Stesura Seveso 199Archivio Italiano di Urologia e Andrologia 2022; 94, 2 ORIGINAL PAPER No conflict of interest declared. altering DNA and the acrosomal reaction, and other fac- tors (8-14). However, these results should be interpreted cautiously because the semen of both fertile and infertile men contains microbiota, mainly bacterial (8). Urogenital infections in men are less frequent than in women, although they can trigger chronic inflammatory processes such as prostatitis (15). Clinically, prostatitis is classified into four types: i) acute bacterial prostatitis; ii) chronic bacterial prostatitis; iii) chronic pelvic pain syn- drome; and iv) asymptomatic inflammatory prostatitis (16-18). Chronic bacterial prostatitis is responsible for 5 to 10% of total prostatitis cases, and at least 30% of those involve recurrent urinary infections (18). It is estimated that 5 to 10% of acute genitourinary infectious and inflammatory processes end in chronic prostatitis (16). Therefore, this work aimed to evaluate the effect of the presence of microorganisms in the semen on seminal quality and inflammatory markers. MATERIALS AND METHODS Study participants This project was approved by the Bioethics Committee for research in humans at the Institute of Medical Research, Medical School, University of Antioquia (Act number 006, April/2018). Ten subjects with chronic prostatitis-like symptoms and eleven fertile donors asymptomatic for uro- genital infections volunteered to participate to the study. The National Institute of Health of chronic prostatitis symp- toms index (NIH-CPSI) (19) translated and validated into Spanish (20) was employed to select the volunteers according to the criteria reported by Nickel et al. (21). The questionnaire contains 13 items that are scored in three discrete domains: pain, urinary symptoms, and the impact on quality of life. We considered as fertile donors those who had children under two years or their partner in pregnancy at study recruitment. To be included in the study they should have no history of any genitourinary symptoms, instru- mentation, or surgery, and NIH-CPSI total score lesser than 3. On the other hand, the inclusion criteria for the chronic prostatitis-like group were aged > 18 years and presence of prostatitis-like syndrome longer than three months (pain and/or discomfort in the perineum or on ejaculation) with a score in the pain domain of the NIH- Objective: Chronic genitourinary infections can alter male fertility and even promote carcinogenic processes. This study aimed to evaluate the effect of the presence in the semen of microorganisms on semen quality. Materials and methods: Clinical symptoms and conventional and functional seminal parameters of eleven fertile donors and ten volunteers with prostatitis-like symptoms were evaluated. Nitric oxide, antioxidant capacity, and pro-inflammatory cytokines in semen and seminal plasma samples were also quantified. Finally, the expression of the ROR-γT, FoxP3, and T-bet genes in semen and the presence of DNA of microorganisms associated with prostatitis in urine and semen were evaluated. Results: When compared with fertile donors, volunteers with chronic prostatitis-like symptoms reported erectile dysfunction (0% vs. 10%, p = 0.2825) and premature ejaculation (0% vs. 40%; p = 0.0190). No statistically significant differences were observed in seminal parameters, cytokine measurement, antioxi- dant capacity, nitric oxide concentration and ROR-γT, FoxP3, T-bet. Microorganisms responsible for sexually transmitted infections and some bacteria associated with the microbiota and infections in the prostate gland were detected. In the semen from the subjects with prostatitis-like symptoms T. vaginalis DNA was detected; in addition, N. gonorrhoeae DNA was also detect- ed in semen and urine samples. S. pyogenes was detected in the urine samples from the control group. Conclusions: Prostatitis-like symptoms are a common finding in young men that affect sexual and reproductive health, but not always the seminal parameters or