seminal fluid oxidative stress than fertile men [4, 7]. Consequently, antioxidants are often used for the treatment of idiopathic oligo-astheno-teratozoospermia due to their ability to counteract the deleterious effects of oxidative stress on sperm parameters and function [24]. Indeed, high levels of oxidative stress can damage the main sperm molecular components (lipid, protein, and DNA). The plasma membrane of spermatozoa plays a key role in successful fertilization because of its fluidity, flexibility, and functional activity. It consists of high levels of polyunsaturated fatty acids. Double bonds of membrane lipids can be oxidized by ROS. This causes a process called lipid peroxidation which affects membrane fluidity and, consequently, alters sperm motility [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, mitochondria are a major source of ROS in spermatozoa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], mainly produced by oxidative phosphorylation. Sperm mitochondrial function is closely related to the motility of these cells. Therefore, an imbalance between oxidative and antioxidant systems may reduce sperm motility by impairing sperm mitochondrial function [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, ROS can directly damage sperm DNA and spermatozoa cannot repair DNA due to the deficiency of the cytoplasmic enzyme systems involved in the molecular mechanisms of DNA repair [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor this reason, several molecules with antioxidant properties are used to counteract the negative effects of oxidative stress on sperm function. Among them, coenzyme Q10, carnitine, and myoinositol have been shown to improve sperm motility and protect DNA from oxidative damage [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR23\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, coenzyme Q10 has shown beneficial effects on the pregnancy rate [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Treatment with CoQ10 increases mitochondrial DNA stability and counteracts the damage caused by increased ROS production to its specific RNA polymerase and mitochondrial transcription factor-A. These molecules play a fundamental role in mitochondrial transcription. These results suggest that antioxidant treatment could improve sperm motility by preserving the sperm transcription system from ROS damage [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, a mixture of antioxidants was investigated. Often during the treatment, the synergistic effect of the various molecules with antioxidant properties is exploited [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For example, carnitine associated with ubiquinol improves sperm mitochondrial function. Co-administration of vitamin C and vitamin E correlates positively with the improvement of motility and normal morphology, reduces the SDF rate, and increases the pregnancy rate [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A significant sperm concentration improvement has been reported after the administration of folic acid and zinc sulfate [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In contrast, Raigani and colleagues conducted a double-blind, randomized, placebo-controlled study on subfertile patients with oligo-astheno-teratozoospermia to evaluate the effects of oral folic acid and zinc sulfate supplementation on both sperm motility and MMP. The results of this study showed that zinc sulfate and folic acid supplementation did not improve sperm quality in infertile patients with severely impaired sperm parameters. Therefore, further studies are needed to define the effects of these molecules and to identify patients who may benefit from supplementation with these molecules [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile the currently available Cochrane meta-analyses [5,40] and the Society\u0026rsquo;s guidelines [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] do not identify, among all male infertile patients, those who could benefit most from therapy, the meta-analytical evidence suggests the positive impact of antioxidants in patients with varicocele. A meta-analysis of six studies, including 576 patients, reported a significant increase in sperm concentration, progressive and total motility, and normal morphology in the antioxidant-treated group, compared with controls, 3 months after varicocelectomy [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. More recently, another meta-analysis reported a significant improvement in sperm concentration, progressive and total motility, and seminal volume in patients with varicocele, who did not undergo surgical repair, after oral antioxidant supplementation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These latter findings are in line with those reported in the present study and suggest varicocele as one of the indications of treatment with antioxidants in infertile patients.