Archivio Italiano di Urologia e Andrologia 2017; 89, 3212 ORIGINAL PAPER Effect of superoxide dismutase supplementation on sperm DNA fragmentation Luciano Negri 1, Renzo Benaglia 1, Emanuela Monti 1, Emanuela Morenghi 2, Alessandro Pizzocaro 3, Paolo E. Levi Setti 1 1 Humanitas Research Hospital, Department of Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Fertility Center, Rozzano, Milan, Italy; 2 Biostatistics Unit, Humanitas Research Hospital, Rozzano, Milan, Italy; 3 Endocrinology Unit, IRCCS, Humanitas Research Hospital, Rozzano, Milan, Italy. Background: antioxidants supplementation improves sperm quality, but few trials have analyzed the effects on sperm DNA fragmentation (SDF). This study compares the effectiveness of SOD-based antioxi- dant supplementation plus hydroxytyrosol and carnosol in reducing SDF with other antioxidants without SOD, hydroxy- tyrosol, and carnosol. Materials and methods: men with high SDF at baseline were selected in our clinical database. The patients taken into account had a 2-month control. SDF was measured by Sperm Chromatin Dispersion test (SCD). Untreated men were used as a control group. The remaining subjects received some oral antioxidant supplements (12 different combinations of both hydrophilic and lipophilic antioxidants), with some of them receiving nutritional support with a SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol. Results: 118 men were selected for a retrospective study. Mean age 39.3 ± 5.4 years. Fifteen had no treatment, 55 were treated with a SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol, and 48 took some antioxidant supplements for 2 months. Clinically, variations of at least 10% in baseline values of classic semen parameters and sperm DNA fragmentation were taken into consideration. Classic seminal parameters did not vary significantly in the three groups, with the exception of viability (p = 0.001). We assessed which of the active substances (no. 19) in differ- ent formulations were associated with variations in SDF. In the multivariable analysis of the 7 active substances that passed the univariable analysis, only the SOD molecule appeared to be linked to an improvement in SDF (< 0.0001). In detail, only one patient in the control group showed a spon- taneous improvement in SDF (6%), compared to 16/48 (33%) of those taking various oral antioxidant supplements, and 31/55 (56%) of those taking a SOD-based antioxidant supple- mentation plus hydroxytyrosol and carnosol. Conclusions: SOD-based antioxidant supplementation plus hydroxytyrosol and carnosol seems to provide a better chance of improving sperm DNA integrity than other classical antiox- idant molecules. KEY WORDS: Sperm DNA fragmentation: Male infertility; Superoxide dismutase; Hydroxytyrosol; Carnosol. Submitted 19 July 2017; Accepted 3 August 2017 Summary No conflict of interest declared. with pregnancy in natural cycles (1), intrauterine insemi- nations (2), and in-vitro procedures (3). SDF is also asso- ciated with recurrent miscarriage (4), both during in-vivo and in-vitro procedures. It is well known that SDF may be present in men with both normal and abnormal semen analysis (5) and that infertile men have higher proportions of sperm with DNA damage compared to fertile men (6). Cohen-Bacrie et al. (2009) (7) found elevated levels of sperm DNA damage in over 60% of men attending fertili- ty clinics, with 30% being severe. Because conventional semen analysis is a poor predictor of sperm DNA damage (8), SDF assays have been suggested in selected cases of infertility, e.g. unexplained infertility, recurrent miscar- riages, and asthenoteratozoospermia. Abortive apoptosis, infection, defective spermatogenesis, and oxidative stress (OS) are thought to be causes of SDF (9), with the latter being the most