Stesura Seveso Archivio Italiano di Urologia e Andrologia 2020; 92, 4362 ORIGINAL PAPER No conflict of interest declared. DOI: 10.4081/aiua.2020.4.362 Antioxidant treatment of increased sperm DNA fragmentation: Complex combinations are not more successful Cevahir Ozer Department of Urology, Baskent University, Adana, Turkey. Oxidative stress (OS) is one of the major causes of DNA fragmentation in spermatozoa (8). This makes antioxi- dants a part of treatment in male infertility. Since OS can be caused by vastly different oxidants, it is possible to assume that combination therapy will be better than a single agent in antioxidant therapy (9). The purpose of this study was to compare the effective- ness of different antioxidant combinations in infertile men with increased sperm DNA fragmentation. MATERIALS AND METHODS Patients We retrospectively reviewed the records of 637 patients who underwent antioxidant support therapy for increased sperm DNA damage between 2014 and 2019. The base- line clinical evaluation for each patient included a com- prehensive history and a complete physical examination. Semen samples were collected after 2-7 days of sexual abstinence in a specially designated room in our embryol- ogy laboratory, with the aid of audiovisual stimulation. Conventional semen analysis was performed according to the criteria of World Health Organization (WHO). DNA fragmentation in spermatozoa was measured using the terminal deoxyribonucleotidyl transferase-mediated dUTP nick- end labelling (TUNEL) assay (Cell Death Detection Kit, Roche Biochemicals, Mannheim, Germany) according to the manufacturer's instructions with minor modifica- tions. All semen tests were repeated at 3 months of treat- ment. Blood samples were taken in the morning to measure fol- licle-stimulating hormone (FSH), luteinizing hormone (LH) and total testosterone levels at baseline and at the end of 3 months of antioxidant treatment. Inclusion and exclusion criteria Patients with DNA damage of 30% or more were includ- ed study. Presence of varicocele, leukocytospermia, known genetic abnormality, history of chemotherapy and/or radiotherapy, history of malignancy, history of orchiectomy and/or orchiopexy and patients receiving hormonal therapy were accepted as exclusion criteria. Ethical consideration The study was approved by Institutional Review Board (Project no: KA19/250). Objective: Oral antioxidant supplementa- tion is part of the treatment of infertility associated with oxidative stress-related sperm damage. It is possible to assume that the combined use of antioxidants will be better than single agent use. The purpose of this study was to compare the effectiveness of different antioxidant combinations in infertile men with increased sperm DNA fragmentation. Materials and methods: We retrospectively reviewed the records of 637 patients who underwent antioxidant support therapy for increased sperm DNA damage between 2014 and 2019. Patients with DNA damage of 30% or more were included study. Result: A total of 163 patients with follow-up data and who fulfilled the study criteria were included in the study. There were four different treatment groups. No statistically significant differences were found between the groups. After 3 months of antioxidant treatment, there was a statistically significant decrease in sperm DNA damage in all treatment groups. However, there was no statistically significant difference between the treatment groups. Conclusions: The complexity of the antioxidant combination may not contribute to the success of the treatment or may cause possible side effects, increase the cost of treatment and decrease patient compliance. KEY WORDS: Infertility; Oxidative stress; Antioxidants. Submitted 3 August 2020; Accepted 8 September 2020 INTRODUCTION The conventional semen analysis remains the main diag- nostic tool for evaluating male factor infertility (1). However, difficulties resulting from the methodology of the conventional semen analysis significantly reduces the potential diagnostic power of this test. Furthermore, the conventional semen analysis cannot clearly identify all cases of male infertility. Although conventional semen analysis identifies some features of sperm func- tion, it does not fully assess functional sperm compe- tence (2). The search for a high-diagnostic method that could better predict the etiology and reproductive out- comes of male infertility resulted in a focus on sperm DNA integrity and fragmentation (3-5). The level of sperm DNA fragmentation seems to correlate negatively with pregnancy and delivery in both natural and assist- ed conceptions. It is