fertility. The presence of pro- statitis-like symptoms does not affect seminal quality. However, it appears to be associated with an increased likelihood of erec- tile dysfunction and premature ejaculation. Thus, affecting the quality of life and sexual and reproductive health. KEY WORDS: Prostatitis; Fertility; Infection; Inflammation; Seminal quality; Sexual health. Submitted 17 February 2022; Accepted 7 April 2022 INTRODUCTION Frequent exposure of the prostate to infectious processes can promote chronic inflammation (1), alter fertility (2- 4), and even promote cancer (5-7). The role of infection, microbiota and inflammation on male fertility is still con- troversial (8). In vitro studies have shown how microor- ganisms can affect sperm function by altering motility, inducing apoptosis, increasing reactive oxygen species, Molecular analysis of microorganisms in the semen and their impact on semen parameters Jenniffer Puerta Suárez 1, Juan Carlos Hernandez 2, Walter Dario Cardona Maya 1 1 Grupo Reproducción, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad de Antioquia - UdeA, Medellín, Colombia; 2 Infettare, Facultad de Medicina, Universidad Cooperativa de Colombia, Medellín, Colombia. DOI: 10.4081/aiua.2022.2.199 Summary Archivio Italiano di Urologia e Andrologia 2022; 94, 2 J. Puerta Suárez, J.C. Hernandez, W.D. Cardona Maya 200 CPSI greater than 4. After agreeing with their participa- tion in the study, all individuals were required to sign an informed consent. Each volunteer gave a semen sample and a urine mid-stream sample. A blood sample was also taken by qualified personnel in a red Vacutainer tube (Becton Dickinson, NJ, USA) to obtain the serum. Finally, participants also filled out a survey including infor- mation on sociodemographic factors, lifestyle, urinary symptoms, and relevant other aspects of sexual and repro- ductive health that allowed us to identify factors associated with prostatitis symptoms. Semen collection and analysis Semen samples were collected into a sterile sample cup through masturbation after sexual abstinence for 2 to 5 days. Conventional seminal parameters Volume, progressive motility, concentration, and sperm morphology were evaluated according to parameters established by the World Health Organization in the fifth edition of its Human Semen Processing Manual (22, 23). The sperm concentration was evaluated using the Makler chamber (22, 23). Functional seminal parameters Sperm mitochondrial membrane potential (24), sperm membrane integrity (25), chromatin structure assay (26), sperm membrane lipoperoxidation (27), and intracellular levels of reactive oxygen species (ROS) (24) were evaluated by flow cytometry (Fortessa-Becton Dickinson, NJ, USA), according to previously established protocols in our lab (24, 28, 29), and analyzing between 5,000 and 10,000 sperm cells. Data were plotted and processed using the FlowJo 7.6 (Tree Star, Inc. Oregon, USA). Seminal plasma total antioxidant capacity evaluation Three mL of DPPH (2,2-diphenyl-1-picrylhydracil) were mixed with 200μL of the sample. After one hour of incu- bation, the sample was read in a spectrophotometer (Spectronic 20 Spectrophotometer®; Genesys, Rochester, NY, USA) at 515 nm, used ascorbic acid as a positive control (28, 29). Nitric oxide determination Nitric oxide quantification was performed using the com- mercial Griess Reagent Kit for nitrite determination (Molecular probes, Oregon, USA) according to the manu- facturer's instructions and after deproteinization of the semen and serum samples according to the Serafini method (30) as previously reported (29). Cytokine quantification Quantification of IL-12p70, IL-10, IL-1a, IL-6, IL-8, TNF, IL-2, IL-4, IL-17, and IFN-a was performed by BD Cytometric Bead Array (CBA) in semen samples (Human Inflammatory Cytokines Kit, and Human Th1/Th2/Th17 Cytokine Kit, Becton Dickinson, NJ, USA), and the analysis was carried out in the FlowJo 7.6 as previously reported (29). Forkhead box P3 transcription factor (FoxP3), T-box 2 (T-bet), and retinoid-related orphan receptor