\u003c/p\u003e \u003cp\u003eTrials have been carried out to evaluate the benefits of administering antioxidants in patients with idiopathic infertility [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. A prospective study on 50 patients with idiopathic infertility and 35 fertile control, evaluated the effects of CoQ10 on conventional sperm parameters and antioxidant indices in the seminal fluid, reporting an increased total and progressive motility and oxidative stress [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. A randomized controlled study on 73 patients with idiopathic infertility and 70 controls found a significant improvement in sperm concentration, motility, and normal morphology in the group treated with L-carnitine. The administration of L-carnitine resulted in greater efficacy compared to CoQ10 and vitamin E [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, despite the already existing evidence, most of the studies claiming to enroll patients with idiopathic infertility provide neither a careful description of the diagnostic workup nor of the inclusion and exclusion criteria, which may suggest the presence of misdiagnosis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, in line with the current literature, our findings support the efficacy of antioxidants in patients with idiopathic infertility.\u003c/p\u003e \u003cp\u003eInterestingly, we found no efficacy in eugonadal patients with mild testicular hypotrophy (i.e., anamnesis positive for cryptorchidism or for apparently unexplained reasons). There are no studies in the literature aimed at evaluating the effects of antioxidants on previously cryptorchidic oligozoospermic patients with borderline-low testicular volume or those with unexplained borderline-low testicular volume. Testicular volume may represent a phenotypic sign that could help in decision-making. Indeed, while idiopathic patients with normal testicular volume appear to respond to antioxidants, those with idiopathic infertility and borderline-low testicular volume may not, due to primary testicular dysfunction. This should come as no surprise, as monogenic causes of oligozoospermia have been discovered in recent times, some of which have a minor impact on testicular volume [\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLastly, patients with chronic MAGI did not respond to antioxidants. MAGI is a complex entity that can recognize a bacterial etiology, even when the microbiological examination is negative [54,55]. Microbes are capable of triggering a cascade of events that increase oxidative stress [56]. Therefore, the eradication of germs should be a prerequisite before the treatment with antioxidants is started.\u003c/p\u003e \u003cp\u003eThe results of the present study should be taken with caution, due to some limitations, given by the limited size of the sample (including that of the subgroups), and by the retrospective design, which does not admit any causal relationship between the therapy and the observed findings. However, one of the strengths of the present study is the evaluation of biofunctional sperm parameters. Conversely, few studies have actually evaluated the effects of antioxidant treatment on biofunctional sperm parameters. A recent review describes that resveratrol, a natural antioxidant, significantly decreased the percentage of spermatozoa with abnormal chromatin compactness and high lipoperoxidation in spermatozoa incubated \u003cem\u003ein vitro\u003c/em\u003e [57]. Furthermore, the rigorous diagnostic workup outlined in Materials and Methods used to include or exclude patients and the robust statistical approach are other strengths of the study.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn conclusion, the results of this single-center retrospective study, which was undertaken to evaluate the best combination of nutraceuticals among those we prescribed most frequently, as well as which infertile patients could benefit most from treatment, indicate that the addition of fibrinolytic or other nutraceuticals to a mixture of carnitine-based antioxidants does not improve the effectiveness of the treatment obtained with the use of the latter alone. In fact, patients on the latter therapeutic regimen showed a significant increase in sperm progressive motility, TMSC, percentage of alive spermatozoa, and a good pregnancy rate. Furthermore, the treatment was effective in infertile patients with varicocele or idiopathic infertility, who showed a significant improvement in sperm progressive motility and TMSC, while patients with mild testicular hypotrophy or chronic MAGI did not benefit from this type if treatment. Prospective studies are needed to further confirm the conclusions of the present study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/strong\u003e: This study was approved by the internal Institutional Review Board of the Division of Endocrinology, Metabolic Diseases and Nutrition of the University-Teaching hospital Policlinico \u0026ldquo;G. Rodolico \u0026ndash; San Marco\u0026rdquo;, University of Catania (Catania, Italy).