common cause (10). Oxidative stress occurs when reactive oxygen species (ROS) overcome the semen’s natural antioxidant defens- es. In physiological conditions, oxidative stress is suit- ably balanced by the action of endogenous enzymatic antioxidants, including superoxide dismutase (SOD), catalase, and glutathione peroxidase⁄reductase, as well as non-enzymatic antioxidants such as ascorbate, urate, vitamin E, pyruvate, glutathione, albumin, vitamin A, ubiquinol, taurine, and hypotaurine (11). These endoge- nous antioxidants scavenge both intracellular and extra- cellular superoxide radicals, preventing the lipid peroxi- dation of plasma membranes (12). Pathological stressors that generate endogenous ROS include infections, varic- ocele, aging, cancer, drugs, cigarette smoking, obesity, pharmaceutics, industrial chemicals, radio-frequency electromagnetic radiation and, lastly, abnormal sperma- tozoa (13, 14). All these stressors decrease sperm motil- ity and viability, while stimulating DNA base adduct for- mation and, ultimately, DNA fragmentation. Physiologically, homeostasis between free radicals and antioxidant substances is guaranteed by very complex systems. The most efficient seems to be the system medi- ated by the Nrf2 (Nuclear factor [erythroid-derived 2]-like 2 transcription factor) pathway. The latter regulates a wide variety of antioxidant cytoprotective enzymes through a promotion sequence known as ARE (antioxi- dant response element) (15). DOI: 10.4081/aiua.2017.3.212 INTRODUCTION Sperm DNA fragmentation (SDF) is an important factor in the etiology of male infertility. SDF negatively correlates Negri_Stesura Seveso 28/09/17 10:23 Pagina 212 213Archivio Italiano di Urologia e Andrologia 2017; 89, 3 Effect of superoxide dismutase supplementation on sperm DNA fragmentation At present, several tests have been developed to evaluate sperm DNA fragmentation. These include the Sperm Chromatin Dispersion test (SCD) (16), a simple and inex- pensive test for basic diagnosis in clinical practice. When no clear SDF etiological factors exist, antioxidants drugs are empirically prescribed (17, 18). Numerous combina- tions of hydrophilic and lipophilic antioxidants are avail- able in drugstores and online, e.g. vitamin C, vitamin E, folic acid, DHA, L-acetyl carnitine, L-carnitine, astaxan- thin, ethyl cysteine, coenzyme Q10, zinc, and selenium. In view of the fact that recent in-vitro and in-vivo trials support the theory that Nrf2 activation strategies could effectively combat oxidative stress, the purpose of our retrospective study is to match the effectiveness of SOD- based antioxidant therapy plus hydroxytyrosol and carnosol (FertiPlus® SOD) in reducing sperm DNA frag- mentation with other antioxidants without SOD, hydroxytyrosol, and carnosol. MATERIALS AND METHODS Selection of participants and data collection Male partners of infertile couples referred to our Fertility Center were selected by a query in our clinical database (June 2014 - November 2016). Inclusion criteria were oligo-normozoospermia, according to the 2010 World Health Organization criteria for the Evaluation of Human Semen (5th Edition) (19); Sperm DNA fragmentation > 15%; no current seminal infections according to sperm culture and/or seminal leucocyte (< 106 x ml); no sperm antibodies (Sperm Mar IgG; Ferti Pro, N.V., Origio, Florence, Italy); no varicocele. These patients had i) mild- ly or severely high levels of DNA fragmentation at base- line examination and ii) SCD control after 2 months. The medical histories of all patients were taken into con- sideration and physical examinations plus ultrasonography of reproductive apparatus were conducted by three clinical andrologists (LN, RB and AP). Semen analyses, as well hor- mone profiles, were evaluated in our hospital. The