gt (RORg- T) mRNA expression. Total RNA extraction was performed from 200 µL of semen sample using a commercial kit (Qiagen RNeasy Mini Kit, QIAGEN, Hilden, Germany). The RNA was used to synthesize cDNA using the commercial RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). With the cDNA obtained, polymerase chain reactions were performed in real-time for the FoxP3 (Forward: 5-CAGCA- CATTCCCAGAGTTCCTC-3; Reverse: 5-GCGTGT- GAACCAGTGGTAGATC-3); ROR-gt (Forward: 5-TTTTC- CGAGGATGAGATTGC-3; Reverse: 5- CTTTCCACAT- GCTGGCTACA-3), and T-bet (Forward: 5-GCCTACA- GAATGCCGAGATTACT-3; Reverse: 5-GGATGC TGGT- GTCAACAGATG-3) genes. The gene expression levels were normalized using LCt with b-actin (31). Bacterial detection in semen specimens by PCR assays DNA extraction DNA extraction was performed using the phenol-chloro- form technique using 500 µL of the semen sample and the 10 mL urine pellet. Briefly, the semen samples were cen- trifuged at 200g for 10 minutes, and the urine samples were centrifuged at 22000 g for 10 minutes. For each urine or semen sample, 0.5 mL of lysis solution (1M Tris, 0.5M EDTA, 5M NaCl, 10% SDS, and 0.1% triton x-100) and 5 μL of proteinase K were added for 12 hours at 54°C. Subsequently, 1 mL of phenol-chloroform-isoamyl was added, and it was centrifuged at 5000 g for 10 min. Then, 1mL of absolute ethanol (-20°C), 50 µL of 3M sodium acetate was added to the recovered supernatant, and it was left at -20°C overnight to precipitate the DNA. Finally, it was washed with 1 mL of 70% ethanol; the ethanol was allowed to dry, the DNA was diluted in 100 µL of DNAse/RNAse-free water and quantified in a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Massachusetts, USA). Polymerase chain reaction The final 25μL reaction volume contained 12.5μL of Master Mix (Thermo-Scientific, Massachusetts, USA), a solution containing 0.025 U/L of Taq DNA polymerase, 2 mM of MgCl2, and 0.2 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), 0.2M of each primer, 2 µL of DNA (200 ng), and 9.3 µL of water were added to each reac- tion. The PCR was carried out in a T3000 thermal cycler (Whatman, Biometra, Goettingen, Germany); cycling condi- tions consisted of an initial denaturation step at 94-95ºC for 5 min, followed by 35-40 cycles of specific conditions as previously, and a final elongation of 5-10 min at 72°C, using primers and following PCR conditions previously described (32) for b-actin (33), Chlamydia trachomatis (34), Escherichia coli (35), Klebsiella pneumnoniae (36), Lactobacillus spp (37), Mycoplasma genitalium (34), Neisseria gonorrhoeae (34), Ochrobatrum atrophy (38), Pseudomonas aeruginosa (36), Staphylococcus aureus (36), Staphylococcus epidermidis (39), Streptococcus agalactiae (40), Streptococcus pneumoniae (41), Streptococcus pyogenes (42), Treponema pallidum (34), Trichomonas vaginalis (34), Universal bacteria 27F y 1942R (43), Ureaplasma ure- alyticum (44), Herpes simplex virus I (34) and II (34), and Human papillomavirus (34). DNA extracted from each bacterial strain or clinical isolates obtained from patients was a positive reaction control. Lactobacillus spp. DNA 201Archivio Italiano di Urologia e Andrologia 2022; 94, 2 Prostatitis, semen parameters, and microorganisms was obtained from a woman's vaginal smear on day 14 of her menstrual cycle. Serum prostate-specific antigen (PSA) quantification According to the manufacturer's instructions, total serum PSA quantification was performed using the commercial total PSA kit (DiaMetra, Perugia, Italy). PSA values greater than 4 ng/mL were considered positive, as previously reported (29). Statistical analysis A chi-square and a Mann Whitney test were used to com- pare both groups' dichotomous and numerical variables. The data were analyzed using the statistical program Graph Pad Prism 6.0 (GraphPad, San Diego, CA, USA), and a value of p < 0.05 was considered significant. RESULTS Eleven fertile donors (median age of 32 years) and ten chronic volunteers with prostatitis-like symptoms (medi- an age of 39.5 years) (p = 0.5219) were