Stesura Seveso Archivio Italiano di Urologia e Andrologia 2025; 97(1):13128 1 ORIGINAL PAPER development are also evaluated (5-7). A high sperm DNA fragmentation index (DFI) has been associated with decreased male fertility and an increased risk of sponta- neous abortions (8, 9). Therefore, it has become an essen- tial parameter in fertility studies for evaluating sperm quality. Sperm DNA fragmentation can occur for different rea- sons, such as oxidative stress, DNA lesions, infections, and other factors (10). Different techniques are used to evaluate the sperm DFI, including the single-cell gel elec- trophoresis (comet) assay, the sperm chromatin structure assay, DNA breakage detection-fluorescence in situ hybridization (DBD-FISH), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay, among others. These method- ologies have shown promising results but require special- ized equipment and high-cost reagents, making those tests unavailable for all populations (11). In contrast, the sperm chromatin dispersion assay (SCD) offers many advantages, such as its low cost compared to other tech- niques and its simplicity, practicality, speed, and repro- ducibility (11-13). The most explored treatment for reducing sperm DFI is the chronic use of oral antioxidants, based on the evi- dence that DNA fragmentation is frequently associated with states of oxidative stress (14-16). However, the pres- ence of bacteria in semen can also affect semen quality, and consequently, identifying the presence of bacteria in couples with fertility problems could be beneficial for improving semen parameters and increasing the chances of successful conception (17). Therefore, antibiotics could also improve sperm DFI in some instances in which DNA fragmentation is caused by a bacterial infection in the male reproductive tract; treat- ment with appropriate antibiotics could clear the infec- tion and reduce inflammation, which could help improve sperm DNA integrity (18). This study aimed to determine the effects of oral antibi- otics and anti-inflammatory agents on semen parameters and the sperm DFI. MATERIALS AND METHODS Ninety-eight men with the diagnosis of primary or sec- ondary infertility, either directed for advanced and non- Aim: To determine the effects of oral antibi- otics and anti-inflammatory agents on semen parameters and the sperm DNA fragmentation index (DFI). Methods: Ninety-eight men with infertility diagnosis were included. The participants submitted two semen samples, before and at least two months after treatment. Macroscopic and microscopic semen parameters were evaluated following the World Health Organization (WHO) guidelines. In addition, the sperm DFI was evaluated using the sperm chromatin dispersion (SCD) technique. Subsequently, a treatment regimen was administered, including daily oral doses of ciprofloxacin (1000 mg) and doxycycline (100 mg) for 21 and 10 days, respectively. In addition, non-steroidal anti-inflammatory drugs (15 mg of meloxicam) were used for 10 days. After treatment, the same parameters were re-evaluated for new semen samples taken under the same initial conditions. Results: After treatment, significant increases in pH, sperm count, total concentration, and normal sperm morphology were observed, but no significant differences were found in the semi- nal volume parameter or progressive motility. After treatment, a significant decrease in the concentration of immature cells was observed, and although not statistically significant, a reduction in the concentration of leucocytes was observed. After treatment, the mean sperm DFI significantly decreased from 28.24 ± 12.39% to 16.2 ±7.1%. Conclusions: Treatment with antibiotics and anti-inflammatories significantly reduced the sperm DFI and improved semen quality. KEY WORDS: Male fertility; Infertility; DNA fragmentation index; Semen; Spermatozoa. Submitted 19 September 2024; Accepted 6 October 2024 INTRODUCTION Infertility is a problem that affects an increasing number of people around the world, regardless of gender (1, 2). When studying semen to evaluate and diagnose