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. 2016 Feb;33(2):291-300.
doi: 10.1007/s10815-015-0635-7. Epub 2016 Jan 16.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using bench top flow cytometer for evaluation of sperm DNA fragmentation in fertility laboratories: protocol, reference values, and quality control

Rakesh Sharma  1 , Gulfam Ahmad  2   3 , Sandro C Esteves  4 , Ashok Agarwal  5
Affiliations

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using bench top flow cytometer for evaluation of sperm DNA fragmentation in fertility laboratories: protocol, reference values, and quality control

Rakesh Sharma et al. J Assist Reprod Genet. 2016 Feb.

Abstract

Purpose: The purpose of this study is to provide a detailed protocol and quality control steps for measuring sperm DNA fragmentation (SDF) by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay using a new bench top flow cytometer, determine the reference value of SDF, and assess sensitivity, specificity, and distribution of SDF in infertile men and controls with proven and unproven fertility.

Methods: Semen specimens from 95 controls and 261 infertile men referred to a male infertility testing laboratory were tested for SDF by TUNEL assay using Apo-Direct kit and a bench top flow cytometer. Percentage of cells positive for TUNEL was calculated. Inter- and intraobserver variability was examined. TUNEL cutoff value, sensitivity, specificity, and distribution of different cutoff values in controls and infertile patients were calculated.

Results: The reference value of SDF by TUNEL assay was 16.8 % with a specificity of 91.6 % and sensitivity of 32.6 %. The positive and negative predictive values were 91.4 and 33.1 %, respectively. The upper limit of DNA damage in infertile men was significantly higher (68.9 %) than that in the controls (19.6 %).

Conclusions: TUNEL assay using flow cytometry is a reproducible and easy method to determine SDF. At a cutoff point of 16.8 %, the test showed high specificity and positive predictive value. The results of this test could identify infertile men whose sperm DNA fragmentation does not contribute to their infertility and confirm that a man who tests positive is likely to be infertile due to elevated sperm DNA fragmentation.

Keywords: Male infertility; Sensitivity; Specificity; Sperm DNA fragmentation; TUNEL assay.

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Figures

Fig. 1
Fig. 1
Schematic of the DNA staining procedure using the TUNEL assay
Fig. 2
Fig. 2
Set up of the BD Accuri C6 bench top flow cytometer
Fig. 3
Fig. 3
a Receiver operator characteristic (ROC) curve showing TUNEL assay cutoff and the area under the curve. Values within the parentheses represent the 95 % confidence interval. b Distribution of TUNEL test values between controls and infertile men
Fig. 4
Fig. 4
Panel showing interobserver variability for a absolute, b percent difference, and intraobserver variability for c absolute d percent difference between individual measurements and final values
See this image and copyright information in PMC

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