also strongly associated with recur- rent spontaneous abortion (6, 7). Summary 363Archivio Italiano di Urologia e Andrologia 2020; 92, 4 Treatment of increased sperm DNA fragmentation Data interpretation and statistical analysis There were 193 patients who met the study criteria, had follow-up results and used 4 different antioxidant treatment protocols. Age, infertility period, history of varicoc- electomy, cigarette smoking, alcohol con- sumption, serum FSH, serum LH and serum testosterone levels, initial conventional semen parameters, and sperm DNA damage rate before and after treatment were deter- mined. We used propensity score-matched analysis to balance differences in age, dura- tion of infertility, smoking and alcohol use among antioxidant treatment groups. Power value was 76.0% for an effect size value (0.179) that was calculated for sperm DNA fragmentation at 3 month of study. With this power value, it was found that the sample size was suffi- cient. Statistical analysis was performed using the statistical package SPSS software (Version 25.0, SPSS Inc., Chicago, IL, USA). If continuous variables were normal, they were describle as the mean±standard deviation [(p > 0.05 in Kolmogorov-Smirnov test or Shapira-Wilk (n < 30)], and if the continuous variables were not normal, they were described as the median. Comparisons between groups were made using one way ANOVA for normally distrib- uted data and Krukal Wallis test were used for the data not normally distributed. Since analysis of variance was significant, comparisons were made using the Post Hoc test or Mann-Whitney U test. The catagorical variables between the groups was analyzed by using the Chi square test. Values of p < 0.05 were considered statisti- cally. G-power (Version 3.1, Department of Psychology, University of Düsseldorf, Germany) was used for post hoc power analysis. RESULTS After the treatment groups are homogenized, a total of 163 patients with follow-up data and who fulfilled the study criteria were included in the study. All patients had no known medical problems. The medical history of the patients revealed varicocelectomy in 41 patients. Patient characteristics are presented in Table 1. No statistically significant differences were found between the groups with regard to age, infertility period, smoking, alcohol consumption, history of varicocelecto- my, the initial semen parameters and initial sperm DNA damage rate (Table 1). There were four different treatment groups that met the criteria. Group A received 500 mg daily vitamin C (Ester- C plus, Solgar, USA), 400 IU daily vitamin E (Evicap fort, Kocak Farma, Turkey) and 600 mg daily N-acetylcysteine (NAC) (Assist plus, Bilim, Turkey). Group B received 100 mcg daily selenium (Selenium, Solgar, USA) and 100 mg daily coenzyme Q10 (coQ10) (Coenzyme Q-10, Solgar, USA) in addition to vitamin C, vitamin E and NAC. Group C received commercial multiantioxidant supple- ment (NeoFortil M, Tani Pharma, Turkey) in addition to vitamin C, vitamin E and NAC. Group D received anoth- er commercial multiantioxidant supplement (Promotil men, Centax Pharma, Turkey) in addition to vitamin C, vitamin E and NAC. The multiantioxidant supplement used in Group C (NeoFortil M) contains a daily dose of 600 mg L-Carnitine, 250 mg L-Arginine, 120 mg vitamin C, 72 mg vitamin E, 15 mg coQ10, 60 mcg selenium, 40 mg zinc sulfate and 800 mcg folic acid and the other one used in Group D (Promotil men) contains a daily dose of 2000 mg L-carnitine, 500 mg L-arginine, 200 mg vita- min C, 120 mg vitamin E, 100 mg coQ10, 60 mcg sele- nium, 60 mg magnesium, 15 mg zinc sulfate, 400 mcg folic acid, 2 mg vitamin B6, 6 mcg vitamin B12, 10 mcg vitamin D, 1 mg vitamin A and 2 mg beta-carotene. Table 1. Clinical characteristics of patients. Group A Group B Group C Group D Total p (n: 39) (n: 57) (n: 42) (n: 25) (n: 163) value Age (years) 34.91 ± 5.94 36.16 ± 5.01 34.10 ± 4.91 36 ± 5.61 35.31 ± 5.32 0.237 Infertility period (years) 5 (1-13) 6 (1-21) 5 (1-5) 7 (1-14) 5 (1-21) 0.211 Smoking 15 (44.1) 26 (45.6) 29 (69) 11 (44) 81 (51.3) 0.064 Alcohol 0 (0) 4 (7) 3 (7.1) 4 (16) 11 (7) 0.127 Varicocelectomy 11 (32.4) 15 (26.3) 8 (19) 7 (28) 41 (25.9) 0.609 Semen parameters Volume (mL) 3 (1-6) 3 (1-7) 2.5 (1-7) 3 (1-6) 3 (1-7) 0.764 Concentration (million/mL) 20 (8-178) 52 (5-282) 63 (3-188) 76 (0-143) 53 (0-282) 0.413 Motility (%) 55 (5-75) 55 (0-84) 52.5 (11-79) 55 (0-84) 50 (0-84) 0.983 DNA damage (%) 47.72 ± 17.29 50.77 ± 11.26 46.71 ± 10.84 51.12 ± 18.32 49.06 ± 14.03 0.408 Table 2. The effect of oral antioxidants on sperm DNA damage. Initial DNA damage (%) 3th month DNA damage (%) p Group A 