The incidence and effect of bacteriospermia and elevated seminal leukocytes on semen parameters

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1 ORIGINAL ARTICLES: ANDROLOGY The incidence and effect of bacteriospermia and elevated seminal leukocytes on semen parameters Trustin Domes, M.D., a Kirk C. Lo, M.D., a Ethan D. Grober, M.D., a John Brendan M. Mullen, M.D., b Tony Mazzulli, M.D., b and Keith Jarvi, M.D. a a Division of Urology, Department of Surgery and b Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada Objective: To determine the incidence of bacteriospermia and elevated seminal leukocytes (ESL) in a subfertile male population and correlate these results with semen parameters. Design: Retrospective cohort study. Setting: Canadian tertiary-level male infertility clinic and university-affiliated andrology and microbiology laboratories. Patient(s): Four thousand nine hundred thirty-five nonazoospermic subfertile men. Intervention(s): Analysis and concurrent culture of 7,852 semen samples. Main Outcome Measure(s): Incidence of bacteriospermia and ESL and comparison of semen parameters between these groups. Result(s): The rate of bacteriospermia was 15% (22 species), and the rate of ESL was 19%, with no statistical correlation between these groups. Bacteriospermic patients (without ESL) had a statistically significant deterioration in DNA fragmentation index (DFI) only, compared with patients without bacteriospermia and ESL (24.1 vs. 21.8%). ESL alone was associated with a statistically significant deterioration in sperm concentration (20.6 vs /ml), motility (21.8 vs. 26.9%), normal morphology (12.3 vs. 17.4%), and DFI (26.5 vs. 21.8%), with no additional deterioration identified with bacteriospermia. Conclusion(s): Bacteriospermia and ESL were prevalent, but not statistically associated, in subfertile men. Bacteriospermia alone was associated with an increase in DFI only, but the presence of ESL was the dominant factor associated with deterioration in semen parameters. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Bacteriospermia, seminal leukocytes, semen parameters, DNA fragmentation index Acute and chronic infections of the genitourinary (GU) tract play an important causative role in male factor infertility. Infectious etiologies are involved in up to 15% of male factor infertility cases (1). Multiple microbes have been implicated, including numerous bacterial, viral, fungal, and protozoan species as outlined in reviews by Ochsendorf (2) and Keck et al. (3). These microbes can gain access to the normally sterile GU tract through sexual transmission, intracanicular spread of infected urine, or hematogenous seeding of genital organs. Infections of the testicle (4), epididymis (5, 6), and prostate (7, 8) can lead to local and systemic symptoms and have been documented to negatively affect spermatogenesis and fertility potential. Although GU tract infection can lead to elevated seminal leukocytes (ESL), there are multiple other causes of ESL independent of infectious etiologies. Exposures to environmental toxins, vaginal products used during intercourse, tobacco products, alcohol, marijuana, and certain medications Received August 10, 2011; revised January 23, 2012; accepted January 26, 2012; published online February 16, T.D. has nothing to disclose. K.C.L. has nothing to disclose. E.D.G. has nothing to disclose. J.B.M.M. has nothing to disclose. T.M. has nothing to disclose. K.J. has nothing to disclose. Reprint requests: Keith Jarvi, M.D., Murray Koffler Urologic Wellness Centre, Joseph and Wolf Lebovic Building, Mount Sinai Hospital, 60 Murray Street, 6th Floor, Toronto, Ontario, M5T 3L9, Canada ( kjarvi@mtsinai.on.ca). Fertility and Sterility Vol. 97, No. 5, May /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert can cause ESL (9, 10). A history of GU surgery (vasovasostomy and uretheroplasty), presence of varicoceles, and autoimmunity are also potential noninfectious causes of ESL (11). Lastly, defective spermatogenesis and poor sperm viability can lead to ESL, as the seminal leukocytes scavenge the flawed sperm (11, 12). Therefore, although men with ESL should undergo microbiological testing to rule out infectious causes, the other potential etiologies for ESL should also be considered. Bacteriospermia and the recruitment of seminal leukocytes can affect male fertility through multiple proposed mechanisms, including deterioration of spermatogenesis, impairment of sperm function, and genital tract dysfunction and/or obstruction (Fig. 1). Bacterial cellular interactions, adhesion, and/or agglutination can affect the motility of 1050 VOL. 97 NO. 5 / MAY 2012