pres- ence of previous cryptorchidism, long-term medication use (e.g. selective serotonin reuptake inhibitors, tranquilizers, antihypertensives, substances for the prevention of fatty acid metabolism disorders, mesalazine), and idiopathic hypogonadism were not considered exclusion factors as they are representative of everyday real-life clinical prac- tice. Table 1 shows the clinical characteristics of patients. This retrospective study was approved by our hospital’s Institutional Ethical Committee and all patients provided written informed consent for the scientific use of their clinical data. The primary endpoint of the study was to analyze the improvement in sperm integrity (SDF reduc- tion) after antioxidant oral supplementation. The second- ary endpoints were the effects on classic semen parameters (sperm count, progressive motility, normal forms, viabili- ty) and the presence of adverse events. Pregnancy, miscar- riage, and live birth rates were not considered due to the short duration of treatment (2 months). Semen processing Semen samples were obtained in a collection room locat- ed in the same facility as the andrology laboratory after 3-5 days of sexual abstinence. After liquefaction at 37°C in sterile cups, seminal volume and pH, sperm concen- tration, motility, morphology, and viability were evaluat- ed according to World Health Organization guidelines (2010) (19). We analyzed the total sperm count instead of the concentration/ml, as it is more representative of actual testicular function. SCD test The method used was Halosperm G2® (Halotech, Madrid, Spain), in keeping with the manufacturer’s protocol (http://www.halotechdna.com/wp-content/uploads/2015/ 04/IU-halosperm-G2_10det_v2.pdf). The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. Sperm cells with very small halos or without halos are to be considered as containing fragmented DNA. The extent of DNA damage for each semen sample is expressed as the sperm DNA fragmentation index (SDF). In humans, a threshold of 30% SDF is suggested as a cut- off to distinguish between a potentially fertile vs infertile semen sample, although a threshold of 18% has been sug- gested as predictive of a poor fertilization rate. In order to understand whether the patient achieved a clinically useful improvement in SDF, we arbitrarily selected a cut-off of 10% DNA fragmentation percentage change, calculated as: Statistics The data were described as number and percentage, or mean and standard deviation, as appropriate. Differences were explored with the Wilcoxon test for paired data when comparing data at baseline and 2 months control, or the Kruskal-Wallis test when comparing improve- ments in SDF or integrator type. The association between the percentage variation and the commercial product Table 1. Conventional seminal parameters and clinical characteristics of patients - TPMC means total progressively motile sperm count [(volume x sperm concentration x progressive motility)/10-8]. At baseline 2 Months control P Patients 118 118 Infertility duration (months) 32.9 ± 24.8 Male age (yrs.) 39.4 ± 5.4 Female age (yrs.) 35.1 ± 4.3 Male BMI 25.7 ± 3.0 Total orchidometry (ml) 28.7 ± 8.2 Active smoking 27 (22.88%) FSH (mu/ml) 5.43 ± 3.07 Comorbidities 35 (29.66%) Medication use 25 (21.19%) Semen volume (ml) 3.52 ± 1.51 3.43 ± 1.55 0.393 Total sperm count (106) 70.4 ± 63.2 80.9 ± 78.4 0.159 TPMC (106) 13.9 ± 15.4 17.2 ± 20.0 0.160 Progressive motility (%) 18.3 ± 10.2 19.3 ± 11.4 0.738 Normal forms (%) 3.49 ± 2.09 3.64 ± 1.86 0.334 Viability (%) 63.6 ± 11.9 66.6 ± 10.4 0.014 Negri_Stesura Seveso 28/09/17 10:23 Pagina 213 Archivio Italiano di Urologia e Andrologia 2017; 89, 3 L. Negri, R. Benaglia, E. Monti, E. Morenghi, A. Pizzocaro, P.E. Levi Setti 214 was explored with an univariable linear regression; all