included in the study (Table 1). Mean body mass index was similar in the two groups (fertile donors 25.7 vs. prostatitis-like sub- jects 23.4, p = 0.2299). Only 40% of the prostatitis-like group were married or living with a partner, compared to 100% of the control group (p = 0.0099). Erectile dys- function (10%) and premature ejaculation (40%) were self-reported by the subjects with prostatitis-like symp- toms (p = 0.2825 and p = 0.0190, respectively). In addi- tion, 50% of subjects with prostatitis-like symptoms reported a history of chronic diseases and stress (p = 0.0072 and p = 0.0382), and 70% reported feeling anxi- ety (p = 0.0166) associated with their symptoms. Three subjects with prostatitis-like symptoms were excluded from the seminal quality analysis because they reported being vasectomized. No statistically significant differ- ences were found on other sexual health and reproduc- tive aspects evaluated, nor on conventional or functional seminal parameters evaluation. No differences were found between groups in seminal plasma antioxidant capacity, plasma/serum nitrites con- centration, or PSA determinations (Table 2). The IL-12p70, IL-10, IL-1b, IL-6, IL-8, TNF, IL-2, IL-4, IL- 17, IFN-g cytokines concentrations were evaluated in serum and seminal plasma samples without finding differ- ences (Table 3). Table 1. Sociodemographic characteristics. Characteristics Control group n = 11 Prostatitis group n = 10 P-value Children 100 40 0.0034 Education level 0.1005 High school 9.1 0 Technician 0 10 University 18.2 60 Postgraduate 72.7 30 Marital status 0.0099 Unmarried 0 50 Married 100 40 Divorced 0 10 Number of sexual partners 0.5250 None 0 10 One to three 36.4 40 More than three 63.6 50 Type of sex Masturbation 63.6 70 0.7574 Vaginal 100 60 0.0197 Oral 63.6 60 0.8639 Anal 0.2568 Insertive 36.4 40 0.5366 Receptive 0 20 0.1189 Condom use 0.3580 Always 0 10 Frequently 18.2 20 Rarely 64.6 30 Never 18.2 40 Chi-square. Data indicate percentage. Table 2. Seminal parameters, nitric oxide concentration and serum PSA. Parameters Control group Prostatitis group P-value Volume (mL) 2.0 (1.5-4.7) 3.5 (1.5-11.8) 0.4556 Progressive motility (%) 49.0 (19.0-81.0) 49.0 (6.0-67.0) 0.7414 Concentration/mL 100.0 (40.5-270.0) 182.0 (7.0-254.0) 0.3269 Viability (%) 79.0 (76.0-91.0) 82.0 (49.0 -85.0) 0.8485 Normal morphology (%) 5.2 (4.2-8.7) 4.6 (4.2-7.0) 0.3874 Teratozoospermia index 1.25 (1.10-1.52) 1.18 (1.12-1.37) 0.4091 High mitochondrial membrane potential (%) 61.3 (33.5-73.5) 66.6 (12.3-75.5) 0.3167 Plasma membrane integrity (%) 63.9 (37.8-84.4) 63.1 (12.1-70.0) 0.8095 ROS production (%) 63.0 (34.8-86.2) 56.9 (17.7-66.8) 0.3612 DNA fragmentation index (%) 10.9 (10.4-14.9) 10.6 (10.3-14.3) 0.5795 Membrane lipoperoxidation (%) 66.8 (9.1-93.3) 71.1 (44.9-96.9) 0.3269 Total antioxidant seminal plasma capacity (%) 61.0 (22.1-81.4) 62.3 (9.5-69.7) > 0.9999 Seminal plasma nitric oxide concentration (Nitritos µM) 1.25 (0.55-11.7) 0.55 (0.23-2.16) 0.1384 Serum nitric oxide concentration (Nitritos µM) 4.6 (1.6-13.0) 2.8 (1.2-7.4) 0.1728 Serum PSA (ng/mL) 0.0 (0.0-18.1) 0.45 (0.0-120.0) 0.3292 Mann Whitney test. Data presented as median and range. ROS: Reactive oxygen species; PSA: Prostatic-specific antigen. Table 3. Detection of cytokines in seminal plasma and serum. Cytokine Control Prostatitis-like P-value pg/mL symptoms Seminal plasma IL-12p70 0 (0-68.9) 8.5 (0-107.5) 0.5907 IL-1β 0 (0-36.8) 2.7 (0-31.53) 0.7260 IL-6 8.9 (0-86.9) 7.1 (0-101.9) 0.9159 IL-8 1808 (0-4202) 1692 (680.4-4334) > 0.999 TNF 0 (0-83.8) 24.7 (0-127.5) 0.1553 IL-2 3.9 (0-23.5) 11.4 (4.3-45.7) 0.2940 IL-4 0 (0-13.7) 0 (0-19.3) 0.1454 IL-10 0 (0-26.7) 1.3 (0-19.3) 0.9113 IL-17 9.3 (0-73.1) 6.6 (0-429.9) 0.8749 IFN-γ 0 (0-5.9) 0 (0-33.5) 0.6084 Serum IL-12p70 0 (0-304.3) 0 (0-95.1) > 0.9999 IL-1β 0 (0-54.3) 0 (0-2.7) 0.3246 IL-6 0 (0-15.6) 0 (0-14.9) 0.2479 IL-8 23.6 (0-301.3) 9.6 (0-66.1) 0.6668 TNF 0 (0-92.7) 0 (0-30.4) 0.3128 IL-2 4.9 (2.1-36.0) 8.2 (4.1-47.3) 0.2439 IL-4 0 (0-19.4) 0 (0-27.0) 0.3128 IL-10 0 (0-10.5) 0 (0-13.1) 0.5573 IL-17 0 (0-182.7) 9.9 (0-85.6) 0.2757 IFN-γ 0.3 (0-12.8) 1.3 (0-6.3) 0.6476 Mann Whitney. Data presented as median and range. Archivio Italiano di Urologia e Andrologia 2022; 94, 2 J. Puerta Suárez, J.C. Hernandez, W.D. Cardona Maya 202 Figure 1. ROR-gT, FoxP3 and T-bet genes expression. Figure 2. STIs DNA detection. Figure 3. DNA detection from other bacteria associated. 