Rakesh Sharma1, Gulfam Ahmad2,3, Sandro C Esteves4, Ashok Agarwal5. 1. American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, 44195, USA. sharmar@ccf.org. 2. American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, 44195, USA. gulfam.uh@gmail.com. 3. Department of Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan. gulfam.uh@gmail.com. 4. ANDROFERT, Andrology and Human Reproduction Clinic, Campinas, SP, Brazil. s.esteves@androfert.com.br. 5. American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, 44195, USA. agarwaa@ccf.org.
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.
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.
Entities:
Keywords:
Male infertility; Sensitivity; Specificity; Sperm DNA fragmentation; TUNEL assay
Authors: Sheena E M Lewis; R John Aitken; Sarah J Conner; Geoffry De Iuliis; Donald P Evenson; Ralph Henkel; Aleksander Giwercman; Parviz Gharagozloo Journal: Reprod Biomed Online Date: 2013-07-11 Impact factor: 3.828
Authors: U Punjabi; H Van Mulders; I Goovaerts; K Peeters; E Roelant; D De Neubourg Journal: J Assist Reprod Genet Date: 2019-05-14 Impact factor: 3.412
Authors: Sandro C Esteves; Armand Zini; Robert Matthew Coward; Donald P Evenson; Jaime Gosálvez; Sheena E M Lewis; Rakesh Sharma; Peter Humaidan Journal: Andrologia Date: 2020-10-27 Impact factor: 2.775