the products with a p less than 0.1 were then subjected to a multivariable linear regression. All analyses were made with stata13 software (StataCorp LP, 4905 Lakeway Drive, College Station, TX 77845, USA). A p < 0.05 was considered as significant. RESULTS 118 male partners of infertile couples treated from June 2014 - November 2016 were selected for a retrospective study. Mean age was 39.3 ± 5.4 years, infertility duration was 32.9 ± 24.8 months. Female age on examination was 35.1 ± 4.3 years. Conventional seminal parameters and clinical characteristics of patients are reported in Table 1. None of the patients reported any adverse events after oral antioxidant supplementation. Of these men, 15 had received no medical or surgical treatment and were used as a control group. The remaining 103 had received some oral antioxidant supplementation (12 different combinations of both hydrophilic and lipophilic antioxidants), whose compo- sition is shown in Table 2. Fifty-five were treated with SOD-based antioxidant FertiPlus® SOD, whose formula- tion contains ORISOD®, Extramel®, α-lipoic acid, glutathione, folic acid, zinc, and vitamins B2, B3, B6, B12. FertiPlus® SOD is a balanced combination of enzy- matic and non-enzymatic antioxidants (SOD micro encapsulated [Extramel®]), alpha lipoic acid, glutathione (low dose), zinc, B vitamins, a micronutrient complex (ORISOD®) containing substances of plant origin, hydroxytyrosol, and carnosol, identified as substances able to activate the antioxidant system and detoxify intracellular endogenous NRF-2 (nuclear transcription factor-erythroid 2). Five patients had a clinical history of juvenile orchidopexy and one had a history of previous unilateral seminoma without chemoradiotherapy; one had testicular microlithi- asis, one reported a low birth weight, one had unilateral absence of the vas deferens and ipsilateral renal agenesis, and one had unilateral testicular torsion without anti- sperm antibodies. Another 25 patients were taking med- ication for anxiety and depression (n. 1), asthma (n. 6), Behçet’s disease (n. 1), gastroesophageal reflux (n. 5), nasal polyposis (n. 1), hypertension (n. 3), hypothy- roidism (n. 1), Crohn’s disease (n. 1), juvenile diabetes (n. 1), epilepsy (n. 1), mild chronic renal insufficiency (n. 1), hypercholesterolemia (n. 1), and pudendal nerve entrapment (n. 1). One patient was using cannabis. We then assessed which of the active substances were associated with variations in DNA fragmentation, indi- vidually considered and corrected on an individual basis for the statistically significant cases. The results are shown in Table 2. In the multivariable analysis of the seven active substances that passed the univariable analysis, only the SOD molecule appears to be linked to an improvement in SDF. Clinically, variations of at least 10% in baseline values of classic semen parameters and sperm DNA fragmentation were taken into consideration. Variations between the two limits were not considered clinically relevant. Table 3a shows the clinical and seminal parameters compared to the percentage change in SDF. No seminal parameters were associated with the variation in DNA fragmentation, except for the improvement in sperm viability (p = 0.001) and, to a slight extent, the improvement in progressive motility (p = 0.07). The distribution of comorbidities and medication use is homogeneous in the three analyzed groups. Furthermore, no differences in age, BMI, active smoking, FSH, and total testicular volume were observed in the three groups. Classic seminal parameters (total sperm count, progressive motility, and morphology) do Table 2. The table lists the substances contained in 12 commercial products. The second column shows the number of prescriptions in the 103 patients evaluated in the