Stesura Seveso Archivio Italiano di Urologia e Andrologia 2025; 97(1):13750 1 REVIEW infertility is caused by defective sperm parameters (sper- matogenic failure), including total absence (azoosper- mia), low count (oligozoospermia), abnormal morpholo- gy (teratozoospermia), and/or impaired motility (astheno- zoospermia). The failure in dealing with male infertility is mostly because of the multifaceted etiology that arises from the interaction of genetics, lifestyle choices, envi- ronmental influences, and concomitant conditions (2). Reactive oxygen species (ROS) are normal products of cel- lular metabolism, mainly produced in the mitochondria during oxidative phosphorylation. Free radicals form during oxygen reduction for energy production. When oxidants increase, the balance shifts toward oxidative stress, which is linked to over 100 disorders, including infertility. ROS can induce protein degradation, lipid per- oxidation, DNA damage and apoptosis. Evidence indi- cates that ROS-mediated damage to sperm significantly contributes to 30%-80% of all cases (3, 4). N-acetyl-cysteine (NAC) is a dietary supplement and mucolytic drug utilized in the treatment of acetamino- phen and paracetamol overdoses. As a thiol-based deriv- ative of the amino acid L-cysteine and a precursor to glu- tathione peroxidase, it has potent anti-inflammatory, mucolytic, and antioxidant properties. Numerous studies indicate that NAC supplementation can elevate sperm counts, improve motility, diminish abnormal morpholo- gy, minimize DNA fragmentation, promote acrosomal activity, and function as a potent semen antioxidant (5). This review investigates the potential of NAC supplemen- tation in the latest studies on seminal parameters and its safety in male with infertility or impaired semen parame- ters. METHOD Search strategy Two authors independently searched for and reviewed all randomized controlled trial describing the efficacy and safety of NAC in male with infertility or impaired semen parameters from inception until February 21, 2024 from several databases comprised of PubMed, ScienceDirect, Introduction and objectives: N-acetyl-cys- teine (NAC) is one of the oldest and most powerful antioxidants used to treat various diseases. It plays an important role in protecting cells against oxidative damage and has the potential to improve seminal parameters in male with infertility. This systematic review and meta-analysis aim to comprehensively evaluate the efficacy and safety profile of antioxidant supplementation with NAC in male with infertility or impaired semen parameters. Materials and methods: This systematic review and meta-analy- sis adhered to Cochrane Handbook guidelines. A literature search across PubMed, ScienceDirect, Cochrane Library and Scopus on February 21, 2024 of studies evaluating NAC supple- mentation for male infertility or impaired semen parameters was conducted. Study quality was assessed using Revised Cochrane's risk of bias (RoB 2.0) and RevMan 5.4 was used for meta-analysis. Results: Search yielded 1.106 articles and 5 studies were includ- ed in this meta-analysis. Our study showed that patients who received NAC had statistically significant results in improving sperm volume [MD: 0.69 (0.26-1.12), P = 0.002], sperm concen- tration [MD: 4.43 1.50-7.36), P = 0.003], sperm total motility [MD: 9.69 (6.61-12.77), P < 0.00001], and normal sperm mor- phology [MD: 1.36 (0.70-2.03), P < 0.0001] compared to con- trol. Additionally, patients given NAC had no reported side effects based on our included studies. Conclusions: We found NAC supplementation significantly improves seminal parameters and has a favorable safety profile. These findings highlight the potential role of NAC as a safe sup- plementation for male with infertility or in male with impaired semen parameters. KEY WORDS: Antioxidant; Sperm parameters; Safety; Meta-analysis; N-acetylcysteine. Submitted 18 February 2025; Accepted 24 February 2025 INTRODUCTION The male component significantly accounts for around 50% of all infertility cases (1). Male fertility is primarily influenced by spermatogenesis, the process of spermato- zoa formation from spermatogonia within the testes. Male Unlocking the potential of antioxidant supplementation with N-acetylcysteine to improve seminal parameters and analysis of its safety: A systematic review and meta-analysis of randomized controlled