203Archivio Italiano di Urologia e Andrologia 2022; 94, 2 Prostatitis, semen parameters, and microorganisms We also found no statistical difference in the expression of ROR-gT, FoxP3, and T-bet genes in semen samples (Figure 1). Finally, we detected microorganisms respon- sible for STIs (Figure 2) and some bacteria associated with the microbiota and infections of the prostate gland (Figure 3). T. vaginalis DNA was detected in 40% of the semen sam- ples of the subjects with prostatitis-like symptoms (p = 0.0197). Furthermore, N. gonorrhoeae DNA was detected in in 50% and 40% of semen and urine samples of this group (p = 0.0072 and p = 0.0197). S. pyogenes was detected in 45.5% of the urine samples from the control group volunteers (p = 0.0146). DISCUSSION Male factor is responsible in 50% of infertility cases, high- lighting urogenital infections as the leading causes (8, 45). Urinary tract infections are the most common type of infec- tion in humans, with an estimated annual prevalence of 150 million, representing a high financial impact (46). In men, urogenital infections are a risk factor for prostatitis development, a disease that dramatically impacts mental and sexual health and quality of life (19, 47). Chronic pro- statitis is a common but poorly understood disease that affects men of any age regardless of their geographical ori- gin (15, 21). Prostatitis has been associated with detri- ments in seminal quality and affects male fertility (4). In the present study, the seminal quality of fertile donors asymptomatic for urogenital infections was compared with that of men with symptoms of chronic prostatitis without finding significant differences in the conventional or func- tional parameters. Volunteers with prostatitis-like symp- toms had 75 and 82% greater semen volume and concen- tration than the control group of fertile donors, although the difference did not reach statistical relevance. Similar findings were obtained by Shang et al. (18). N. gonorrhoeae and T. vaginalis were detected more fre- quently in volunteers with prostatitis-like symptoms. Both were also observed in the semen of infertile men being globally prevalent although easily treatable (48). In addition, the genome of other microorganisms, as Propionibacterium acnes, was frequently observed in the semen and urine of patients with recurrent urinary tract infections (49), and the presence of T. vaginalis in the uro- genital tract was also associated with an increase of the risk of prostate cancer (50). In fact, there is a close relationship between urogenital infections and prostate cancer. It is estimated that one in five neoplasms could be attributed to microorganisms (1), and prostatitis was considered as a risk factor for can- cer development (7, 51). However, microorganisms are not a synonymous of dis- ease, because it has been described that the microbiota modulates the immune system; for example, Lactobacillus spp. is a protective factor in prostatitis (52). On the other hand, in chronic pelvic pain syndrome ani- mal models, infiltration of macrophages and CD4+ T cells has been observed, which according to the local microen- vironment, can differentiate into Th1, Th2, Th17, or Treg (regulatory) cells (7). Chronic prostatitis patients show specific Th1 and Th17 immune responses to prostate anti- gen associated with chronic inflammation of the male gen- ital tract, which may be the basis for the induction and development of chronic pelvic pain (53). Among the T cell subpopulations are Treg cells that secrete transforming growth factor b (TGF-b) and IL-10 and express the FoxP3 transcription factor. Th17 lymphocytes secrete IL-17 and IL-22 and express the transcription