study. The fourth and fifth columns report the percentage variation in DNA fragmentation for each individual active substance. Data are expressed as mean ± SD. Ingredients Commercial No. patients Absent Present P P products (n. 12) (n. 118) (mean ± SD) (mean ± SD) (univariable) (multivariable) Vit. E 7 28 6.96 ± 27.83 -4.66 ± 28.47 0.1189 Vit. C 6 33 7.21 ± 26.90 -3.57 ± 30.69 0.0920 0.2505 Zinc 6 82 -7.54 ± 28.93 9.36 ± 26.60 0.0024 0.3565 Arginine 6 23 6.18 ± 28.64 -3.96 ± 25.84 0.1404 Selenium 5 16 5.87 ± 28.44 -6.43 ± 25.74 0.1141 L-carnitine 5 17 5.57 ± 28.11 -3.91 ± 28.90 0.2902 Folic acid 5 71 -7.36 ± 27.23 11.86 ± 26.50 0.0002 0.6156 Coenzyme Q10 3 17 5.3 ± 29.63 -3.70 ± 17.11 0.1341 Inositol 3 8 5.18 ± 27.90 -9.30 ± 32.36 0.2702 Vit. B 2 57 -6.54 ± 26.55 15.69 ± 25.65 < 0.0001 0.2226 Astaxanthine 2 8 4.77 ± 28.85 -3.56 ± 18.80 0.2941 α-lipoic acid 2 57 -6.54 ± 26.55 15.69 ± 25.65 < 0.0001 -* DHA 2 8 4.33 ± 28.04 2.47 ± 33.80 0.4799 SOD 1 55 -7.13 ± 26.65 17.18 ± 24.46 < 0.0001 < 0.0001 L-taurine 1 12 4.81 ± 27.78 -1.14 ± 33.47 0.7997 Aspartic acid 1 10 5.02 ± 28.95 -4.60 ± 18.90 0.2308 Glutathione 1 60 -7.06 ± 25.45 15.09 ± 26.78 < 0.0001 0.6537 Tryptophan 1 2 4.71 ± 28.11 -25.06 ± 33.09 0.1961 Maca 1 6 4.22 ± 27.98 3.93 ± 36.88 0.5648 Any treatment 103 -16.15 ± 22.39 7.17 ± 27.93 0.0028 * α-lipoic acid omitted from multivariable analysis for collinearity. Negri_Stesura Seveso 28/09/17 10:23 Pagina 214 215Archivio Italiano di Urologia e Andrologia 2017; 89, 3 Effect of superoxide dismutase supplementation on sperm DNA fragmentation not vary significantly in the three groups, except for SDF, showing an improvement in the group receiving SOD (Table 3b). In particular the post-hoc evalution of SDF variation test power (by Cohen’s d) is greater than 0.98 confirming the adeguacy of sample size. In greater detail, only one patient in the control group showed a sponta- neous improvement in SDF (6%), compared to 16/48 (33%) of those taking various oral antioxidant supple- Table 3a. Clinical and seminal parameters compared to the percentage change in SDF. The Δ are calculated as (post-pre)/pre and expressed as a percentage. Table 3b. Clinical and seminal parameters in the three groups. Classic seminal parameters (total sperm count, progressive motility, and morphology) do not vary significantly in the three groups, except for viability and SDF, showing an improvement in the group receiving SOD- The Δ are calculated as (post-pre)/pre and are expressed as a percentage. SDF variation > 10% Unchanged > 10% P deterioration improvement N 36 34 48 Male age (yrs.) 38.6 ± 4.2 39.9 ± 4.8 39.6 ± 6.5 0.640 Female age (yrs.) 35.2 ± 4.3 34.8 ± 4.2 35.1 ± 4.4 0.923 Male BMI 25.5 ± 2.9 26.1 ± 2.8 25.5 ± 3.2 0.364 Total orchidometry (ml) 30.6 ± 7.9 27.8 ± 8.8 27.8 ± 7.9 0.302 Infertility duration (months) 32.8 ± 28.6 33.7 ± 22.7 32.5 ± 23.7 0.587 Active smoking 6 (16.67%) 7 (20.59%) 14 (29.17%) 0.419 FSH (mu/ml) 5.20 ± 2.90 5.87 ± 3.53 5.28 ± 2.87 0.512 Medication use 6 (16.67%) 6 (17.65%) 13 (27.08%) 0.471 Comorbidities 8 (22.22%) 9 (26.47%) 18 (37.50%) 0.296 Antioxidants < 0.001 None 8 (22.22%) 6 (17.65%) 1 (2.08%) Other 21 (58.33%) 11 (32.35%) 16 (33.33%) SOD 7 (19.44%) 17 (50.00%) 31 (64.58%) Baseline semen volume (ml) 3.47 ± 1.40 3.65 ± 1.68 3.45 ± 1.50 0.918 Δ Semen volume (%) 11.5 ± 28.7 2.5 ± 34.7 -5.6 ± 28.1 0.025 Baseline total sperm count (106) 80.2 ± 79.1 57.1 ± 52.2 72.4 ± 56.3 0.299 Δ Total sperm count (%) 68.4 ± 163.1 18.3 ± 77.5 16.0 ± 68.9 0.307 Baseline TPMC (106) 17.1 ± 19.7 10.3 ± 9.5 14.1 ± 14.8 0.4971 Δ TPMC (%) 98.6 ± 179.1 27.0 ± 124.1 83.9 ± 269.7 0.204 Baseline progressive motility (%) 19.8 ± 11.5 17.0 ± 7.7 18.3 ± 10.8 0.6110 Δ Progressive motility (%) 11.8 ± 55.0 -3.4 ± 43.4 43.3 ± 115.6 0.074 Baseline normal forms (%) 3.36 ± 2.17 3.32 ± 