male fer- tility, it is considered that male factors are responsible for 50% of infertility cases (3), and specialists focus primari- ly on the macroscopic and microscopic parameters of semen (4). However, although these parameters can be considered essential for determining the success of a pregnancy, it is crucial that, in the presence of infertility, sperm functional alterations that could affect embryonic Sperm DNA fragmentation: Focusing treatment on seminal transport fluid beyond sperm production Moises Abraham Adel Domínguez 1, Walter D. Cardona Maya 2, Andrés Mora Topete 1 1 Centro de Uro-Andrología S.C., Guadalajara, Jalisco, México; 2 Grupo Reproducción, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia. DOI: 10.4081/aiua.2025.13128 Summary Archivio Italiano di Urologia e Andrologia 2025; 97(1):13128 M.A. Adel Domínguez, W.D. Cardona Maya, A. Mora Topete 2 advanced reproductive techniques were included (Centro de Uro-Andrología S.C., Guadalajara, Jalisco, México). Each patient was invited to participate in the study, and authorization was requested under current ethical stan- dards; each participant also provided informed consent. The study participants provided two semen samples under similar conditions of sexual abstinence (3-5 days), one before treatment and one at least two months after treatment. Both semen samples were analyzed in the same laboratory by the same expert technician. Basic macroscopic and microscopic sperm parameters were evaluated following the WHO guidelines (19), and the sperm DFI was determined using the sperm chro- matin dispersion test (SCD, Sperm DNA kit, Obi biotek) following the manufacturer's instructions. In addition, the concentrations of leukocytes and immature cells were also evaluated. After the initial semen evaluation, the patients were invit- ed to receive daily oral doses of ciprofloxacin (1000 mg) or doxycycline (100 mg) for 21 or 10 days, respectively. In addition, non-steroidal anti-inflammatory drugs (15 mg of meloxicam) were used for 10 days. Statistical analysis The data distribution was evaluated with the normality test of residuals, and the descriptive analysis was carried out according to the distribution of the variables. Paired t tests were carried out for related samples when the data met normality, and the Wilcoxon test was used for non- normal distributed samples. A p value of < 0.05 was con- sidered to indicate statistical significance. RESULTS Ninety-eight men with infertility diagnosis participated in the study. The median age was 36 ± 6.7 years, with an interquartile range (25-75) of 32-41 years. As shown in Table 1, no significant differences were found regarding seminal volume or progressive motility. However, after treatment, a significant increase in pH, sperm count, total concentration, and normal sperm morphology was observed. In addition, a decrease, although not statistically signifi- cant, in the concentration of immature cells and leuco- cytes was observed after treatment: 1.4 ± 2.0 million/mL vs. 0.85 ± 1.6 million/mL (p = 0.2613) and 1.9 ± 2.2 vs. 1.2 ± 1.8 million/mL (p = 0.4541), respectively. Finally, after treatment, the mean sperm DFI significantly decreased from 27.2 ± 10.7% to 16.9 ± 7.6% (p < 0.001) (Figure 1). DISCUSSION The results of this study support the effectiveness of an antibiotic and anti-inflammatory treatment approach in male patients diagnosed with infertility, consistent with previously published preliminary results (20). Initial evaluation of semen samples revealed various parameters essential for determining sperm quality, such as volume, pH, viscosity, sperm concentration and sperm DFI (7). We hypothesize that the sperm DFI problem lies not in the quality of the sperm itself during spermatogenesis but in the factors that promote the degradation of its integri- ty once it has reached maturity and in the seminal tract. Following this approach, we have implemented a treat- ment plan based on dual-scheme antibiotics accompa- nied by anti-inflammatories to improve the quality of the transport medium rather than the spermatogenesis