factor ROR-gT with a critical role in infections and tumors. Th1 cells secreting IFN-g cytokines, IL-2 and TNF-a express the transcription factor T-bet and are essential in developing autoimmune prostatitis (7). Activation of Th1 and Th17 profiles inhibits Treg cells' action, promoting the appearance of chronic pelvic pain (7). Therefore, we assessed in semen samples the mRNA expression of lineage-specifying tran- scription factors FoxP3, T-bet, and RORg-T. Furthermore, although we evaluated several essential aspects of sexual and reproductive health and lifestyle, other variables not evaluated in this study including the impact of ejaculation delayed and intercourse interrup- tions, were described as risk factors for chronic prostati- tis (54). Even urinary retention and anxiety are risk fac- tors for chronic prostatitis (7). However, this is an excel- lent approach to evaluating prostatitis's effect on male fer- tility and understanding the relationship between the urogenital microbiota, infection, and inflammation. The present study is an interesting approach, as a base- line, to understand the impact on the fertility of chronic prostatitis. Although prostatitis does not seem to alter the seminal quality, it seems to impact on fertility by pro- moting the appearance of other diseases such as erectile dysfunction and premature ejaculation. However, a limi- tation of the present study is the limited number of sub- jects included in the study could explain the lack of dif- ference observed in the comparison of microbiology and immune response between controls and subjects with prostatitis-like symptoms. CONCLUSIONS Although chronic prostatitis is a disease that affects the quality of life, it does not appear to affect seminal param- eters. However, chronic prostatitis seems to be related to alterations in sexual function, such as premature ejacula- tion and erectile dysfunction. ACKNOWLEDGMENTS The authors acknowledge the valuable contributions of the volunteers. Puerta-Suárez J was supported by a fel- lowship from MINCIENCIAS (785-2017). REFERENCES 1. Miyake M, Ohnishi K, Hori S, et al. Mycoplasma genitalium infec- tion and chronic inflammation in human prostate cancer: detection using prostatectomy and needle biopsy specimens. Cells. 2019; 8:212. 2. Xu C, Sun GF, Zhu YF, Wang YF. The correlation of Ureaplasma urealyticum infection with infertility. Andrologia. 1997; 29:219. 3. Verze P, Cai T, Lorenzetti S. The role of the prostate in male fer- tility, health and disease. Nat Rev Urol. 2016; 13:379. 4. Zhao Q, Yang F, Meng L, et al. Lycopene attenuates chronic pro- Archivio Italiano di Urologia e Andrologia 2022; 94, 2 J. Puerta Suárez, J.C. Hernandez, W.D. Cardona Maya 204 statitis/chronic pelvic pain syndrome by inhibiting oxidative stress and inflammation via the interaction of NF-kappaB, MAPKs, and Nrf2 signaling pathways in rats. Andrology. 2020; 8:747. 5. Fujita K, Ewing CM, Sokoll LJ, et al. Cytokine profiling of prosta- tic fluid from cancerous prostate glands identifies cytokines associat- ed with extent of tumor and inflammation. Prostate. 2008; 68:872. 6. Zhang Q, Liu S, Parajuli KR, et al. Interleukin-17 promotes prostate cancer via MMP7-induced epithelial-to-mesenchymal tran- sition. Oncogene. 2017; 36:687. 7. Chen J, Zhang H, Niu D, et al. The risk factors related to the sever- ity of pain in patients with Chronic Prostatitis/Chronic Pelvic Pain Syndrome. BMC Urol. 2020; 20:154. 8. Farahani L, Tharakan T, Yap T, et al. The semen microbiome and its impact on sperm function and male fertility: A systematic review and meta-analysis. Andrology. 2021; 9:115. 9. Puerta-Suárez J, Giraldo M, Cadavid A, Cardona-Maya W. Infecciones bacterianas del tracto reproductivo masculino y su papel en la fertilidad. Rev Chil Obstet Ginecol 2014; 79:209. 10. Galarzo S, Cano-Cháves A, Puerta Suárez J, et al. Efecto de los factores solubles de Staphylococcus aureus, Staphylococcus capitis y Staphylococcus epidermidis sobre la fisiología espermática. Aprobado para publicación Rev Chil Obstet Ginecol 2015; 80:316-323. 