2.40 3.71 ± 1.82 0.2039 Δ Normal forms (%) 10.2 ± 54.0 25.0 ± 70.0 54.6 ± 161.0 0.717 Baseline viability 63.4 ± 11.6 65.5 ± 8.2 62.4 ± 14.2 0.8646 Δ Viability (%) 2.9 ± 21.9 -0.4 ± 12.5 20.0 ± 47.5 0.001 No drugs Other drugs SOD P N 15 48 55 Male age (yrs.) 38.7 ± 4.4 38.9 ± 5.5 40.0 ± 5.6 0.454 Female age (yrs.) 34.3 ± 5.2 35.1 ± 3.5 35.2 ± 4.6 0.709 Male BMI 24.8 ± 2.1 25.6 ± 2.7 26.0 ± 3.4 0.441 Total orchidometry (ml) 30.7 ± 7.4 29.6 ± 8.9 27.3 ± 7.7 0.421 Infertility duration (months) 34.7 ± 21.5 32.3 ± 24.8 33 ± 26.1 0.754 Active smoking 1 (6.67%) 14 (29.17%) 12 (21.82%) 0.202 FSH (mu/ml) 4.39 ± 1.16 5.72 ± 3.59 5.46 ± 2.91 0.464 Medication use 3 (20.00%) 12 (25.00%) 10 (18.18%) 0.703 Comorbidities 3 (20.00%) 13 (27.08%) 19 (34.55%) 0.521 Baseline SDF 34.9 ± 12.5 37.0 ± 9.5 40.0 ± 12.6 0.3728 Δ SDF (%) 16.1 ± 22.4 4.3 ± 27.5 -17.2 ± 24.5 < 0.001 Baseline semen volume (ml) 3.64 ± 1.26 3.60 ± 1.55 3.40 ± 1.56 0.639 Δ Semen volume (%) 8.7 ± 27.5 2.5 ± 31.4 -0.5 ± 31.5 0.596 Baseline total sperm count (106) 76.4 ± 90.0 62.4 ± 50.8 75.7 ± 64.9 0.825 Δ Total sperm count (%) 52.1 ± 78.2 45.5 ± 145.4 16.1 ± 75.7 0.165 Baseline TPMC (106) 15.1 ± 15.7 11.8 ± 11.7 15.5 ± 17.9 0.857 Δ TPMC (%) 74.6 ± 144.5 63.1 ± 155.4 79.0 ± 263.4 0.644 Baseline progressive motility (%) 22.2 ± 13.4 17.7 ± 8.5 17.9 ± 10.6 0.473 Δ Progressive motility (%) 11.1 ± 57.1 9.2 ± 49.2 32.2 ± 111.7 0.529 Baseline normal forms (%) 2.53 ± 1.41 3.60 ± 2.16 3.65 ± 2.15 0.229 Δ Normal forms (%) 45.5 ± 98.1 15.1 ± 58.9 44.9 ± 150.3 0.483 Baseline viability 68.1 ± 11.0 63.6 ± 10.1 62.4 ± 13.5 0.161 Δ Viability (%) -5.8 ± 11.4 6.8 ± 17.8 14.6 ± 46.4 0.029 Negri_Stesura Seveso 28/09/17 10:23 Pagina 215 Archivio Italiano di Urologia e Andrologia 2017; 89, 3 L. Negri, R. Benaglia, E. Monti, E. Morenghi, A. Pizzocaro, P.E. Levi Setti 216 ments and 31/55 (56%) of those taking oral antioxidant supplements with SOD. Nevertheless, it should be con- sidered that although fragmentation can also improve spontaneously in patients with risk factors for comorbidi- ties or drug therapy, the positive impact of the integrator administration persists, succeeding in combatting the oxidative damage caused by free radicals and highly reac- tive oxygen species, which have been identified as the agents responsible for sperm DNA damage. DISCUSSION It is believed that about 80 million people worldwide are affected by the inability to have children (20), with male factor subfertility accounting for up to 50% of these cases (21). Some 30-80% of male factor subfertility cases are believed to be due to the damaging effects of oxidative stress (21). Oral supplementation with antioxidants is thought to improve sperm quality by reducing oxidative stress (22) and these products are widely available and inexpensive when compared to other fertility treatments. This suggestion is so widely spread by the media that, cur- rently, a high percentage of couples turning to our Fertility Center are already taking antioxidants, prescribed by gyne- cologists, general practitioners, or even self-prescribed. At present, several tests have been developed to evaluate sperm DNA fragmentation, e.g. TUNEL (TdT-mediated dUTP nick-end labeling) (23), Comet Assay (24), Sperm Chromatin Structure Assay (SCSA) (25) and Sperm Chromatin Dispersion test (SCD) (16). While TUNEL and Comet Assay directly detect DNA damage (the latter also finding single and double strand breaks), SCSA and SCD measure DNA fragmentation after a mild denaturation process. TUNEL and SCSA employ flow cytometry, with little intra-technician variability. However, they are com- plex, time consuming, and expensive (flow cytometer). Comet Assay is not suited for rapid diagnosis and requires highly specialized personnel to analyze the results. The SCD test is a simple and