process in the seminal fluid used for spermatogenesis. A few years ago, there was no consensus on how to man- age high prostate-specific antigen (PSA) levels that have occasionally been detected during PSA screening, because PSA levels can increase for several reasons, including trau- ma, ejaculation, rectal and urethral procedures, and numerous noncancerous etiologies such as benign prostat- ic hyperplasia, inflammation, and infection (21); therefore most urologists make decisions based on their training and experience, and the use of antibiotics to reduce high PSA is Table 1. Changes in semen parameters before and after treatment. Pre-treatment Post-treatment P value Volume, mL 1 2.9 ± 1.6 3.0 ± 1.5 0,529 pH 2 7.6 ± 0.6 7.7 ± 0.4 0.007 Progressive motility, percentage 2 33 (10-50) 34 (21-50) 0.763 Concentration, 106/mL 2 21 (8.7-50.8) 22 (9.2-58) 0.046 Total concentration, 106/ejaculate 2 60 (20-126) 83.3 (19.1-172.5) 0.006 Normal morphology, percentage 2 1 (1-2) 2 (1-4) < 0.01 1 Paired t test, data show means ± standard deviations. 2 Wilcoxon test, data show medians and their interquartile range (p25-75). Figure 1. Changes in the sperm DFI after treatment. Archivio Italiano di Urologia e Andrologia 2025; 97(1):13128 3 Antibiotic treatment and sperm DNA fragmentation an alternative (22), although it is not safe to discard biop- sies in patients who achieve a satisfactory PSA response to antibiotics (22), because if it has been observed that the use of antibiotics delays the diagnosis of prostate cancer. In the same way, most of the seminal fluid is composed of by secretions of accessory glands, which comprise the prostate, the seminal vesicles, and the bulbourethral glands, and it is possible that the presence of bacteria in this fluid and in its places of origin (23, 24) could nega- tively affecting the semen and sperm quality. Therefore, as bacteria are present in the semen, safe from its origin, the use of antibiotics will eradicate the local inflammatory effect that promotes tissue damage and release of more prostate antigen, as is happening in patients with increased PSA. However, given that the fragmentation of sperm DNA don’t occur during its for- mation, eliminating bacteria from the seminal pathways would allow the elimination of local inflammation and its deleterious effects on sperm DNA. Bacteria may release soluble factors, such as lipopolysac- charides, hemolysins, and other soluble spermatotoxic fac- tors that can affect sperm physiology (23, 25). The effects of bacterial infection may be multiple, comprising reduc- tion in motility, induction of teratozoospermia (abnormal sperm morphology), apoptosis, DNA fragmentation, sperm agglutination, and exposure to oxidative stress through forming reactive oxygen species (ROS) (23, 25). Altogether, the use of low-potency steroids is necessary because antibiotics do not affect the mucosa of the urothelium or the urethra, but steroids can generate an anti-inflammatory effect on mucous membranes (26). Therefore, using antibiotics and steroids can impact both the tissue of the epididymis, prostate and seminal vesicles as well as the urothelial mucosa of the urethra and pro- static urethra, where the semen is expelled. A key finding in this study was the high sperm DFI observed in the samples, with a mean of 28.24 ± 12.39%. This result highlights the presence of significant sperm DFI in the infertile patient population. According to the litera- ture, a cutoff of 20% can differentiate between fertile and infertile men (27). Previous evidence from a comparative study carried out with fertile and infertile patients showed that the average sperm DFI was 29.95% (26.6-34.3%) in infertile patients and 19.90% (15.6-24.4%) in fertile patients; moreover, a sperm DFI > 26.1% was associated with a 2.8-fold increased risk of infertility (28). Furthermore, orally administered ciprofloxacin and etori- coxib for 15 days reduced the sperm DFI from 36 ± 3% to 24.9 ± 1% (20). The implemented treatment, which included antibiotics and anti-inflammatories, led to significant changes in semen parameters. A