11. Cano-Cháves A, Galarzo-Pardo S, Puerta-Suárez J, et al. Efecto de las bacterias uropatógenas y de los factores solubles de su metab- olismo sobre la calidad espermática: Escherichia coli y Enterococcus faecalis. Clínica e Investigación en Ginecología y Obstetricia. 2017; 44:106-112. 12. Guerrero Hurtado L, Puerta Suarez J, Cardona Maya W. Papel de los espermatozoides en la transmisión de bacterias uropatógenas: Escherichia coli y Enterococcus faecalis. Clínica e Investigación en Ginecología y Obstetricia 2018:45:2-6. 13. Puerta Suárez J, Cardona Maya W. Evaluación in vitro del efec- to de Neisseria gonorrhoeae y los factores solubles producto de su metabolismo sobre la calidad espermática. Revista chilena de obste- tricia y ginecología. 2016; 81:211. 14. Zuleta-Gonzalez MC, Zapata-Salazar ME, Guerrero-Hurtado LS, et al. Klebsiella pneumoniae and Streptococcus agalactiae: Passengers in the sperm travel. Arch Esp Urol. 2019; 72:939. 15. Mandar R, Punab M, Korrovits P, et al. Seminal microbiome in men with and without prostatitis. Int J Urol. 2017; 24:211. 16. Videcnik Zorman J, Maticic M, Jeverica S, Smrkolj T. Diagnosis and treatment of bacterial prostatitis. Acta Dermatovenerol Alp Pannonica Adriat. 2015; 24:25. 17. Kumar S, Dave A, Wolf B, Lerma EV. Urinary tract infections. Dis Mon. 2015; 61:45. 18. Shang Y, Liu C, Cui D, et al. The effect of chronic bacterial pro- statitis on semen quality in adult men: a meta-analysis of case-con- trol studies. Sci Rep. 2014; 4:7233. 19. Litwin MS, McNaughton-Collins M, Fowler FJ, Jr., et al. The National Institutes of Health chronic prostatitis symptom index: development and validation of a new outcome measure. Chronic Prostatitis Collaborative Research Network. J Urol. 1999; 162:369. 20. Collins MM, O'Leary MP, Calhoun EA, et al. The Spanish National Institutes of Health-Chronic Prostatitis Symptom Index: translation and linguistic validation. J Urol. 2001; 166:1800. 21. Nickel JC, Downey J, Hunter D, Clark J. Prevalence of prostati- tis-like symptoms in a population based study using the National Institutes of Health chronic prostatitis symptom index. J Urol 2001; 165:842. 22. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 2010. 23. Cardona-Maya W, Berdugo J, Cadavid A. Comparación de la concentración espermática usando la cámara de Makler y la cámara de Neubauer. Actas Urológicas Españolas. 2008; 32:443. 24. Mayorga-Torres BJ, Cardona-Maya W, Cadavid A, Camargo M. Evaluation of sperm functional parameters in normozoospermic infertile individuals. Actas Urol Esp. 2013; 37:221. 25. Martinez-Pastor F, Mata-Campuzano M, Alvarez-Rodriguez M, et al. Probes and techniques for sperm evaluation by flow cytometry. Reprod Domest Anim. 2010; 45 Suppl 2:67. 26. Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertil- ity and comparisons with other techniques. J Androl. 2002; 23:25. 27. Aitken RJ, Wingate JK, De Iuliis GN, McLaughlin EA. Analysis of lipid peroxidation in human spermatozoa using BODIPY C11. Molecular human reproduction. 2007; 13:203. 28. 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. 29. Puerta Suárez J, Cardona Maya WD. Microbiota, Prostatitis, and Fertility: Bacterial Diversity as a Possible Health Ally. Advances in Urology. 2021; 2021:1007366. 30. Serafini M, Maiani G, Ferro-Luzzi A. Alcohol-free red wine enhances plasma antioxidant capacity in humans. J Nutr. 1998; 128:1003. 31. Rueda C, Velilla PA, Chougnet CA, et al. HIV-induced T-cell activation/exhaustion in rectal mucosa is controlled only partially by antiretroviral treatment. PloS one. 2012; 7:e30307. 32. Velásquez Rivera V, Cardona Maya WD, Puerta Suárez J. The relationship between sexually transmitted bacteria, microbiota and seminal quality in asymptomatic men. Asian J Urol (in press) 101016/jajur202109004. 2021. 33. Schmittgen TD, Zakrajsek BA, Mills AG, et al. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Annal Biochem. 2000; 285:194. 34. Gimenes F, Medina FS, Abreu AL, et al. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex- PCR and of HPV by single PCR. PloS one. 2014; 9:e98862. 