title: A novel cell-processing method 'AgarCytos' in conjunction with OCT3/4 and PLAP to detect intratubular germ cell neoplasia in non-obstructive azoospermia using remnants of testicular sperm extraction specimens.
abstract: OBJECTIVE
  Can we diagnose intratubular germ cell neoplasia (IGCN) using the immunohistochemical markers placental-like alkaline phosphatase (PLAP) and OCT3/4 using a novel cell-processing method 'AgarCytos', applied to the remnants of testicular sperm extraction (TESE) specimens and what is the prevalence of a testicular germ cell (pre)malignancy in men with a non-obstructive azoospermia (NOA) undergoing TESE for fertility treatment?
  
  
  CONCLUSIONS
  IGCN can be successfully detected by immunohistochemical evaluation of AgarCytos, made of the remnants of TESE biopsies. The observed prevalence of a germ cell (pre)malignancy in this specific population was found to be 4.4%.
  
  
  BACKGROUND
  Infertile men are at higher risk for testicular cancer than the general population. IGCN can be detected by immunohistochemistry using PLAP and OCT3/4 in standard testicular biopsies and, with less accuracy, in semen.
  
  
  METHODS
  Between January 2011 and April 2012 a prospective cohort study was conducted at a Dutch tertiary care academic training hospital. All males with NOA (n = 182) undergoing a urological work-up followed by a diagnostic TESE for fertility treatment (n = 251) were included.
  
  
  METHODS
  After cryopreservation of sperm, if present, an AgarCyto was made of the remnants of the TESE biopsies. Sections were stained with haematoxylin-eosin for pathological examination as well as PLAP and OCT3/4 for immunohistochemistry to detect IGCN.
  
  
  RESULTS
  Eight men (4.4%) were diagnosed with a germ cell (pre)malignancy: six of them had seminoma, two without and four with concomitant IGCN, and two of them had IGCN only. Microscopic evaluation including immunohistochemical analysis of the AgarCytos diagnosed three (1.6%) more cases of a germ cell (pre)malignancy compared with scrotal ultrasound alone (one case of bilateral seminoma with concomitant IGCN and two cases of IGCN alone). No false-positive cytology results were found upon conventional histological evaluation.
  
  
  CONCLUSIONS
  The main limitation of this study is lack of a simultaneously taken standard testicular biopsy, to compare the results of our novel diagnostic method with. Nevertheless, in all but one of our cases orchidectomy followed and the diagnosis was confirmed by histology. In the remaining case repeat TESE showed similar results.
  
  
  CONCLUSIONS
  Simultaneous screening for IGCN is highly recommended to men with NOA undergoing TESE, because of the increased incidence of germ cell (pre)malignancies in this specific population. The principal advantage of our new method is that all available testicular tissue can be used for both sperm recovery and pathological evaluation, increasing the yield of spermatozoa as well as the chance to find (pre)malignant cells. In those cases where the disease is still in a premalignant stage, early diagnosis will allow for timely treatment and reduction of morbidity and mortality in this group of patients.
  
  
  BACKGROUND
  This study was (partially) funded by Merck Serono (the Netherlands). There are no conflicting interests to disclose.
  
  
  BACKGROUND
  N/A.
authors.affiliation: Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, PO Box 9101, Nijmegen HB 6500, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Hessel
authors.forename: L
authors.initials: L
authors.lastname: Ramos
authors.forename: A F C
authors.initials: AF
authors.lastname: Hulsbergen
authors.forename: K W M
authors.initials: KW
authors.lastname: D'Hauwers
authors.forename: D D M
authors.initials: DD
authors.lastname: Braat
authors.forename: C A
authors.initials: CA
authors.lastname: Hulsbergen-van de Kaa
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism*; D053713:Azoospermia / Q000150:complications* / Q000473:pathology; D018929:Cell Culture Techniques; D015331:Cohort Studies; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism*; D016015:Logistic Models; D008297:Male; D009373:Neoplasms, Germ Cell and Embryonal / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D050814:Octamer Transcription Factor-3 / Q000378:metabolism*; D012671:Seminiferous Tubules / Q000378:metabolism / Q000473:pathology; D018239:Seminoma / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology; D053625:Sperm Retrieval
chemical_list: D007527:Isoenzymes; D050814:Octamer Transcription Factor-3; C055791:germ-cell AP isoenzyme; D000469:Alkaline Phosphatase
keywords: IGCN; OCT3/4; PLAP; TESE; screening