inexpensive technique, but could have higher intra-individual variation. There are currently six meta-analyses of antioxidant treat- ment for male infertility available (22, 26-30) and all report improvements in pregnancy rate and sperm quality after therapy. Ross et al. (2010) (29) report improvement in at least one semen variable in 13 out of 17 studies analyzed. In a more recent Cochrane meta-analysis (22), comprising 48 studies, 4.179 men were analyzed; of these, 2.466 received oral antioxidant supplementation and 1.713 received no treatment. The patient population was made up of the male partners of couples who had attended a fer- tility clinic. Surprisingly, only two trials performed on a total of 100 patients (64 + 36) analyzed the effects of oral antioxidant supplementation on SDF (31, 32) and both observed a reduction in SDF when compared to placebo (mean difference: -13.85, 95% CI -17.28 to -10.41, P < 0.00001). One investigator used vitamin C + vitamin E, while the other used docosahexaenoic acid (DHA). Menezo et al. (2007) (33) (not included in the meta-analysis) treat- ed 58 men with an SDF > 15% with oral antioxidant ther- apy (vitamins C and E, beta carotene, zinc and selenium) for 13 weeks and reported a significant improvement in DNA fragmentation (-19.1%, p < 0.0004). Our data are not all consistent with those reported in lit- erature, not providing a significant improvement in clas- sic seminal parameters (total sperm count, progressive motility, and morphology). Basic semen parameters do not vary significantly in the three groups (antioxidants, FertiPlus® SOD, no medication), except as regards viabil- ity. The reasons may be related to the older age of our population (39.4 ± 5.4 yrs.), which reflects the later age at which couples are deciding to have children. Secondly, the selection of patients was as close as possi- ble to everyday real-life clinical practice. Indeed, we only excluded patients with varicocele and seminal infections, as diseases associated with SDF, but susceptible to effec- tive specific treatment (antibiotics and surgery). Patients with antisperm antibodies were excluded as in other studies, although the two available cases did not have a high degree of DNA fragmentation (data not shown). Most of the studies published to date did not enroll men with a considerable number of risk factors, such as smoking, recreational drug use, systemic diseases, long- term medication use, alcohol, oligozoospermia, high serum gonadotropins, previous orchidopexy, and anatomic abnormalities of the genital tract. While this approach permits a better appreciation of the effect of medical treatment, it also drastically reduces the number of candidates for oral antioxidant treatment. At our Fertility Center, perfectly healthy, young patients without any bad habits are really very few. Our selection criteria could, therefore, justify unsatisfac- tory results in terms of classic sperm parameters. Antioxidants not containing SOD led to an improvement of at least 10% in TPMC in 43.8% patients vs 45.5% in men treated with FertiPlus® SOD (n.s.). In the same two groups sperm morphology increased by at least 10% to 31.3% and 36.4%, n.s.), respectively, while oral antioxi- dant supplementation proved effective in reducing sperm DNA fragmentation. As mentioned in the Results section, only one patient in the control group showed a spontaneous improvement in SDF (6%), compared to 16/48 (33%) of those taking various oral antioxidant supplements and 31/55 (56%) of those taking oral antioxidant supplements with SOD (p < 0.0001). From a clinical viewpoint, the possibility to reduce sperm DNA fragmentation in 56% of otherwise untreat- able infertile patients is certainly an ethically and eco- nomically sound approach. We must therefore consider that