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to participate\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;Written informed consent to participate the study was obtained from each participant after a thorough explanation of the purpose and nature of all procedures used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;Written informed consent to publish the study was obtained from each participant.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material: Data will be made available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCode availability\u003c/em\u003e\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, R.C. and A.E.C.; literature search, A.A., L.M.M., F.B.; data management: A.C., S.S.; writing original draft preparation, A.A., R.C., R.A.C., A.C.; statistical analysis, R.C., A.C.; revision and editing, R.S.K., S.K., S.L.V., A.E.C.; supervision, A.E.C. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors have nothing to disclose and declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgarwal, A., Mulgund, A., Hamada, A., Chyatte, M.R. A unique view on male infertility around the globe. Reprod Biol Endocrinol 2015; 13,37.\u003c/li\u003e\n\u003cli\u003eLanzafame, F.M., La Vignera, S., Vicari, E., Calogero, A.E. 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PMID: 33818914; PMCID: PMC8596874.\u003c/li\u003e\n\u003cli\u003eLa Vignera S, Condorelli R, D\u0026apos;Agata R, Vicari E, Calogero AE. Semen alterations and flow-citometry evaluation in patients with male accessory gland infections. J Endocrinol Invest. 2012 Feb;35(2):219-23. doi: 10.3275/7924. Epub 2011 Sep 23. PMID: 21946047.\u003c/li\u003e\n\u003cli\u003eMongioi\u0026rsquo;, L.M., Perelli, S., Condorelli R.A., Barbagallo, F., Crafa, A., Cannarella, R., La Vignera, S., Calogero, A.E.The Role of Resveratrol in Human Male Fertility. Molecules. 2021; 26,2495\u003cem\u003e.\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antioxidants, fibrinolytic, conventional sperm parameters, bio-functional sperm parameters, indications, male infertility, varicocele, idiopathic infertility","lastPublishedDoi":"10.21203/rs.3.rs-2773630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2773630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis single-center real-life study was conducted to evaluate the most effective combination of nutraceuticals and the most appropriate indications for the treatment of male infertile patients. Infertile patients aged 20\u0026ndash;55 years were treated with a combination of antioxidants (Androlen\u0026reg;) (Group 1), with Androlen\u0026reg; and a mixture of fibrinolytic molecules (Lenidase\u0026reg;) (Group 2), or Androlen\u0026reg; and other molecules different from those used for the patients of the Group 2 (Group 3). Patients were also subdivided according to the presence of varicocele, mild testicular hypotrophy, idiopathic infertility, and chronic male accessory gland infection (MAGI).\u003c/p\u003e \u003cp\u003eForty-three patients were enrolled. In the overall analysis, only progressive motility significantly improved after therapy. Subgroup analysis showed a significant increase in progressive motility, total motile sperm count (TMSC), and in the percentage of alive spermatozoa after treatment in the Group A. Progressive motility improved significantly in patients with varicocele, while the TMSC in patients with varicocele and those with idiopathic. The percentage of alive spermatozoa increased in patients with testicular hypotrophy.\u003c/p\u003e \u003cp\u003eTreatment with antioxidants increases progressive sperm motility. The association of fibrinolytic or other nutraceuticals does not improve the efficacy of the treatment with antioxidants alone. The treatment was effective in patients with varicocele or idiopathic infertility.\u003c/p\u003e","manuscriptTitle":"Antioxidants for male infertility: therapeutic scheme and indications. A retrospective single-center real-life study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-08 14:41:30","doi":"10.21203/rs.3.rs-2773630/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69fc2405-ed40-4707-951c-7c6afeb86d32","owner":[],"postedDate":"May 8th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-10T09:54:38+00:00","versionOfRecord":{"articleIdentity":"rs-2773630","link":"https://doi.org/10.23736/S2724-6507.23.04080-0","journal":{"identity":"minerva-endocrinology","isVorOnly":true,"title":"Minerva Endocrinology"},"publishedOn":"2024-01-01 09:54:38","publishedOnDateReadable":"January 1st, 2024"},"versionCreatedAt":"2023-05-08 14:41:30","video":"","vorDoi":"10.23736/S2724-6507.23.04080-0","vorDoiUrl":"https://doi.org/10.23736/S2724-6507.23.04080-0","workflowStages":[]},"version":"v1","identity":"rs-2773630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2773630","identity":"rs-2773630","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}