trials Syarif 1, Moh. Anfasa Giffari Makkaraka 2, Ahmad Taufik Fadillah Zainal 2, Ponco Birowo 3, Widi Atmoko 3 1 Division of Urology, Department of Surgery, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia; 2 Faculty of Medicine, Hasanuddin University, Makassar, Indonesia; 3 Department of Urology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia. DOI: 10.4081/aiua.2025.13750 Summary Archivio Italiano di Urologia e Andrologia 2025; 97(1):13750 Syarif, M.A. Giffari Makkaraka, A. Taufik Fadillah Zainal, et al. 2 Scopus and Cochrane Library. The subsequent keywords were generated by integrating several terms, including “N-acetyl-cysteine” AND “Semen quality” OR “seminal parameters” OR “Semen”. This study applied no restric- tions regarding country or publication year. The protocol of this meta-analysis was registered in PROSPERO (CRD42024516001). This study also followed the guide- line of PRISMA 2020 (6). Eligibility criteria This systematic review and meta-analysis examined studies of randomized controlled trials that met the specified crite- ria. The studies were included into this meta-analysis if they fulfilled the following criteria. (1) Evaluated NAC sup- plementation for male with infertility or impaired semen parameters; (2) Each article provided precise data, mostly involving the subject count and indicator outcomes of: semen volume, sperm concentration, total motility, normal sperm morphology and adverse event (AE) reported; and (3) Full-text article in English language and related data can be obtained. Studies presented as abstracts, review arti- cles, and case reports were removed. Selection process Duplicate studies were identified and excluded after the initial search. The titles and abstracts of the remaining lit- erature were screened by at two independent reviewers to determine eligibility. Studies meeting the criteria were included, while those not meeting requirements were excluded. Conflicts in study classification were resolved through group discussion. The results of the literature screening adhere to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA guidelines). Data extraction The following data were collected for each study by dif- ferent reviewers: (1) First author’s name; (2) Published time; (3) The type of study design; (4) Patients descrip- tion; (5) Patient’s received therapy, dosage and treatment period; (6) Number of patients in each group; (7) age, and (8) Data on semen volume (mL), sperm concentra- tion (106/mL), total motility (%), normal sperm mor- phology (%) and AE reported. Quality assessment Two authors independently assessed all identified inclu- sion studies, and any disagreements that emerged were addressed with the input of a third reviewer. Cochrane Risk of Bias (ROB) Tools 2 was used to assess the quality of each study. ROB 2 instrument was utilized to assess the risk of bias in randomized controlled trials (RCTs), focusing on five domains (7). Overall risk-of-bias judgement of these instruments was classified into 3 groups which low risk of bias (if the study is judged to be at low risk of bias for all domains for this result), some concerns (if there is some concern at least in one domain) and high risk of bias (if the study is judged to be at high risk of bias in at least one domain for this result). Risk-of-bias Visualization (ROBVIS) was used for the visualization of risk of bias graph (8). Statistical analysis The acquired data was analyzed with Review Manager 5.4. (Cochrane Collaboration, UK). Variations between baseline (study entrance) and study completion (end-point meas- ure) were utilized to indicate changes in the results. Mean difference (MD) was used to explain continuous data and odds ratio (OR) for dichotomous results with the corre- sponding 95% confidence interval (CI). I2 value refers to sta- tistical analysis of heterogeneity. The fixed-effects model is used if I2 < 50%, while the random-effects model is used if I2 ≥ 50%. The results will be presented in a forest plot, and the overall effect is considered significant if p < 0.05. Asymmetry tests, including Egger’s test for assessing poten- tial publication bias via funnel plots, will not be performed if the meta-analysis comprises fewer than 10 studies due to their restricted reliability (9). Furthermore, subgroup analysis and sensitivity analysis will also not be conducted if there is a small number