35. Lee C, Kim J, Shin SG, Hwang S. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol. 2006; 123:273. 36. Anbazhagan D, Mui WS, Mansor M, et al. Development of con- ventional and real-time multiplex PCR assays for the detection of nosocomial pathogens. Braz J Microbiol. 2011; 42:448. 37. Fu CJ, Carter JN, Li Y, et al. Comparison of agar plate and real- time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces. Lett Appl Microbiol. 2006; 42:490. 38. Kulkarni G, Gohil K, Misra V, et al. Multilocus sequence typing of Ochrobactrum spp. isolated from gastric niche. J Infect Public Health. 2017; 10:201. 39. Vandecasteele SJ, Peetermans WE, Merckx R, Van Eldere J. Quantification of expression of Staphylococcus epidermidis house- 205Archivio Italiano di Urologia e Andrologia 2022; 94, 2 Prostatitis, semen parameters, and microorganisms keeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J Bacteriol. 2001; 183:7094. 40. Kong F, Ma L, Gilbert GL. Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization. J Med Microbiol. 2005; 54:1133. 41. Kais M, Spindler C, Kalin M, et al. Quantitative detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR. Diagn Microbiol Infect Dis. 2006; 55:169. 42. Zhao X, He X, Li H, et al. Detection of Streptococcus pyogenes using rapid visual molecular assay. FEMS Microbiology Letters. 2015; 362. 43. Frank JA, Reich CI, Sharma S, et al. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl Environ Microbiol. 2008; 74:2461. 44. Rivera Tapia J, Centeno Torres M, Santellan Olea M, Rodríguez Preval N. Prevalencia de Ureaplasma urealyticum en mujeres. Rev Mex Patol Clin. 2004; 51:33. 45. Lundy SD, Sangwan N, Parekh NV, et al. Functional and taxo- nomic dysbiosis of the gut, urine, and semen microbiomes in male infertility. Eur Urol. 2021; 79:826-836 46. Mouraviev V, McDonald M. An implementation of next genera- tion sequencing for prevention and diagnosis of urinary tract infec- tion in urology. Can J Urol. 2018; 25:9349. 47. Eslahi A, Farpour H, Hosseini A, et al. Evaluation of the sympa- thetic skin response in men with chronic prostatitis: a case-control study. Res Rep Urol. 2020; 12:239. 48. Gimenes F, Souza RP, Bento JC, et al. Male infertility: a public health issue caused by sexually transmitted pathogens. Nat Rev Urol. 2014; 11:672. 49. Manente L, Gargiulo U, Gargiulo P, Dovinola G. Propionibacterium acnes in urine and semen samples from men with urinary infection. Arch Ital Urol Androl. 2022; 94:62. 50. Aglamis E, Ceylan C, Akin MM. Is there a correlation between the aggressiveness of chronic asymptomatic prostatitis National Institutes of Health category IV and the Gleason score in patients with prostate cancer? Can Urol Assoc J. 2020; 14:E568. 51. Perletti G, Monti E, Magri V, et al. The association between pro- statitis and prostate cancer. Systematic review and meta-analysis. Arch Ital Urol Androl. 2017; 89:259. 52. Cai T, Gallelli L, Cione E, et al. The use of Lactobacillus casei DG® prevents symptomatic episodes and reduces the antibiotic use in patients affected by chronic bacterial prostatitis: results from a phase IV study. World J Urol. 2021; 39:3433-3440. 53. Zhang M, Liu Y, Chen J, et al. Single-cell multi-omics analysis presents the landscape of peripheral blood T-cell subsets in human chronic prostatitis/chronic pelvic pain syndrome. J Cell Mol Med. 2020; 24:14099. 54. Li HJ, Kang DY. Prevalence of sexual dysfunction in men with chronic prostatitis/chronic pelvic pain syndrome: a meta-analysis. World J Urol. 2016; 34:1009. Correspondence Jenniffer Puerta Suárez jenniffer.puerta@udea.edu.co Grupo Reproducción, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad de Antioquia, Medellín (Colombia) Juan Carlos Hernandez juankhernandez@gmail.com Infettare, Facultad de Medicina, Universidad Cooperativa de Colombia, Medellín (Colombia) Walter Dario Cardona Maya (Corresponding Author) wdario.cardona@udea.edu.co Grupo Reproducción, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad de Antioquia, Medellín (Colombia)