title: Inter- and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation.
abstract: One of the challenges with the sperm DNA fragmentation results is the inconsistency and the large variability in the results obtained by different techniques. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay quantifies the incorporation of fluoresceinated dUTP into single- and double-strand DNA breaks by labeling the 3'-OH terminal with TdT. The goal of this study was optimize the TUNEL protocol for assessment of sperm DNA fragmentation by standardization of the method and comparison of the data across two reference laboratories (i) at Basel, Switzerland and (ii) Cleveland Clinic, Ohio, USA. Semen samples from 31 subjects grouped into three cohorts. Sperm DNA fragmentation was data measured by two experienced operators at two different laboratories using identical semen samples, assay kit, protocol and acquisition settings using identical flow cytometers (BD Accuri C6). No significant differences were observed between the duplicates in any of the experiments performed. By including an additional washing step after fixation in paraformaldehyde, a high correlation was seen between the two laboratories (r = 0.94). A strong positive correlation was observed between the average sperm DNA fragmentation rates (r = 0.719). The mean sperm DNA fragmentation measured in each laboratory was similar. Both flow cytometers were identical in their settings and performance. This inter- and intra-laboratory study establishes that TUNEL is a reproducible assay when utilizing a standardized staining protocol and flow cytometer acquisition settings. Standardization and consensual guidelines for TUNEL validate the assay and establishes TUNEL as a robust test for measuring sperm DNA fragmentation especially in a multicenter setting.
authors.affiliation: Clinic of Gynecological Endocrinology and Reproductive Medicine, University Hospital, University of Basel, Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Ribeiro
authors.affiliation: American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA.
authors.forename: R
authors.identifier: 0000-0002-9664-6978
authors.initials: R
authors.lastname: Sharma
authors.affiliation: American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gupta
authors.affiliation: American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Cakar
authors.affiliation: Clinic of Gynecological Endocrinology and Reproductive Medicine, University Hospital, University of Basel, Basel, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: De Geyter
authors.affiliation: American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA.
authors.forename: A
authors.identifier: 0000-0003-0585-1026
authors.initials: A
authors.lastname: Agarwal
mesh_terms: D053938:DNA Fragmentation; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000592:standards*; D007248:Infertility, Male / Q000175:diagnosis / Q000235:genetics; D008297:Male; D015203:Reproducibility of Results; D055101:Semen Analysis / Q000592:standards*; D013094:Spermatozoa / Q000473:pathology*; D013557:Switzerland
keywords: 
  TUNEL
  ; DNA fragmentation; apoptosis kit; flow cytometer; spermatozoa