of studies (10). RESULTS Literature search, screening results and characteristic of studies From various databases, 1.106 studies were initially iden- tified using keywords. After excluding 82 duplicates, two reviewers independently screened the remaining 1.024 study titles and abstracts, which excluded 1.015 articles according to the inclusion and exclusion criteria. After reviewing only nine full texts, we excluded four studies because they lacked sufficient data or did not meet our study criteria. Finally, 5 RCT studies (11-15) were included in our analysis with a total of 666 patients. Full details of the search and selection process are presented in the PRISMA flow diagram (Figure 1) and the characteristics of these studies are stated in Table 1. Quality assessment result Three studies (11-13) raised concerns regarding the ran- domization process (domain 1), while one study (11) indicated issues related to the lack of personnel and patient blinding, which pertains to deviations from intended interventions (domain 2) and bias in outcome measurement (domain 4). Additionally, one study (13) expressed concerns about missing outcome data due to a significant number of patient withdrawals, resulting in an imbalance in sample sizes between groups (domain 3). Four studies (11, 12, 14, 15) raised concerns about bias in the selection of reported results due to the absence of a published protocol (domain 5). A detailed assessment of the risk of bias is presented in Figure 2. Sperm volume Sperm volume from two studies assessing the efficacy of NAC showed a marked improvement of sperm volume (MD: 0.69; 95% CI: 0.26-1.12; P = 0.002), NAC demon- strated a markedly larger enhancement in sperm volume in relation to the control group (Figure 3A). Sperm concentration Five studies evaluating the efficacy of NAC exhibited that the NAC group had a significant improvement (MD: 4.43; 95% CI: 1.50-7.36; P = 0.003) (Figure 3B), it Archivio Italiano di Urologia e Andrologia 2025; 97(1):13750 3 N-acetylcysteine to improve seminal parameters demonstrated that NAC is more effective in increasing sperm concentration compared to the control (Figure 3B). Total sperm motility Four studies revealed that patients who received NAC intervention had a significant improvement in total sperm motility (MD: 9.69; 95% CI: 6.61-12.77; P < 0.00001) (Figure 4A), which shown that NAC was more effective in enhancing sperm motility than the control (Figure 4A). Normal sperm morphology There were four studies that assessed normal sperm mor- phology. The random effects model showed that there was significant increase in normal sperm morphology in the NAC group (MD: 1.36; 95% CI: 0.70-2.03; P < 0.0001), which demonstrated that NAC resulted in a much higher enhancement in sperm normal morphology relative to the control (Figure 4B). Safety profile The included studies reported that patient receiving NAC supplementation at a dosage of 600 mg/day for twelve weeks or 26 weeks, resulted in improved sperm quality without any reported side effects. DISCUSSION The results of this meta-analysis showed that NAC supple- mentation significantly increased sperm volume, sperm concentration, total sperm motility and normal sperm mor- Figure 1. Flow of literature search and selection based on Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). Archivio Italiano di Urologia e Andrologia 2025; 97(1):13750 Syarif, M.A. Giffari Makkaraka, A. Taufik Fadillah Zainal, et al. 4 Figure 2. Risk of bias assessment using the revised Cochrane risk-of-bias tool algorithm for randomized trials (RoB 2.0). RCT: Randomized controlled trial; NAC: N-acetylcystein. Table 1. Characteristics data of included studies. Archivio Italiano di Urologia e Andrologia 2025; 97(1):13750 5 N-acetylcysteine to improve seminal parameters phology in male with infertility or impaired semen param- eters. Wei et al. previously carried out a meta-analysis assessing the effectiveness of L-carnitine/L-acetyl-carnitine (LC/LAC) and NAC in males with idiopathic astheno- zoospermia. The findings indicated that supplementation with LC/LAC and NAC significantly enhanced sperm motility, normal morphology, sperm concentration, and ejaculate volume (16). Different from that study, this meta- analysis included 5 RCT studies evaluating NAC supple- mentation and providing new insights into the effective- Figure 3. Forest plots of seminal parameters of (A) Semen volume. (B) Sperm concentration. CI: Confidence interval; IV: Inverse variance; SD: standard deviation. Figure 4. Forest plots of seminal parameters of (A) Total motility. (B) Normal sperm morphology. CI: Confidence interval; IV: Inverse variance; SD: standard deviation. Archivio Italiano di Urologia e Andrologia 2025; 97(1):13750 Syarif, M.A. Giffari Makkaraka, A. Taufik Fadillah Zainal, et al. 6 ness of NAC for improving semen parameters. Several pre- vious studies have shown that around 30-80% of male infertile patients have increased ROS in their semen (17). An increase in ROS that surpasses the antioxidant capacity in semen leads to an imbalance between pro-oxidants and antioxidants, resulting in oxidative stress (OS) conditions that damage the spermatozoa plasma membrane and sperm DNA. This explains why increased ROS can have a negative impact on sperm parameters and cause infertility in men (18). One way to prevent OS is to increase the antioxidant capacity of semen by providing antioxidant supplements. NAC is a derivative of the natural amino acid L-cysteine, which has an important role in cellular protection against oxidative damage (19). NAC is one of the oldest and most powerful antioxidants in treating several diseases, includ- ing its application in decreasing the viscosity and flexibil- ity of mucus due to its capacity to break disulfide bonds. In addition, NAC has the potential to interact directly with oxidants and with some thiols, which are excellent hydroxyl radical scavengers (20). The results of this meta- analysis show that NAC administration can improve the quality of sperm parameters. This is thought to be related to the main role of NAC as a stem-form antioxidant, which has the ability to increase intracellular glutathione concentration, the most crucial biothiol responsible for cellular redox imbalance (21). NAC administration can also increase the total antioxidant capacity (TAC) in semen fluid (20). Increased TAC will prevent OS due to overproduction of ROS. This is proven by the study of Barekat et al., who reported that patients given NAC reduced the percentage of ROS in semen (13). Preventing OS in semen can prevent damage to spermatozoa and enhance the integrity of sperm DNA, leading to a signifi- cant increase in the number of sperm in the semen (19). Several studies have reported that the antioxidant effect in semen can also increase the mitochondrial function of spermatozoa, thereby providing more energy and increas- ing their motility (22). Jannatifar et al. indicated that fol- lowing NAC administration, the levels of seminal malon- dialdehyde (MDA) drastically reduced. MDA served as a particular indicator of lipid peroxidation, whereas TAC exhibited a considerable rise. NAC mitigates the intensity of oxidative stress, therefore diminishing lipid peroxida- tion (12). The theoretical basis and mechanisms explain the results of this meta-analysis, which show that NAC is effective and safe in improving sperm parameters. In addition, NAC is relatively safe and well tolerated, even at high doses (21). Of all the included studies, none report- ed any significant side effects of NAC administration on male with infertility or impaired semen parameters. The primary goal of infertility therapy is to achieve preg- nancy. In a study conducted by Barekat et al., NAC was administered to men following varicocelectomy, leading to significant improvements in semen quality and a high- er reported clinical pregnancy rate in the NAC group compared to the control group. This indicates a notable enhancement in fertility outcomes associated with NAC supplementation (13). Unfortunately, our analysis could not assess pregnancy rates because the studies we includ- ed did not provide sufficient data for further evalua- tion.The dosage and duration of NAC supplementation have been extensively evaluated in studies related to male fertility. Most studies have administered NAC at a dosage of 600 mg each day for three months, which has consis- tently resulted in significant improvements in sperm quality and oxidative stress markers. Jannatifar et al. reported enhanced sperm parameters following this regi- men (12), while Rafiee et al. observed similar benefits in men with impaired semen parameters (11). In a larger trial by Safarinejad et al., participants received 600 mg of NAC daily for 26 weeks, leading to significant improve- ments in multiple semen parameters (15). Comhaire et al. also noted improvements in sperm concentration and acrosome reaction at similar dosages (23). These studies suggest that NAC supplementation at dose of 600 mg each day for durations of three to six months should be preferred for enhance seminal parameters. Several studies have reported on the safety profile of NAC supplementation. Erkkilä et al. found no cytotoxic effects of NAC on human male germ cells in vitro, supporting its safety for reproductive health applications (24). A sys- tematic review by Zafarullah et al. highlighted that NAC is also safe in various clinical applications, being generally well-tolerated even at higher doses used for treating con- ditions such as chronic obstructive pulmonary disease and acetaminophen overdose (25). This study suggests that NAC is a safe and effective antioxidant supplement for enhancing male fertility parameters without a signifi- cant risk of adverse events. DECLARATIONS Ethical approval: This study did not need ethical approval. Availability of data and material: All data and materials from this research are available to the researcher and we will provide it upon request if the researcher needs it. Competing interests: The authors certify that there is no con- flict of interest with any financial organization regarding the material discussed in the manuscript. Funding: All funding for this research comes from researchers without receiving reimbursement of research costs or research grants from third parties. Authors' contributions: Contribution Details (to be ticked marked as applicable): SS MAG ATF Concepts √ √ √ Design √ √ √ Definition of intellectual content √ Literature search √ √ Data acquisition √ √ √ Data analysis √ √ √ Statistical analysis √ √ √ Manuscript preparation √ √ √ Manuscript editing √ Manuscript review √ √ √ Guarantor √ Acknowledgments: We as authors would like to thank all parties involved in this study, including the Department of Urology, Faculty of Medicine, Hasanuddin University and also Hasanuddin University Hospital. Conference Presentation: This article has been oral present- ed in the 21st Urological Association of Asia Congress (UAA) on September 7th 2024. Archivio Italiano di Urologia e Andrologia 2025; 97(1):13750 7 N-acetylcysteine to improve seminal parameters We need to acknowledge some limitations of this study. First, there are only a few trials included in our study. However, all studies used in this research are randomized controlled trials, hence augmenting the robustness of the findings. In addition, bias due to deviations from intend- ed interventions may affect the final results of this study. Secondly, this meta-analysis is limited by the limited number of RCT studies. Based on these limitations, it is important to interpret the findings of this review cau- tiously, as they may warrant further studies. Additionally, future studies should analyze and compare the combina- tion of NAC and other types of antioxidant supplements to improve sperm parameters. Based on our analysis, we recommend NAC supplementation for males experienc- ing impairment of semen parameters, either at a dose of 600 mg or at a dose of 200 mg NAC three times a day for a minimum of 12 weeks, and up to 26 weeks. CONCLUSIONS A significantly improvement of seminal parameter was found in male with infertility or impaired semen parameters who received NAC supplementation. NAC supplementa- tion has also been proven to be safe. These findings high- light the potential role of NAC as a safe supplementation for males with infertility or impaired semen parameters. REFERENCES 1. Schlegel PN et al. Diagnosis and Treatment of Infertility in Men: AUA/ASRM Guideline Part I. J Urol. 2021; 205:36-43. 2. Assidi M. Infertility in Men: Advances towards a Comprehensive and Integrative Strategy for Precision Theranostics. Cells. 2022; 11:1711. 3. O’Flaherty C. Reactive Oxygen Species and Male Fertility. Antioxidants. 2020; 9:287. 4. Agarwal A et al. 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Anfasa Giffari Makkaraka fasagifari@gmail.com Ahmad Taufik Fadillah Zainal ahmadtaufik2014004@gmail.com Faculty of Medicine, Hasanuddin University, Makassar, Indonesia Ponco Birowo ponco.birowo@gmail.com Widi Atmoko dr.widiatmoko@yahoo.com Department of Urology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia