title: Autoantibodies against protein disulfide isomerase ER-60 are a diagnostic marker for low-grade testicular inflammation.
abstract: OBJECTIVE
  Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions?
  
  
  CONCLUSIONS
  In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found.
  
  
  BACKGROUND
  Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis.
  
  
  METHODS
  This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (n = 20); male blood donors (n = 14); men with impaired semen quality without (n = 14) or with (n = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (n = 16); men after pharmacotherapy of genital tract infection/inflammation (n = 15) and men with acute epididymo-orchitis (n = 30).
  
  
  METHODS
  Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed.
  
  
  RESULTS
  The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; P < 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; P < 0.05) as compared with healthy normozoospermic men (median 2.13; P < 0.001), male blood donors with unknown fertility status (median 2.72; P < 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; P < 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; P < 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; P < 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%.
  
  
  CONCLUSIONS
  The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level.
  
  
  CONCLUSIONS
  Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances.
  
  
  BACKGROUND
  This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen-WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group 'MIBIE' (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University of Giessen, Aulweg 123, 35392 Giessen, Germany monika.fijak@anatomie.med.uni-giessen.de.
authors.forename: Monika
authors.initials: M
authors.lastname: Fijak
authors.affiliation: DRG Instruments GmbH-R&D Department, 35037 Marburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Zeller
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University of Giessen, Aulweg 123, 35392 Giessen, Germany Clinic for Urology, Pediatric Urology and Andrology, Justus-Liebig-University Giessen, 35385 Giessen, Germany.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Huys
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University of Giessen, Aulweg 123, 35392 Giessen, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Klug
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University of Giessen, Aulweg 123, 35392 Giessen, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Wahle
authors.affiliation: Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, 35385 Giessen, Germany.
authors.forename: Monica
authors.initials: M
authors.lastname: Linder
authors.affiliation: Department of Dermatology, Andrology Unit, University of Bonn, 53127 Bonn, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Haidl
authors.affiliation: Department of Dermatology, Andrology Unit, University of Bonn, 53127 Bonn, Germany.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Allam
authors.affiliation: Clinic for Urology, Pediatric Urology and Andrology, Justus-Liebig-University Giessen, 35385 Giessen, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Pilatz
authors.affiliation: Clinic for Urology, Pediatric Urology and Andrology, Justus-Liebig-University Giessen, 35385 Giessen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Weidner
authors.affiliation: Clinic for Urology, Pediatric Urology and Andrology, Justus-Liebig-University Giessen, 35385 Giessen, Germany.
authors.forename: Hans-Christian
authors.initials: HC
authors.lastname: Schuppe
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University of Giessen, Aulweg 123, 35392 Giessen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Meinhardt
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001323:Autoantibodies / Q000032:analysis*; D053713:Azoospermia / Q000175:diagnosis / Q000276:immunology / Q000473:pathology; D015415:Biomarkers / Q000032:analysis; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D007249:Inflammation / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D008297:Male; D008875:Middle Aged; D019704:Protein Disulfide-Isomerases / Q000276:immunology*; D055101:Semen Analysis; D013737:Testis / Q000473:pathology*; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D015415:Biomarkers; D019704:Protein Disulfide-Isomerases
keywords: ER-60; autoantibodies; immunological infertility; protein disulfide isomerase family A, member 3; testicular inflammation