significant increase in pH and total sperm concentration was observed at the end of treat- ment, suggesting an improvement in sperm quality. Although the semen volume did not significantly change, these other indicators support the effectiveness of the therapeutic approach (29). Furthermore, a reduction in the concentration of leuko- cytes and round cells, which are markers of inflamma- tion, is an important finding. Although the decrease in leukocyte concentration did not reach statistical signifi- cance, the reduction in the number of round cells was sta- tistically significant. Since round cells are a marker of transient inflammation secondary to spermatogenic aggression, their decrease is positive and could be related to a decrease in sperm DNA fragmentation (30). The most striking finding was the significant decrease in the DFI at the end of treatment. This indicates a substantial improvement in sperm DNA integrity and, therefore, in sperm quality. Taken together, these results support the initial hypothesis that male infertility problems do not reside solely in the quality of the sperm themselves but rather in the factors that may promote the degradation of their integrity once they have reached maturity. Antibiotic and anti-inflammatory treat- ments effectively address these factors, substantially improving semen parameters, including significantly reduced sperm DFI (20). The proposed hypothesis is that a significant percentage of sperm DFI can be generated during its passage through the seminal pathway rather than being an exclusive process of spermatogenesis. In essence, factors present in the epi- didymis, seminal vesicles, and prostate can substantially contribute to fragmentation, even in the absence of clinical symptoms or semen culture findings. These factors include inflammatory changes in the epididymis, asymptomatic bacterial colonization, subclinical prostatitis, infrequent ejaculation and others. Evaluating and managing these conditions in the seminal pathway could improve, to a cer- tain extent, the quality of the seminal sample before decid- ing on Intracytoplasmic sperm injection (ICSI). Therefore, in this way, the possibility of optimizing sperm quality through a therapeutic approach targeting the sem- inal pathway, as a strategy before considering assisted reproduction techniques, has to be considered, intending to improve natural pregnancy rates or reduce the need for ICSI in some cases. CONCLUSIONS These findings are promising and may have significant implications for managing male infertility aiming to the quality of the seminal transport fluid beyond spermatoge- nesis, in order to obtain a non-hostile environment where sperm DNA fragmentation is reduced. However, it is essen- tial to highlight the need for additional research and long- term studies to validate these results and better understand the underlying mechanisms involved in this treatment. DECLARATIONS Ethical approval: Authorization was requested under current ethical standards; each participant also provided informed consent. Availability of data and material: All inquiries can be direct- ed to the corresponding author. Competing interests: None. Funding: None. Authors’ contributions: M.A.A.D., W. D. C.M., A. M.T. Acknowledgments: None. Archivio Italiano di Urologia e Andrologia 2025; 97(1):13128 M.A. Adel Domínguez, W.D. Cardona Maya, A. Mora Topete 4 REFERENCES 1. Eisenberg ML, Esteves SC, Lamb DJ, et al. Male infertility. Nat Rev Dis Primers. 2023; 9:49. 2. Kimmins S, Anderson RA, Barratt CLR, et al. Frequency, morbid- ity and equity - the case for increased research on male fertility. Nat Rev Urol. 2024; 21:102. 3. Schlegel PN, Sigman M, Collura B, et al. Diagnosis and Treatment of Infertility in Men: AUA/ASRM Guideline Part I. J Urol. 2021; 205:36. 4. Abayomi BA, Afolabi BM, Victor DA, Oyetunji I. Semen parame- ters associated with male infertility in a subsaharan black population: The effect of age and body mass index. J Gynecol Infertility. 2018; 1:1. 5. Gil-Villa AM, Cardona-Maya W, Agarwal A, et al. Role of male factor in early recurrent embryo loss: do antioxidants have any effect? Fertil Steril. 