Male accessory gland infection and subfertility: a diagnostic challenge - Chapter 5: Cytokine concentrations IL-6, IL-8, TNF-alfa and IFN-gamma) in seminal fluid of infertile

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Male accessory gland infection and subfertility: a diagnostic challenge

Trum, J.W.

Publication date

1999

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Citation for published version (APA):

Trum, J. W. (1999). Male accessory gland infection and subfertility: a diagnostic challenge.

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Y Pannekoek, J W Trum, OP Bleker, F van der Veen, L Spanjaard and J. Dankert

Abstract

The inflammatory response to the presence of Ureaplasma urealyticum or Mycoplasma

hominis in the lower genital tract of infertile men was investigated by measuring the

con-centrations of interleukin-6 (IL-6), inter- leukine-8 (IL-8), tumor necrosis factor alpha (TNFalfa) and interferon-gamma (IFNgamma) in seminal plasma of 30 infected patients and compared with 23 control samples obtained from non-infected patients. By using enzyme-linked immunosorbent assays it was found that IL-8 was present in relative high concentrations (0.12-4.8 ng ml-1) in all semen samples investigated. In contrast, the other cytokines were only detectable in 72% (IFNg), 44% (IL-6) and 19% (TNFalfa) of the samples and were found in relative lower concentrations (1-410 pg ml-1).

No statistically differences in the cytokine concentrations were found between samples originating from the infected versus the control group. Our data strongly indicate that the presence of U. urealyticum or M. hominis is not accompanied by an inflammatory response as indicated by elevated levels of the cytokines investigated. The measurements of these cytokines in seminal fluid is not helpful for the identification of U. urealyticum or M.

Introduction

Mycoplasmas are a unique group of bacteria, characterized by their small cell size (0.3 to 0.8 um), lack of a bacterial cell wall and a small genome size (Razin). Among the 16 spe-cies that have been isolated from human beings, Mycoplasma hominis and Ureaplasma

urea-lyticum are frequently cultured from the genital tract with reported colonization values of

between 5 to 30% for M. hominis and 30 to 85% for U. urealyticum (Samra, Soffer, Moskowitz ,Trum).

The presence of these genital mycoplasmas in a large proportion of healthy men and women complicates the assessment of the pathogenic roles of these organisms, but several studies have indicated that vaginal colonization with genital mycoplasmas can be associa-ted with an increased risk of developing certain pathogenic conditions and pregnancy abnormalities, e.g., bacterial vaginosis, pelvic inflammatory disease, postpartum septicemia, premature rupture of membranes and preterm labor and birth (Hill, Hoist, Krohn,

McDonald, Taylor-Robinson).

The pathogenic role of M hominis and U. urealyticum in males is less clear. It has been proposed that M. hominis, as an important component of the bacterial vaginosis flora, could have a role in non-gonococcal urethritis (NGU), but on its own it seems to behave as an commensal in males (Taylor-Robinson). U. urealyticum has been isolated from men with primary and recurrent N G U but repeated infections result in colonization without subsequent disease (Taylor Robinson lecture). This might explain the frequent isolation of mycoplasmas from the urethra of men without any genito-urinary complaints.

(Moskowitz, Trum).

The role of genital mycoplasmas in male infertility is also still a matter of debate. Although some studies have shown that U. urealyticum may directly adversely affect sperm function

(Busoio, Upadhyaya, Weidner), antibiotic treatment trials of infected infertile males have reported controversial results concerning improved conception rates and sperm parameters (Bar-Chama). In addition, the presence of high numbers ( > 1 x 106 ml-1) of leukocytes

in semen (leukocytospermia) does not seem to be related to the isolation of these bacteria, suggesting colonization rather than active infection with inflammation (Trum).

Inflammation mediators, among which interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor alpha (TNFalfa), interferon-gamma (IFNgamma) do not only mediate the host immune response but are also involved in several aspects of reproductive physiology and fertility regulation, including gonodal and sperm functions (Anderson, Ben Rafael, Estrada, Eiserman). The inflammatory response as accompanied by urogenital infection with pathogenic bacteria may lead to a misbalance between various seminal cytokines that can result in immunologic infertility (Hill, Hill, Wang). Whether the silent presence of

U. urealyticum or M. hominis in the male lower genital tract of infertile patients, in the

absence of any other bacterial pathogens is characterized by fluctuating seminal cytokine concentrations is unknown.

The purposes of this study were firstly to evaluate the inflammatory response to the pre-sence of U. urealyticum and M. hominis in the lower genital tract in order to investigate the possible pathogenic potential of these microorganisms in a male infertility population and, secondly, to evaluate the value of measuring these inflammation mediators for the diagnosis of male accessory gland infection. We therefore determined the levels of IL-6, IL-8, TNFalfa and IFNgamma, cytokines most widely studied for routine diagnosis of male infertility, in seminal fluid of mycoplasma infected and non-infected infertile males.

Materials and Methods

Patients and baC. trachomatiserial culture

The studied population consisted of 53 men that presented to the Center for Reproductive Medicine at our hospital and who were selected from a larger group (n =

184) that participated in a study evaluating sexually transmitted disease as a cause of male infertility (Trum). Permission to conduct this study was obtained from our hospital's Institutional Review Board and all patients gave their consent to participate.

All men underwent digital prostatic massage after which two cotton urethral swabs were taken to investigate the presence of bacteria in accordance with standard methods. An urethral smear was gram stained and examined for the presence of Neisseria

gonorrhoeae. One cotton swab was placed in a Stuart medium and used for bacterial

cultu-re. Culture for A^ gonorrhoeae was done using a modified Thayer-Martin plate and culture of U. urealyticum and M. hominis was done using Shepard broth and a Shepard plate. The presence of C. trachomatis was investigated with a DNA hybridization assay (Genprobe Inc, San Diego, CA). A positive bacterial culture or chemiluminescence signal was conside-red to indicate bacterial infection. According to culture results, 53 men were selected from which 23 were culture negative, and 30 were infected with either U. urealyticum or M.

hominis.

Cytokine measurements

Semen was collected by masturbation after at least 72 hours of abstinence, liquefied and centrifuged at 1,000 x g for 10 min to remove cells and debris. So obtained seminal plas-ma was aspirated and stored at -80°C until cytokine assays. Cytokine levels were measured using commercial enzyme-linked immunosorbent assays (ELISAs), according to the instructions of the manufacturer (CLB, Amsterdam, The Netherlands). In brief, semen

samples were added to the wells of microtiterplates precoated with anti-cytokine monoclo-nal antibodies. After incubation at room temperature for 1 h, the unbound components from the samples were removed by washing. Second anti-human cytokines antibodies were added and incubated for 1 h at room temperature. After washing of the wells a third anti-body conjugated with peroxidase was added and incubated for 30 min at room temperatu-re. Finally, the reaction was developed after adding the substrate and stop solution and measured with an ELISA reader at 450 nm. Samples were assayed twice in duplicate and individual levels are expressed as means, group levels as medians and ranges.

Statistical analysis

Differences in the levels of IL-6, IL-8, TNFalfa and IFNgamma between patients with-and without a positive culture for mycoplasmas, were assessed using the non-parametric test (Mann Whitney U-test, Wilcoxon Rank Sum W test). Values of P<0.05 were conside-red significant.

Results

Prevalence and levels of IL-6, IL-8, TNFalfa and IFNgamma in seminal plasma from mycoplasma infected and non-infected patients. None of the patients indicated genito-urinary complaints.

A total of 53 semen samples were available for cytokine analysis. These samples were deri-ved from 53 patients from which 24 were infected with U. urealyticum and 6 with M.

hominis. The remaining 23 samples were obtained from patients who tested culture

negati-ve for all bacteria cultured and sernegati-ved as non infected controls

Analysis by enzyme-linked immunosorbent assays for cytokines indicated that IL-8 was present in all samples tested (n=53). In contrast, the other assessed cytokines were not detectable

in all samples. IFNgamma was found in 38 out of 53 (72%) samples, IL-6 in 24 out 53 (44%) andTNFalfa in only 10 out of 53 (19%) samples (Figure 1). IL-8 was found to be present in relative high concentrations, ranging from 0.12 to 4.8 ng ml-1.

