Stesura Seveso 465Archivio Italiano di Urologia e Andrologia 2021; 93, 4 ORIGINAL PAPER No conflict of interest declared. be ≥ 1.5 mL. The sperm concentration is reported as the number of sperm per mL of semen that should be ≥ 15 million per mL. Total sperm number (also known as ‘total sperm count’) is described as the total number of sperm in the ejaculate, calculated by multiplying the semen vol- ume by the sperm that should be ≥ 39 million. Sperm progressive motility should be ≥ 32% motile within 60 minutes of ejaculation. Sperm vitality should be ≥ 58%. Sperm normal morphology is described as the percentage of the total number of sperms that should be ≥ 4% (4). COVID-19 may affect male fertility through virus divi- sion, cytotoxic effects on testicular tissue and immunopathological effect (6). Coronavirus can directly affect testicular tissue as well as some sperm parameters by altering the expression of the angiotensin-converting enzyme 2 gene pattern (ACE-2) (7) because seminiferous cells, spermatogonia, Leydig cells and Sertoli cells express this enzyme (8). Studies have shown that ACE-2 is pres- ent in the post-acrosomal region, neck and middle part of normal sperm (9). Sperm ACE binds to the glycosyl-phos- phatidyl-Inositol (GPI) portion of the oocyte Zona Pellucida and is therefore involved in fertilization (10). N-acetylcysteine (NAC), a derivative of amino acid L-cys- teine, is used mainly as an antioxidant (11). NAC has free radical scavenging activity (12). In addition, daily treat- ment with NAC results in a significant improvement in sperm motility in comparison to placebo (13). NAC improved sperm concentration and acrosome reaction while reducing ROS and oxidation of sperm DNA (14). This study investigates the effect of NAC on abnormal sperm parameters in men with COVID 19 infection. MATERIALS AND METHODS Design This interventional study was conducted from March 2020 to July 2021. All couples whose infertility treatment cycle were canceled due to COVID-19 pandemic were included in this study. Male patients with abnormal sperm analysis before COVID-19 were excluded from the study. In total, 273 male patients were included in this study, 47 patients did not present to the infertility center to continue treatment during the time of this study and Male infertility is an important factor accounting for 40-50% of infertility cases that may be due to disturbance in one of the parameters as con- centration, motility and morphology observed in one or two semen analysis with an interval of 1 and 4 weeks. COVID-19 may affect male fertility through virus division, cytotoxic effects on testicular tissue and immunopathological effect. N-acetyl cys- teine (NAC) improved sperm concentration and acrosome reac- tion while reducing reactive oxygen species (ROS) and oxidation of sperm DNA. This interventional study was conducted on 200 men who were referred to private infertility clinics for female factor (their previous semen analysis was normal) and got COVID-19 infection in the last 3 months showing an impair- ment of the latest semen analysis due to COVID. Men were placed in two groups of control (n = 100) and intervention (NAC consumption). Subjects who got COVID-19 infection had a sig- nificant impairment of sperm quality (sperm concentration, sperm motility, and normal sperm morphology) compared to their semen analysis evaluated before the COVID-19 infection. NAC consumption significantly improved sperm total motility, sperm morphology and sperm concentration. COVID-19 infec- tion has a negative effect on sperm parameters. NAC supple- mentation may have positive effect on sperm parameters. KEY WORDS: COVID-19; Sperm; Morphology; Infection. Submitted 25 September 2021; Accepted 12 October 2021 INTRODUCTION According to the International Committee for Monitoring Assisted Reproductive Technology of World Health Organization (WHO), infertility is a reproductive disorder that prevents clinical pregnancy after 12 months or more of unprotected intercourse (1). Studies have shown that nearly 72.4 million couples worldwide are involved in infertility problems (2). Male infertility is an important factor accounting for 40-50% of infertility cases that may be due to disturbance in one of the parameters of con- centration, motility and morphology in one or two semen analysis with an interval of 1 and 4 weeks (3). Normal sperm parameters are assessed based on WHO criteria (4). Semen analysis is still a powerful and essen- tial tool with a sensitivity of 89.6% that can identify 9 out of 10 men with infertility problems (5). Normal semen volume is the total amount of fluid ejaculated that should The effect of N-acetyl cysteine consumption on men with abnormal sperm parameters due to positive history of COVID-19 in the last three months Bahare Rafiee 1, Seyed Mohammad Bagher Tabei 2 1 PhD Student of Reproductive Biology, Department of Reproductive Biology, School of Advanced medical Sciences and Applied Technologies, Shiraz, Iran; 2 Genetics Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. DOI: 10.4081/aiua.2021.4.465 Summary Archivio Italiano di Urologia e Andrologia 2021; 93, 4 B. Rafiee, S.M. Bagher Tabei 466 26 patients presented to continue the treatment more than two months after positive PCR and were excluded from the study. Other exclusion criteria were diabetes, hypertension, mumps history, sexually transmitted dis- eases, varicocele, and chronic diseases history. All the 200 eligible COVID-19 patients were willing to participate in the study and were randomly allocated in two groups of controls and treated subjects. All the cases gave a semen sample after completing written informed consent. Subjects having a positive nasopharyngeal swab test for COVID-19 (ESwab collection kit, Copan diagnostics) or positive Immunoglobulin (Ig) M and IgG antibodies were considered positive for COVID-19 (15). Intervention and assessment The subjects took NAC 600 mg/day by oral route for 3 months (16). Variables including seminal parameters were measured before and after the intervention. Semen sampling method Semen samples were collected once, at the beginning of study and at the end of the intervention. Sperm samples were taken by masturbation after 3-5 days of sexual absti- nence and kept in a plastic container. Then the samples were incubated at 37°C for 30 minutes and analyzed after one hour. The sperms were counted by light microscope with a magnification of 400. Different characteristics of semen including appearance, volume, pH, color, viscosi- ty, liquefaction time, sperm concentration and sperm motility were investigated. Statistical analysis For statistical analysis was used the Statistical Package for Social Science (SPSS Inc, Chicago, Illinois, USA) version 16.0. P value significance was set at 0.05 and confidence interval was at 95%. Paired t-test was used to compare the results before and after interventions. Independent t test was used to compare between control and intervention group. RESULTS The average time between initial normal sperm analysis and COVID-19 infection was 2 months (range 1-5 months). Average time from COVID-19 infection and sperm analysis re-evaluation was 6 weeks (range 3-8 weeks). Patients who presented more than 2 months after COVID-19 infection were excluded. Subjects suffering from COVID-19 infection had a statis- tically significant impairment of sperm quality (sperm concentration, sperm motility, and normal sperm mor- phology) compared to their semen analysis before the COVID-19 infection (Table 1). In Table 2, results of sperm analysis after NAC treatment were not significantly different from initial results of sperm analysis (before COVID). In Table 3 the results of sperm analysis during the follow- up of controls are described. Results of the initial sperm analysis (before COVID) and the last sperm analysis at 3 month follow-up were signifi- cant different for sperm motility (p = 0.04) and sperm concentration (p = 0.03). DISCUSSION Coronavirus disease 2019 (COVID-19) is a highly trans- missible infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), a single- strand enveloped RNA virus belonging to the family of coronaviridae (17). This virus enters host cells mainly through ACE-2 and transmembrane protease serine 2 (TMPRSS2) (18). Recent advances suggest a possible infection of the endocrine system in COVID-19 patients (19-21). In the present study, we evaluated semen parameters after acute SARS-CoV-2 infection. Subjects suffering from COVID-19 infection had a statistically significant impair- ment of sperm quality (semen volume, sperm concentra- tion, sperm motility, and normal sperm morphology) compared to their semen analysis before the covid infec- tion. Our findings are in accordance with other studies which are available in the literature (22-24). It is recom- mended that men with positive history of SARS-CoV-2 who are interested in fertility should be evaluated by a fertility specialist. N-acetylcysteine (NAC) is a thiol-based antioxidant that plays an important role in the protection of cellular con- Table 1. Age, body mass index and semen parameters for individuals before and after COVID-19 infection. Before COVID After COVID P value Age 36.1 ± 4.1 36.1 ± 4.1 0.96 BMI (kg/m2) 23.12 ± 2.5 21 ± 3.1 0.04* Volume (ml) 3.5 ± 0.9 2.9 ± 0.6 0.05* Sperm concentration (106) 115.1 ± 35.1 68.7 ± 53.6 0.01* Total motility (%) 69.9 ± 32.7 30.1 ± 29.6 0.01* Morphology 4 ± 1.2 2 ± 0.9 0.03* Table 2. Comparison of semen parameters before and after COVID and after 3 months treatment with NAC. NAC consumption significantly improved sperm total motility, sperm morphology and sperm concentration. Sperm Before After COVID After NAC P value before vs parameters COVID before NAC after NAC Volume (ml) 3.10 ± 0.56 2.3 ± 0.6 4.02 ± 0.18 0.03* Total motility (%) 81.1 ± 23.2 35.1 ± 29.6 71.6 ± 25.3 0.01* Sperm concentration (106/mL) 115 ± 32.5 61.7 ± 53.6 98.7 ± 44.5 0.04* Morphology (%) 4 ± 0.6 2 ± 0.9 4 ± 1.5 0.03* Table 3. Comparison of semen parameters before and immediately after COVID infection and after 3 months of follow-up in the control group. Sperm Before After After 3 P value after parameters COVID COVID 3 months COVID vs 3 of follow-up months follow-up Volume (ml) 4.13 ± 0.61 2.5 ± 0.3 4.44 ± 1.32 0.04* Total motility (%) 65.10 ± 19.63 37.5 ± 25.1 43.3 ± 21.43 0.06 Sperm concentration (106/mL) 110 ± 21.42 58.8 ± 25.1 63.54 ± 54.21 0.071 Morphology (%) 3 ± 0.71 2 ± 2.1 3 ± 0.9 0.06 467Archivio Italiano di Urologia e Andrologia 2021; 93, 4 N-acetyl cysteine and abnormal sperm parameters after COVID-19 stituents against oxidative damage. The hypothetical action of NAC comes from the ability to stimulate and sustain intracellular levels of reduced glutathione and to detoxify ROS. NAC is one of the oldest and most powerful antioxi- dants that treat various diseases, including respiratory dis- orders, heart disease, heavy metal poisoning, overdose with acetaminophen and epilepsy (25). Safarinejad et al. also reported significant improvements in all semen parameters in subjects receiving selenium or NAC (13). 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Effects of Nacetylcysteine on semen parameters and oxidative/antioxidant status. Urology. 2009; 74:73-76. Correspondence Bahare Rafiee, PhD Student of Reproductive Biology (Corresponding Author) medicalarticle2020@yahoo.com Department of Reproductive Biology, School of Advanced medical Sciences and Applied Technologies, Shiraz (Iran) Seyed Mohammad Bagher Tabei Genetics Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz (Iran)