2009; 92:565. 6. Gil-Villa AM, Cardona-Maya W, Agarwal A, et al. Assessment of sperm factors possibly involved in early recurrent pregnancy loss. Fertil Steril. 2010; 94:1465. 7. McQueen DB, Zhang J, Robins JC. Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis. Fertil Steril. 2019; 112:54. 8. Álvarez JG. Aplicaciones clínicas del estudio de fragmentación del ADN espermático. Revista Internacional de Andrología. 2007; 5:354. 9. Robinson L, Gallos ID, Conner SJ, et al. The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta- analysis. Hum Reprod. 2012; 27:2908. 10. Evenson DP. Sperm chromatin structure assay (SCSA®) for fer- tility assessment. Current Protocols. 2022; 2:e508. 11. Cicaré J, Avila A, Caille A, Munuce MJ. Incorporación del test de dispersión de la cromatina espermática al laboratorio andrológico. Revista Internacional de Andrología. 2016; 14:137. 12. Fernández JL, Muriel L, Goyanes V, et al. Simple determination of human sperm DNA fragmentation with an improved sperm chro- matin dispersion test. Fertil Steril. 2005; 84:833. 13. Fernández JL, Muriel L, Rivero MT, et al. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl. 2003; 24:59. 14. Aitken RJ, De Iuliis GN. Origins and consequences of DNA dam- age in male germ cells. Reprod Biomed Online. 2007; 14:727-33 15. Greco E, Iacobelli M, Rienzi L, et al. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl. 2005; 26:349. 16. Saldarriaga Monsalve LJ, Cardona Maya WD. Efecto del zumo de sandía (Citrullus lanatus) en el estrés oxidativo en espermato- zoides humanos. Revista chilena de obstetricia y ginecología. 2020; 85:423. 17. Ocampo Flórez GM, Calle Correa E, Carvajal Obando A, Cardona Maya WD. En búsqueda de la fertilidad, tratamiento con antibiótico resuelve el problema de una pareja infértil. Reporte de caso. Revista Médica de Risaralda. 2022; 28:138. 18. Gallegos G, Ramos B, Santiso R, et al. Sperm DNA fragmenta- tion in infertile men with genitourinary infection by Chlamydia tra- chomatis and Mycoplasma. Fertil Steril. 2008; 90:328. 19. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen,. 6th ed. ed2021. 20. Bibancos M, Rocha AM, Hassun PA, et al. Sperm DNA frag- mentation decreases after oral anti-inflammatory and antibiotic treatment. Fertil Steril. 2008; 90:S467. 21. Schaeffer AJ, Wu SC, Tennenberg AM, Kahn JB. Treatment of chronic bacterial prostatitis with levofloxacin and ciprofloxacin low- ers serum prostate specific antigen. J Urol. 2005; 174:161. 22. Atalay HA, Canat L, Alkan I, et al. Prostate-specific antigen reduction after empiric antibiotic treatment does not rule out biopsy in patients with lower urinary tract symptoms: prospective, con- trolled, single-center study. Prostate Int. 2017; 5:59. 23. Zuber A, Peric A, Pluchino N, et al. Human Male Genital Tract Microbiota. Int J Mol Sci. 2023; 24. 24. La Vignera S, Condorelli RA, Vicari E, et al. Markers of semen inflammation: supplementary semen analysis? J Reprod Immunol. 2013; 100:2. 25. Tvrda E, Duracka M, Benko F, Lukac N. Bacteriospermia - A formidable player in male subfertility. Open Life Sci. 2022; 17:1001. 26. Jayakumar S, Pringle K, Ninan GK. Idiopathic urethritis in chil- dren: Classification and treatment with steroids. J Indian Assoc Pediatr Surg. 2014; 19:143. 27. 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. 28. Wiweko B, Utami P. Predictive value of sperm deoxyribonucleic acid (DNA) fragmentation index in male infertility. Basic Clin Androl. 2017; 27:1. 29. Sergerie M, Laforest G, Bujan L, et al. Sperm DNA fragmenta- tion: threshold value in male fertility. Hum Reprod. 2005; 20:3446. 30. Zhou J, Chen LI, Li J, et al. The semen pH affects sperm motility and capacitation. PloS one. 2015; 10:e0132974. Correspondence Moises Abraham Adel Domínguez, MD moises.adel@gmail.com Andrés Mora Topete, MD drandresmora@hotmail.com Centro de Uro-Andrología S.C., Guadalajara, Jalisco, México Walter D. Cardona Maya, PhD wdario.cardona@udea.edu.co Grupo Reproducción, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia