ORIGINAL ARTICLE

Male immunity to the chlamydial 60 kDa heat shock protein (HSP 60) – associated with semen quality? W. Eggert-Kruse, K. Batschulat, T. Demirakca & T. Strowitzki Department of Gynecological Endocrinology and Reproductive Medicine, Women‘s Hospital, University of Heidelberg, Heidelberg, Germany

Keywords Antisperm antibodies—Chlamydia trachomatis—sperm function—sperm/mucus interaction Correspondence Waltraud Eggert-Kruse, Department of Gynecological Endocrinology and Reproductive Medicine, Women’s Hospital, University of Heidelberg, Im Neuenheimer Feld 440, 69120 Heidelberg, Germany. Tel.: +49 6221 567910; Fax: +49 6221 564099; E-mail: Waltraud_Eggert-Kruse@med. uni-heidelberg.de Accepted: November 15, 2013 doi: 10.1111/and.12224

Summary The role of Chlamydia trachomatis for male infertility is a matter of constant debate. It is assumed that in its persistent form this pathogen may produce high levels of 60 kD heat shock protein (Chlam HSP60). Cross-reactivity between epitopes of the bacterial and human HSPs, involved in many steps of the reproductive process, might induce an autoimmune response with potential impairment of semen quality and sperm fertilising capacity. This prospective study included asymptomatic males of a total of 128 unselected subfertile couples (median duration of infertility 3 years) to determine the clinical relevance of male immunity to Chlam HSP60 during infertility investigation. After medical history and clinical examination of both partners, serum antibodies (Ab) to Chlam HSP60 were determined. Same day semen quality evaluation included microscopical standard sperm analysis, determination of seminal white blood cells (WBC) and of antisperm Ab (ASA) of the Ig G- and Ig-A class (mixed antiglobulin reaction, MAR), microbial screening and examination of sperm functional capacity. Sperm/mucus interaction was tested in vitro and in vivo. Simultaneously, patients′ female partners were tested for Chlam HSP60 Ab and results were compared with a standard serology evaluation for antichlamydial IgG Ab. The presence of ChlamHSP60 Ab (positive in 24% of males) was not significantly associated with semen quality, seminal WBC and antisperm AB of the IgG- or Ig A-class, the outcome of the microbial screening nor with sperm functional capacity and results of sperm/mucus interaction testing in vitro and in vivo. Chlam HSP60 Ab were significantly more frequent in female partners of Chlam HSP60 Ab-positive men, and results correlated with the outcome of standard chlamydial serology evaluation. In conclusion, when serum Chlam HSP60 Ab are used as marker, male immunity to the chlamydial 60 kD heat shock protein is not associated with semen quality, sperm functional capacity and other clinically relevant parameters of male fertility.

Introduction The ‘heat shock response’ is a vital cellular survival mechanism. As evolutionary highly conserved chaperones, heat shock proteins (HSP) are important mediators of cell division and apoptosis and key regulators of both the innate and the adaptive immune system (Calderwood et al., 2007; Van Eden et al., 2007; Cappello et al., 2009). Human HSPs also play a fundamental role during germ cell differentiation and development (Neuer et al., 1999). However, apart from their physiological properties, 66

these proteins may also exert pathogenic effects on reproductive processes, for example, by inducing a persistent inflammatory response, and HSP molecules may serve as antigenic targets for the immune system. Heat shock proteins, particularly members of the 60 kiloDalton (kD) HSP family (HSP60) have also been recognised as immunodominant antigens of many genital tract microorganisms with increased amount of bacterial HSP expression at sites of an infection. Humoral as well as cellular immune responses against microbial HSPs have been shown to cross-react with human HSPs (Van Eden et al., 2007). © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 66–76

W. Eggert-Kruse et al.

Chlamydia trachomatis is considered the most common sexually transmitted bacterial pathogen in industrial countries. Chlamydial (Chlam) HSP60 shares about 50% amino acid sequence homology with the human HSP60 (Shinnik, 1991; Morrison et al., 1992); as a consequence of molecular mimicry the development of an autoimmune response to the human HSP60 in susceptible individuals has been suggested (Yi et al., 1993; Domeika et al., 1998; Witkin, 2002). Cross-reactivity may result in autoimmune-mediated reproductive failure with, for example, in males, the induction of antisperm antibodies (ASA). The impact of C. trachomatis infection on female fertility has been established. However, the potential consequences for the fertility of the male are subject of constant debate (Paavonen & Eggert-Kruse, 1999; Hosseinzadeh et al., 2004; Idahl et al., 2004; Cunningham & Beagley, 2008; Al-Mously et al., 2009; Mazzoli et al., 2010; Motrich et al., 2012). Screening studies in asymptomatic subfertile couples have shown a low prevalence rate of C. trachomatis in genital secretions, swabs and/or urine, when amplification methods are used for identification of these pathogens (Fujisawa et al., 1999; Eggert-Kruse et al., 2003; Pannekoek et al., 2003; Rosemond et al., 2006). Therefore, serologic markers are used to detect previous or persistent, chronic infection with potential induction of an inflammatory response. Some relationship of chlamydial HSP60 Ab expression with adverse IVF treatment outcome and a tubal factor has been suggested in subfertile women (Dieterle & Wollenhaupt, 1996; Tiitinen et al., 2006; Sziller et al., 2008; Linhares & Witkin, 2010), but there is only scarce information concerning the role of Chlam HSP60 Ab for male infertility. Thus, the aim of this prospective study was to evaluate the clinical significance of immunity to ChlamHSP60 Ab as potential indicator of an adverse microbial stress response in asymptomatic males of subfertile couples with regard to semen quality, sperm function and other relevant determinants of fertilising capacity. Materials and methods

Male immunity to Chlam HSP 60

presented with primary infertility in 76.3% and with secondary infertility in 23.7%. All men, as well as their female partners, were without symptoms of genital tract infection. During the time of the study, none of the patients was treated with antibiotics, corticosteroids or antiphlogistics. The study was approved by the Institutional Review Board (Ethics Committee of the Medical Faculty, University of Heidelberg). Informed consent was obtained from all patients. Basic infertility investigation and andrological examination A detailed medical history was obtained, and physical examinations were performed on both partners. Investigation for female infertility factors (hormonal analyses in the early follicular and the luteal phase, examination of tubal patency and of the uterine and cervical factor) was carried out as described previously (Eggert-Kruse et al.,1997). Ejaculates were obtained in the hospital (in a room near the laboratory) (Division of Andrology, Department of Dermatology) after 5 days of sexual abstinence and were examined directly after liquefaction (at room temperature). Routine semen evaluation included: determination of ejaculate volume (calibrated Falcon tube), measurement of seminal pH by means of paper strips (pH-Indikatorpapier Merckâ, Darmstadt, Germany), evaluation of sperm concentration using a standard (Neubauer improved) haemocytometer and of sperm progressive motility (%) directly after liquefaction and additionally after 2 h (h) and after 4 h. Sperm vitality was examined using eosin testing (World Health Organisation, 1999). For sperm morphology evaluation, standard criteria (percentage (%) of pathological forms) (World Health Organisation, 1992), with further differentiation for anomalies of the sperm head, neck and tail, were used. All seminal parameters, which were analysed in the present investigation (apart from postcoital testing (PCT) results) were determined in aliquots of the same ejaculates.

Patients

Seminal white blood cells (WBC)

A total of 128 subfertile couples, who presented for their first investigation at our outpatient infertility clinic, were enrolled in this prospective study. Couples were unselected for infertility factors, but men with azoospermia had been excluded. The median duration of infertility was 3 (range 1–15) years. The median age of the male patients was 34 (range 24–57) years, the age of their female partners 32 (range 19–42) years. Couples

Round cells in semen were differentiated in leucocytes (LC) and cells of the germ cell line using an immunocytochemical method with monoclonal Ab and a streptavidin/biotin system described previously (Eggert-Kruse et al., 1992). The mean of triplicate counting was used for analysis. Human peripheral leucocytes, obtained from a healthy donor and separated with a Ficoll-technique, were used as positive controls. Positive and negative

© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 66–76

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Male immunity to Chlam HSP 60

(phosphate-buffered saline (PBS) pH 7.4) controls were included in all of the test series. Thresholds were set at 5%, 10%, 15% and 20% LC ratio. Determination of antisperm antibodies (ASA) For screening of antisperm Ab (ASA) in semen samples, the direct mixed antiglobulin reaction (MAR) was used. MAR was performed in parallel with IgG- and IgA-coated erythrocytes and specific antiserum (Jager et al., 1978). Reading was performed in triplicate, and the mean was taken. A percentage of ≥30% of motile spermatozoa involved in the mixed agglutinates was considered MAR positive (MAR ≥60% a strong positive outcome), based on previous findings (Eggert-Kruse et al., 1991). Data were also considered at additional cut-offs at MAR′s ≥10% and ≥50% (WHO, 2010). Microbial examination To screen semen samples for colonising microorganisms, swabs were obtained and inoculated into a universal transport medium (Port-a-Cul Universalâ, Becton Dickinson Heidelberg, Germany); additionally, 10 ll of semen was transferred with a disposable sterile plastic loop into Shepard′s medium for the detection of mycoplasmas. Microbial prevalence was identified with standard methods (Institute of Microbiology, University of Heidelberg). Microbial evaluation was also simultaneously performed in the lower genital tract of patients′ female partners (potentially pathogenic bacteria, mycoplasmas, yeasts, vaginal pH and amine testing). Evaluation of sperm functional capacity Sperm/cervical mucus penetration test (SCMPT) As parameter of sperm functional capacity, the ability to penetrate the cervical mucus (CM) barrier was evaluated by means of the standardised in vitro sperm/CM penetration test (SCMPT) described previously (Eggert-Kruse et al., 1989a). Briefly, the penetration of spermatozoa within capillaries, filled with fresh samples of CM, obtained from patients’ female partners under hormonally controlled conditions, was observed microscopically after 30-min, 2-h and 6-h incubation. Penetration density, migration distance, quality and duration of motility were examined, graded and summarised in a cumulative score, which served to select two groups with inadaequate (score

Male immunity to the chlamydial 60 kDa heat shock protein (HSP 60) - associated with semen quality?

The role of Chlamydia trachomatis for male infertility is a matter of constant debate. It is assumed that in its persistent form this pathogen may pro...
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