almost one quarter of our patients had untreatable diseases, requiring long-term treatment; 13 had high FSH (7.6-21.1 mu/ml), 17 had a testicular volume of less than 12 ml, 10 had class 1 obesity, and 27 were active smokers. Nevertheless, we were surprised to observe that the presence of co-morbidities, signs of testicular impair- ment and bad habits did not affect the chances of improving DNA fragmentation. While oral SOD supplementation seems to work better than any other antioxidant molecules analyzed, it remains unclear why the benefit is observed in just over half of the cases treated. One could assume that 2 months intake are insufficient to fully express the therapeutic effect. Another possible explanation may be that antioxidant therapy could be ineffective if given to males whose subfertility is not caused by oxidative stress (34) and, in this respect, no Negri_Stesura Seveso 28/09/17 10:23 Pagina 216 217Archivio Italiano di Urologia e Andrologia 2017; 89, 3 Effect of superoxide dismutase supplementation on sperm DNA fragmentation patients underwent an objective test indicating that oxida- tive stress was the key factor behind their condition. Our study has a number of limitations. Firstly, it is retrospec- tive, meaning that neither a causality hypothesis nor mechanistic models can be drawn up due to the nature of our study. Secondly, the data derive from patients enter- ing an IVF-ICSI program, who could have different char- acteristics from the general male population. In addition, another limitation is the low number of subjects exam- ined. Lastly, the seminal OS levels were not assessed. Although to be confirmed in a randomised trial this result is a new and relevant data in patient’s counselling. CONCLUSIONS Oral SOD supplementation appears to produce a better reduction in sperm DNA fragmentation in the infertile population than other commonly used antioxidant for- mulations. When used in unselected infertile patients, representative of daily clinical practice, FertiPlus® SOD reduces DNA fragmentation in 56% of cases compared to 33% of cases using other antioxidant formulations. Therefore, given the absolute tolerability of the product and the affordable cost, this approach is to be considered clinically and ethically acceptable. AUTHOR CONTRIBUTIONS NL provided the study design concept, drafted the arti- cle and interpreted the data. NL, RB and AP recruited the subjects, compiled the med- ical records, performed physical examinations and the color-Doppler ultrasound evaluations. EM performed the SCD test and semen analyses EM performed the statistical analyses PELS made a substantial contribution to critically revis- ing the article. ETHICS APPROVAL AND CONSENT TO PARTICIPATE We declare that our study has been conducted according to the Helsinki Declaration on clinical research and to the Ethical Code on animal research set forth by WHO (WHO Chronicle 1985; 39:51) and that has been approved by IRCCS Istituto Clinico Humanitas INDEPENDENT ETHICS COMMITTEE, reference number 1/17, on January 17, 2017. A written informed consent was obtained from each participant before study. REFERENCES 1. Spano M, Bonde J, Hjøllund HI, et al. Sperm chromatin damage impairs human fertility. Fertil Steril. 2000; 73:43-50. 2. 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Levi Setti, MD paolo.levi_setti@humanitas.it Humanitas Research Hospital, Department of Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Fertility Center, Rozzano-Milan, Italy Emanuela Morenghi, MD emanuela.morenghi@humanitas.it Biostatistics Unit, Humanitas Research Hospital, Rozzano-Milan, Italy Alessandro Pizzocaro, MD alessandro.pizzocaro@humanitas.it Endocrinology Unit, IRCCS, Humanitas Research Hospital, Rozzano-Milan, Italy Negri_Stesura Seveso 28/09/17 10:23 Pagina 218