2 Fertility and Sterility FIGURE 1 Schematic outlining the possible mechanisms leading to impaired male fertility from bacteriospermia and elevated seminal leukocytes. sperm and impair sperm-oocyte interaction (3). Huwe et al. demonstrated that Escherichia coli (E. coli), but not Enterococcus or Staphylococcus saprophyticus, negatively affected sperm motility in an in vitro assay (13). Additionally, Kohn and colleagues showed that bacteriospermia can impact sperm function, including the inducibility of the acrosome reaction (14). ESL can affect sperm function and integrity through formation of cytokines and reactive oxygen species (ROS) (15). ROS can cause lipid peroxidation of the sperm plasma membrane (16) and have been linked to increases in DNA fragmentation index (DFI) (17). Bacteriospermia and ESL can lead to genital tract obstruction (3) and dysfunction as illustrated by changes in the constituents of the seminal plasma (18, 19). Additionally, if there is a breach in the blood-testis/genital tract barrier from a significant infectious or inflammatory process, anti-sperm antibodies (ASA) may be formed (20, 21), which may impair sperm motility (22) and fertilization ability (23). ESL has also independently been implicated in ASA formation (24). The importance of treating symptomatic genital infections in infertile men is clear, however, the majority of patients with culturable bacteria and/or ESL within the semen are asymptomatic (25). In these men, there is significant controversy whether these infections truly impact male fertility potential and whether subsequent treatment of these men improves semen parameters. In this study, we determine the incidence of bacteriospermia and ESL in a large nonazoospermic subfertile male population and evaluate the individual impact that bacteriospermia and/or ESL have on semen parameters. MATERIALS AND METHODS Patient Population A total of 4,935 consecutive nonazoospermic subfertile men (mean age years) attending a tertiary-level male factor infertility clinic in Toronto, Canada, between 2001 to 2010 who underwent concurrent semen analysis and semen culture were included in the study. These men were deemed subfertile by virtue of attending the infertility clinic as the male partner of an infertile couple. There were no exclusion criteria for patients included in the study, as long as there was an antegrade ejaculate sample that did not demonstrate azoospermia on semen analysis. Sample Collection, Semen, and Microbiological Analyses A total of 7,852 semen samples were obtained for semen analysis and culture from the 4,935 infertile men. After patients had been instructed to void urine and cleanse their hands and genitalia with soap and warm water, semen specimens were provided through masturbation after hours of sexual abstinence. After liquefaction of ejaculates, computer-assisted semen analysis was performed following the 1999 World Health Organization (WHO) guidelines (26), with the exception of sperm morphological analysis, which was assessed using the 1992 WHO guidelines (27). ESL was defined in this study as one or more polymorphonuclear (PMN) leukocytes per 100 sperm via direct visualization of cellular morphology using a modified Papanicolaou stain. This concentration of PMNs has previously been shown by Berger and colleagues to negatively impact sperm function (28). The DFI was calculated using the DNA fragmentation assay (24) in 23% (n ¼ 1,806) of samples. An aliquot of semen was used for culture of aerobic and facultative anaerobic microorganisms. The following four culture conditions were used: blood agar media incubated in CO 2 at 35 C for 48 hours, chocolate agar media incubated in CO 2 at 35 C for 72 hours, Martin-Lewis agar incubated in CO 2 at 35 C for 72 hours, and MacConkey agar incubated in O 2 at 35 C for 48 hours. Bacteriospermia was defined VOL. 97 NO. 5 / MAY

3 ORIGINAL ARTICLE: ANDROLOGY FIGURE 2 The four most common bacterial species identified in the seminal fluid. E. fecalis (56%), E. coli (16%), GBS (13%), and S. aureus (5%) account for 90% of all bacterial isolates identified. as R10 3 bacteria/ml, as described by McGowan et al. (29) and the WHO (27). The semen and microbiological analyses data were prospectively updated, and these databases were merged for this study. Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics 18.0 (SPSS). Results are reported as means SD, unless otherwise specified. The c 2 -test and Pearson s correlation analysis was used to determine the relationship between bacteriospermia and ESL. The analysis of variance test with Fisher s least significant difference post hoc analysis was used to statistically compare mean semen parameter results among the four distinct patient groups (bacteriospermia and ESL, ESL without bacteriospermia, bacteriospermia without ESL, and neither bacteriospermia nor ESL). P<.05 was considered statistically significant. RESULTS The rate of bacteriospermia in this male factor infertility population was 15% (1,200/7,852), incorporating 22 bacterial species. The four most common species accounted for 90% of bacterial isolates: Enterococcus fecalis (E. fecalis) (56%), E. coli (16%), group B Streptococcus (GBS) (13%), and Staphylococcus aureus (S. aureus) (5%; Fig. 2). Four other bacteria accounted for at least 1% of all bacterial isolates: Klebsiella pneumoniae (2.2%), Proteus mirabilis (1.7%), Citrobacter koseri (1.5%), and Morganella morganii (1.3%). There were no statistically significant differences in sperm concentration, motility, morphology, or DFI in the semen containing E. fecalis, E. coli, GBS, or S. aureus compared with nonbacteriospermic samples. The rate of ESL was 19% (1,498/7,852), and there was no statistical correlation between bacteriospermia and ESL (18.2% ESL associated with bacteriospermia vs. 19.2% ESL not