5 10 0: 10 - 0 = 1

3

1

The other investigated cytokines were found in relative lower levels 1 to 410 pg ml-1 , (Table 1). More importantly, as clearly illustrated in Figure 1, a wide range of overlapping cytokine concentrations was found between the infected and control group, suggesting that the presence and the concentrations of the cytokines investigated do not seem to be related to the presence of mycoplasmas in the genital tract. Indeed, statistically analysis of the data indicated that the levels of IL-6, IL-8, TNFalfa and IFNgamma were not signifi-cantly different in seminal plasma of men with a positive culture for U. urealyticum or M.

hominis and of men who tested negative for the presence of these micro organisms in the

Table I Median and ranges of the seminal cytokine concentrations of mycoplasma infected And non-infected subfertile men

IL-6 (pg/ ml) IL-8 (ng/ml) TNFalfa (pg/ml) IFNgamma (pg/ml) Infected group (n=30) Median Range 0 0-103 1.05 0.16-4.7 0 0-29 5.5 0-280 Control group (n=23) Median Range 0 0-410 1.52 0.12-3.6 0 0-76 5.5 0-100 Discussion

Cell-mediated immunity is a highly complex immunological phenomenon involving numerous subsets of leukocytes and multiple phases of responses that are mediated by cytokines. There is considerable evidence that cytokines play a role in infertility (Anderson, Ben-Rafael), and numerous studies clearly indicate that mycoplasmas are able to modulate the activities of monocytes/macrophages and natural killer cells, thus trigger the producti-on of a wide variety of up and down regulating cytokines. (Razin)

The aim of the present study was firstly to investigate whether a silent genital tract infecti-on with U. urealyticum or M. hominis infectiinfecti-on is accompanied by elevated levels of cytok-ines that are likely to be involved in inflammation and infertility. Our data show that indi-viduals display a considerable heterogeneity in the various cytokine levels measured but that there are no statistically differences in these levels between mycoplasma infected and

inflammatory response.

Elevated concentrations of cytokines, among which IL-6 and IL-8 have been measured in seminal fluids of men and related to bacterial infections (Comhaire, Depuydt,

Koumantakis, Matalliotakis). In contrast, other investigators found no differences in these cytokine concentrations and TNFalfa between infected and non-infected men (Dousset, Huleihel, Hussenet). Comparison of these data is however hampered because different patient populations were studied and, in neither of these studies the relative contribution of the different bacterial species to cytokine concentration was analyzed. Since we only included patients with either a U. urealyticum or a M. hominis infection and found no marked differences in the cytokine levels analyzed, this is first direct demonstration that the presence of only U. urealyticum or M. hominis does not seem to be accompanied with elevated levels of the cytokines studied. The absence of any inflammatory indications on the cytokine level, and the complete absence of any genito-urinary complaints strongly point to the view to that the presence of these bacteria in the genital tract of this particular group of patients reflects a colonization.

The male reproductive tract is an immunologically dynamic tissue capable of initiating an immune response from which the magnitude, especially in the case of unexplained inferti-lity, can be attributed to a heterogeneous group of unknown etiologies (Anderson). Taken this in consideration, this might explain why we observed such a wide range of cytokine levels, apparently independent of the microbiological status of the genital tract. In addi-tion, it should be taken in account that there are major differences in the capacity of vari-ous mycoplasma species and strains to induce and/or suppress the production of cytokines. This may have a major impact on the resulting virulence of infecting mycoplasmas and on disease manifestation in hosts. Typing of the strains might provide additional information

in order to investigate whether certain strains are typically found in infertile patients wit-hout any genito-urinary complaints and are associated with high or low levels of certain cytokines that not only facilitate bacterial persistence, but also might lead to immunologi-cally infertility.

Based on our findings we conclude that the measurement of IL-6, IL-8, TNFa and IFNg cytokines appears not to be relevant in order to select infertile patients infected with U.

urealyticum or M. hominis. Still, detection is of importance since these bacteria are sexually

transmitted and may cause severe damage in the female genital tract (Taylor-Robinson). In order to elucidate whether mycoplasmas are indeed involved in the pathogenesis of male infertility future studies should aim on more detailed characterization of the strains invol-ved.

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