47.72 ± 17.29 29.51 ± 12.99 0.0001 Group B 50.77 ± 11.26 25.12 ± 13.81 0.0001 Group C 46.71 ± 10.84 23.88 ± 16.07 0.0001 Group D 51.12 ± 18.32 26.28 ± 18.20 0.0001 Total 49.06 ± 14.03 26.03 ± 14.98 0.0001 Figure 1. Change in DNA damage according to treatment groups. Archivio Italiano di Urologia e Andrologia 2020; 92, 4 C. Ozer 364 After 3 months of antioxidant treatment, there was a sta- tistically significant decrease in sperm DNA damage in all treatment groups (Table 2 and Figure 1). However, there was no statistically significant difference between the treatment groups (p = 0.230). None of the patients had side effects requiring discon- tinuation of antioxidant support treatment. DISCUSSION DNA fragmentation of spermatozoa occurs during sper- matogenesis and maturation processes. Increased sperm DNA fragmentation appears to be associated with impaired sperm function. Aging, poor lifestyle-related habits (such as smoking, alcohol consumption, environ- mental radiation and pollution), diseases, drugs, inflam- mation and infection in the external genital tracts and varicocele may cause an increase in sperm DNA fragmen- tation (8,10-12). All these factors induce sperm DNA breaks by three main mechanisms: apoptosis, impairment of sperm chromatin maturation and OS (13). OS is a condition that is associated with an imbalance between the production and removal of reactive oxygen species (ROS) and free radicals (14). Antioxidant defense system act as scavengers to neutralize free radicals and overcome the adverse results of OS (14, 15). Antioxidant system including enzymatic factors (superoxide dismu- tase, catalase, and glutathione peroxidase), non-enzymat- ic factors and low-molecular weight compounds (glu- tathione, NAC, vitamin E, vitamin A and C, coQ10, car- nitines, myo-inositol, lycopene etc.) and nutrients (selenium, zinc, and copper) can protect the body against OS (16- 18). Lack of one of these leads to a reduction in the antioxidant capacity of the plasma (17). The rationale behind the use of oral antioxidant therapy is that seminal OS is due to increased ROS production and/or decreased levels of enzymatic and non-enzymatic seminal antioxi- dants (19, 20). To date, several studies have shown that exogenous antioxidants have the capacity to counteract oxidative damage or OS, improving sperm DNA integrity for infertile men with OS (18, 21). Many oral formula- tions of antioxidants are readily available in the market and are commonly used to treat men with infertility. The different oral antioxidants available belong to the exoge- nous antioxidant category and they include Vitamin C, Vitamin E, coQ10, NAC, carnitines, trace elements such as zinc, selenium, pentoxifylline, and a combination of these oral antioxidants (20). Numerous studies have been conducted to assess the effectiveness of oral antioxidant supplementation for the treatment of infertile men with sperm DNA damage. Most of the studies showed an improvement in one or more of seminal fluid parameters, whereas some studies reported no positive effect. Although there is still no consensus on the type, dosage and duration of antioxidants to be used in the treatment, it appears that in the case of OS, doses of antioxidants should be higher than the usual daily dose and because the time required for the development of a mature sperm from spermatogonia is 72 ± 4 days, should be used for at least three months (17, 22, 23). Since OS may be due to multiple sources, it seems rea- sonable to assume that a combination of antioxidants tar- geting the male reproductive system will provide better protection than a single antioxidant (9). However, the complexation of antioxidant therapy may have potential adverse effects including reductive stres (21). Furthermore, complex combinations may increase the cost of treatment and may decrease treatment compliance (24). According to our study including 163 patients, complexity of oral antioxidant combination treatment does not appear to contribute to treatment success. The limitation of our study was its retrospective and non-randomized nature. Although the small sample size in the groups we compared is another limitation, sample size was found to be sufficient in post hoc power analy- sis. The variability of the compounds, especially in com- mercial combinations, is the weakness of our study. Since placebo arm ethics is arguable in such studies, we believe that the lack of placebo arm of the study is not a limitation. Furthermore, the effect of improvement in sperm DNA damage on fertility has not been studied because the female group was not homogenous. CONCLUSIONS The use of