associated with bacteriospermia; P¼.383). Additionally, there was no threshold PMN/100 sperm value that further predicted the presence of bacteriospermia, indicating that there was no correlation between increasing PMN/100 sperm values and the presence of bacteriospermia (r ¼ 0.010). For example, patients with 1 PMN/100 sperm had a bacteriospermia rate of 81/566 (14.3%) versus patients with >10 PMN/100 sperm, who had a bacteriospermia rate of 36/274 (13.1%). To determine the individual effect that bacteriospermia and/or ESL had on semen parameters, the cohort was divided into four groups: bacteriospermia and ESL (218, 2.8%), ESL without bacteriospermia (1,290, 16.3%), bacteriospermia without ESL (982, 12.5%), and neither bacteriospermia nor ESL (5,372, 68.4%). The comparison of semen parameters (sperm concentration, motility, and morphology) and DFI among the four groups is summarized in Table 1 and Figure 3A D. There is a statistically significant deterioration in sperm concentration, motility, and morphology in the two groups containing ESL, with no additional deterioration with bacteriospermia, compared with the two groups not containing ESL (P<.001). There is no statistically significant difference in sperm concentration, motility, and morphology between the group with bacteriospermia without ESL versus the group with neither bacteriospermia nor ESL. The group with bacteriospermia without ESL had a statistically significant deterioration in DFI compared with the group with neither bacteriospermia nor ESL (24.1 vs. 21.8%; P¼.040), however, there was no statistically significant difference with the addition of bacteriospermia in the groups with ESL (28.1 vs. 26.5%; P¼nonsignificant). DISCUSSION The role of bacteriospermia, especially asymptomatic bacteriospermia, in subfertile men remains largely undefined and TABLE 1 Comparison of semen parameters among the four patient groups. Sperm Patient group No. (%) concentration /ml Motility, % Normal morphology, % Bacteriospermia and ESL 218 (2.8) (n ¼ 35) ESL without bacteriospermia 1,280 (16.3) (n ¼ 238) Bacteriospermia without ESL 982 (12.5) (n ¼ 233) Neither bacteriospermia or ESL 5,372 (68.4) (n ¼ 1,300) DFI 1052 VOL. 97 NO. 5 / MAY 2012

4 Fertility and Sterility FIGURE 3 Comparison of the mean sperm concentration (A), sperm motility (B), sperm morphology (C), and DFI (D) among the four groups. Error bars represent the standard error of measure. There is a statistically significant deterioration in sperm concentration, motility, and morphology in the two groups containing ESL, with no additional deterioration with bacteriospermia, compared with the two groups not containing ESL (P<.001). There is no statistically significant difference in sperm concentration, motility, and normal morphology between the group with bacteriospermia without ESL versus the group with neither bacteriospermia nor ESL. The group with bacteriospermia without ESL had a statistically significant deterioration in DFI compared with the group with neither bacteriospermia nor ESL (P¼.040). controversial. The challenge when evaluating the bacteriospermia literature is that there are inconsistent definitions and sample collection procedures for culturing semen, probable contamination from urethral, meatal, and skin organisms; there is difficulty culturing all potential pathogens; and lastly, it is difficult to know which organisms significantly impact male fertility potential (3). Cottell et al. addressed the issue of seminal fluid contamination and determined that 37% of first-catch urine samples, 27% of midstream urine samples, and 51% of semen samples in their 60 subfertile men contained bacteria, mainly gram-positive commensal organisms (30). There was no correlation between bacteriospermia and leukocytospermia, and the authors concluded that the majority of bacteria cultured in the semen represented urethral or skin contamination. Additionally, changes in semen detection techniques can greatly increase the yield of bacteriospermia, especially with the use of polymerase chain reaction assays to detect bacterial DNA (31). These inconsistencies explain the widely reported incidence of bacteriospermia in the literature, which is reported in 10% 85% of male factor infertility patients (3, 32). This study s overall rate of bacteriospermia of 15% is similar (33) and somewhat lower than that in recently published studies (34 36), however, the relative proportion of culturable organisms is similar. In a study by Balmelli et al. (37), which is the second largest study of bacteriospermia in the literature, with 3,196 infertile males, the Enterococcipositive rate of 6.1% and E. coli-positive rate of 1.7% are very similar to those in this study, where the rates are 8.6% and 2.5% of the total study population, respectively. Sperm concentration, motility, and morphology were not significantly affected in this study s bacteriospermic samples, which has been reported in many other studies investigating bacteriospermia in subfertile patients (38 40). Although a study by Rodin et al. demonstrated that samples with Streptococcus viridans and E. fecalis (but not other bacterial species) negatively affected semen quality (34), the current study did not associate any specific bacterial species (specifically E. fecalis and E. coli) with deterioration in sperm count or quality. Although classic semen parameters were not affected, bacteriospermia was independently associated with an increase in DFI. A recent study has also identified bacteriospermia as a cause of increased DFI and identified improvements in DFI after antibiotic treatment (41). The implication of this finding is that patients with elevated DFI should undergo semen culture and antibiotic treatment of positive cultures, even if they are asymptomatic. VOL. 97 NO. 5 / MAY

