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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

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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.

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Acknowledgment

The study was supported by funds from the American Center for Reproductive Medicine. Dr. Gulfam Ahmad was supported by the Fulbright fellowship. The authors are grateful to the Andrology Center technologists for scheduling the study subjects and to Jeff Hammel, Biostatistician, for his contribution to the data analysis.

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Authors and Affiliations

  1. American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, 44195, USA

    Rakesh Sharma, Gulfam Ahmad & Ashok Agarwal

  2. Department of Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan

    Gulfam Ahmad

  3. ANDROFERT, Andrology and Human Reproduction Clinic, Campinas, SP, Brazil

    Sandro C. Esteves

Authors
  1. Rakesh Sharma
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  2. Gulfam Ahmad
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  3. Sandro C. Esteves
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  4. Ashok Agarwal
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Corresponding author

Correspondence to Ashok Agarwal.

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The study was approved by the Institutional Review Board (IRB) of Cleveland Clinic

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Measurement of sperm DNA fragmentation by bench top flow cytometer.

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Sharma, R., Ahmad, G., Esteves, S.C. et al. 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. J Assist Reprod Genet 33, 291–300 (2016). https://doi.org/10.1007/s10815-015-0635-7

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  • DOI: https://doi.org/10.1007/s10815-015-0635-7

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