oral antioxidants can help to reduce OS and to treat OS related sperm DNA damage. The use of antioxidant agent combinations is expected to be more successful in the treatment than a single agent. However, it should be noted that the complexation of the combi- nation may not provide an additional contribution to treatment success. Therefore, possible side effects, treat- ment cost and patient compliance should be kept in mind when designing combination protocols. ACKNOWLEDGEMENTS The author would like to thank Biostatistics specialist Cagla Sariturk for her expertise and assistance in statisti- cal analysis. REFERENCES 1. Komiya A, Kato T, Kawauchi Y, et al. Clinical factors associated with sperm DNA fragmentation in male patients with infertility. Scientific World Journal. 2014; 2014:868303. 2. Santi D, Spaggiari G, Simoni M. Sperm DNA fragmentation index as a promising predictive tool for male infertility diagnosis and treatment management - meta-analyses. Reprod Biomed Online. 2018; 37:315-326. 3. Bungum M. Sperm DNA integrity assessment: a new tool in diag- nosis and treatment of fertility. Obstet Gynecol Int. 2012; 2012:531042. 4. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update. 2003; 9:331- 345. 5. Sakkas D, Mariethoz E, Manicardi G, et al. Origin of DNA dam- age in ejaculated human spermatozoa. Rev Reprod. 1999; 4:31-37. 6. Agarwal A, Cho CL, Esteves SC. Should we evaluate and treat sperm DNA fragmentation? Curr Opin Obstet Gynecol. 2016; 28:164-171. 7. Evgeni E, Charalabopoulos K, Asimakopoulos B. Human sperm 365Archivio Italiano di Urologia e Andrologia 2020; 92, 4 Treatment of increased sperm DNA fragmentation DNA fragmentation and its correlation with conventional semen parameters. J Reprod Infertil. 2014; 15:2-14. 8. Wright C, Milne S, Leeson H. Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod Biomed Online. 2014; 28:684-703. 9. Lewis SE, John Aitken R, Conner SJ, et al. The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment. Reprod Biomed Online. 2013; 27:325-337. 10. Pourmasumi S, Sabeti P, Rahiminia T, et al. The etiologies of DNA abnormalities in male infertility: An assessment and review. Int J Reprod Biomed (Yazd) 2017; 15:331-344. 11. Cai T, Wagenlehner FM, Mazzoli S, et al. Semen quality in patients with Chlamydia trachomatis genital infection treated con- currently with prulifloxacin and a phytotherapeutic agent. J Androl. 2012; 33:615-623. 12. Alargkof V, Kersten L, Stanislavov R, et al. Relationships between sperm DNA integrity and bulk semen parameters in Bulgarian patients with varicocele. Arch Ital Urol Androl. 2019; 91. 13. Moustafa MH, Sharma RK, Thornton J, et al. Relationship between ROS production, apoptosis and DNA denaturation in sper- matozoa from patients examined for infertility. Hum Reprod. 2004; 19:129-138. 14. Pham-Huy LA, He H, Pham-Huy C. Free radicals, antioxidants in disease and health. Int J Biomed Sci. 2008; 4:89-96. 15. Kao SH, Chao HT, Chen HW, et al. Increase of oxidative stress in human sperm with lower motility. Fertil Steril. 2008; 89:1183- 1190. 16. Calogero AE, Condorelli RA, Russo GI, La Vignera S. Conservative nonhormonal options for the treatment of male infer- tility: antibiotics, anti-inflammatory drugs, and antioxidants. Biomed Res Int. 2017; 2017:4650182. 17. Walczak-Jedrzejowska R, Wolski JK, Slowikowska-Hilczer J. The role of oxidative stress and antioxidants in male fertility. Cent European J Urol. 2013; 66:60-67. 18. Arcaniolo D, Favilla V, Tiscione D, et al. Is there a place for nutritional supplements in the treatment of idiopathic male infertil- ity? Arch Ital Urol Androl. 2014; 86:164-170. 19. Zini A, Al-Hathal N. Antioxidant therapy in male infertility: fact or fiction? Asian J Androl. 2011; 13:374-381. 20. Alahmar AT. Role of oxidative stress in male infertility: an updated review. J Hum Reprod Sci. 2019; 12:4-18. 21. Gharagozloo P, Aitken RJ. The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod. 2011; 26:1628-1640. 22. Alahmar AT. The impact of two doses of coenzyme Q10 on semen parameters and antioxidant status in men with idiopathic oligoasthenoteratozoospermia. Clin Exp Reprod Med. 2019; 46:112-118. 23. Agarwal A, Nallella KP, Allamaneni SS, Said TM. Role of antioxidants in treatment of male infertility: an overview of the lit- erature. Reprod Biomed Online. 2004; 8:616-627. 24. McDonald HP, Garg AX, Haynes RB. Interventions to enhance patient adherence to medication prescriptions: scientific review. JAMA. 2002; 288:2868-2879. Correspondence Cevahir Ozer, MD (Corresponding Author) cevahirozer@gmail.com Dadaloglu Mh Serinevler 2591 Sk No: 4/A, 01250 Yuregir, Adana (Turkey)