5 ORIGINAL ARTICLE: ANDROLOGY Along with bacteriospermia, the incidence and effect of ESL was evaluated. Approximately 19% of samples had ESL, which was not statistically associated with bacteriospermia. Multiple previous reports have also demonstrated that leukocytospermia has little diagnostic value in the detection of bacteriospermia (42, 43). In this study, ESL was independently associated with a statistically significant deterioration in sperm concentration, motility, morphology, and DFI. The addition of bacteriospermia to ESL was not synergistic and did not further affect semen quality. Yanushpolsky et al. in their study demonstrated very similar findings in their patients with ESL, including a dose-response relationship where increasing concentrations of leukocytes in the semen further deteriorated semen parameters (44). Additionally, Thomas et al. associated leukocytospermia with decreased sperm motility (45), and Moskovtsev et al. demonstrated higher DFI in leukocytospermic samples (46). As a proof of principle, two studies treating leukocytospermic patients with anti-inflammatory (47) and antioxidant (48) medications demonstrated improvement in the patient s semen parameters after treatment. Although this is the largest study evaluating bacteriospermia, ESL, and semen parameters, it does have limitations. We did not use the standard WHO definition for leukocytospermia ( leukocytes/ml) and instead used a modified definition for ESL (R1 PMN/100 sperm). Although this is an unconventional definition, this threshold did demonstrate a significant deterioration in semen quality and DFI in our patients. The R1 PMN/100 sperm definition for ESL has been shown in a previous study by Berger et al. to be associated with decreased sperm function (28). Multiple other authors have questioned the WHO definition for leukocytospermia, which is an arbitrary cutoff based on expert opinion and has not been sufficiently validated in well designed studies. Menkveld and Kruger determined that a revised definition of leukocytospermia of > leukocytes/ml demonstrated the optimal sensitivity/specificity when predicting abnormal sperm morphology secondary to leukocytospermia (49). Similarly, Punab and colleagues determined that a cutoff of leukocytes/ml had the optimal sensitivity/specificity to predict bacteriospermia (50). In addition, Shy et al. determined that R6 PMN/100 sperm was associated with decreased pregnancy rates in couples with male factor infertility (51). These studies and others question the standard WHO definition. Second, we did not routinely measure DFI in all patients, and therefore we can make comments only on the patients who had DFI measured, which introduces bias to these results. Third, the seminal culture results may include contaminants, and this may explain why we did not demonstrate any deterioration in the classic semen parameters in bacteriospermic samples. Although the standard bacteriospermia definition was used, there is no way to determine whether bacteriospermic samples were merely contaminants or asymptomatic infections. Fourth, specific clinical characteristics of the population are not available to assess whether patients were symptomatic, had risk factors for ESL or bacteriospermia, or underwent treatment for the same. Lastly, given that this study was conducted in subfertile men, it is possible that the association between ESL and diminished semen parameters may be the effect and not the cause of abnormal spermatogenesis. It has been documented that abnormal spermatogenesis and poor sperm viability have been associated with enhanced seminal leukocyte recruitment (11). If this study had had a fertile control group, it might have helped elucidate this association better. In summary, this is the largest study to evaluate bacteriospermia, ESL, and semen parameters in nonazoospermic subfertile men. Bacteriospermia and ESL were commonly diagnosed during the evaluation of these men, however, these conditions were not statistically associated. Bacteriospermia was independently associated with a statistically significant increase in DFI, while ESL was associated with a more profound deterioration in sperm concentration, motility, morphology, and DFI. These findings question the routine treatment of asymptomatic bacteriospermia patients with antibiotics, however, a properly designed randomized controlled trial is needed to definitively answer this important question. Acknowledgments: The authors thank Mr. David Nguyen for his technical assistance regarding database management and Ms. Susan Lau for her assistance with manuscript preparation. REFERENCES 1. 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