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Ashok Agarwal - One of the best experts on this subject based on the ideXlab platform.
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TUNEL Assay-Standardized method for testing sperm DNA fragmentation.
Andrologia, 2020Co-Authors: Rakesh Sharma, Ashok Agarwal, Concetta Iovine, Ralf HenkelAbstract:Sperm DNA integrity is important for normal functions such as fertilization, implantation, pregnancy and fetal development. Sperm DNA fragmentation (SDF) is more common in infertile men and may be responsible for poor reproductive function. Although there are a number of tests available to measure SDF, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling TUNEL) Assay using flow cytometry is becoming more popular to measure the sperm DNA fragmentation. It is a direct test that measures both single- and double- DNA strand breaks. In this review, we describe the protocol, quality control and measurement of sperm DNA fragmentation using a benchtop flow cytometer. We also briefly discuss the factors that can affect the results, challenges and clinical implications of TUNEL in assessing male infertility.
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TUNEL Assay: Establishing a sperm DNA fragmentation cut-off value for Egyptian infertile men.
Andrologia, 2019Co-Authors: Eman Mohamed Hassanen, Ralf Henkel, Khaled Mohamed Elqusi, Hosam Zaki, Ashok AgarwalAbstract:Male factor infertility is responsible for half of all infertility cases. Conventional semen analysis is inadequate to evaluate male fertility. Sperm DNA fragmentation (SDF) test can be done by: direct methods such as Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) and Comet Assay, or indirect like Sperm Chromatin Structure Assay (SCSA) and Sperm Chromatin Dispersion (SCD). TUNEL Assay measures both single- and double-strand breaks and is technically less demanding, while SCSA tests for the susceptibility for nuclear DNA denaturation and samples should be sent to the reference lab. Studies showed that a single cut-off value does not fit all. Therefore, this study aimed at establishing a cut-off value to discriminate between fertile and infertile Egyptian men. We enrolled 354 infertile men and 40 proven fertile volunteers.TUNEL Assay was performed using Apo-Direct kit and bench top flow cytometer.The calculated SDF cut-off value was 20.3% with a sensitivity of 96.6% and specificity of 87.5%, and the overall accuracy of the test was 95.7%. Sperm DNA fragmentation Test using TUNEL Assay is valuable tool for male infertility evaluation, and it assists in offering the best treatment options based on it's results.
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TUNEL Assay by Benchtop Flow Cytometer in Clinical Laboratories
A Clinician's Guide to Sperm DNA and Chromatin Damage, 2018Co-Authors: Rakesh Sharma, Zeynep Cakar, Ashok AgarwalAbstract:A high level of sperm DNA damage is associated with impaired infertility and increased risk of miscarriages. Evaluating semen samples utilizing the advanced sperm markers such as sperm DNA fragmentation (SDF) is important to identify its role in male infertility and the success of subsequent treatment outcome such as in ART. Sperm DNA fragmentation tests are increasingly being used to understand the molecular mechanism of DNA fragmentation and subsequent male infertility. Of these tests, sperm chromatin structure Assay or SCSA has been the most studied and is considered the gold standard. While other tests such as comet and sperm DNA dispersion test are also being increasingly used, their utility has not been widespread. In recent years, the TUNEL or the terminal deoxynucleotidyl transferase dUTP nick end labeling is gaining increasing recognition in the literature as the marker of sperm DNA fragmentation that provides the most relevant information for the clinicians and the patients alike. TUNEL test is fast becoming the Assay of choice. In this chapter, we provide details on the measurement of sperm DNA fragmentation using a simple bench flow cytometer.
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Inter-and Intra-Laboratory Standardization of TUNEL Assay for Assessment of Sperm DNA Fragmentation.
Current protocols in toxicology, 2017Co-Authors: Sajal Gupta, Rakesh Sharma, Ashok AgarwalAbstract:The functional aspects of sperm activity such as sperm chromatin integrity and ability to fertilize cannot be characterized by routine semen parameters. Men with unexplained infertility and idiopathic infertility, as well as men with normozoospermic semen profiles, show high DNA fragmentation. Molecular anomalies in the sperm can be detected by a sperm DNA fragmentation (SDF) Assay which can be used in adjunct to conventional semen analysis. While the sperm chromatin structure Assay (SCSA) remains the "gold standard," the TUNEL Assay using flow cytometry is becoming popular among the different tests that are currently available to measure sperm DNA fragmentation. In this unit, we describe the inter-laboratory and intra-laboratory standardization of the TUNEL Assay using a benchtop cytometer. The article also provides a step-by-step protocol for measuring sperm DNA fragmentation using the TUNEL Assay and a bench-top flow cytometer, and also points out the inherent challenges with this test. © 2017 by John Wiley & Sons, Inc.
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inter and intra laboratory standardization of TUNEL Assay for assessment of sperm dna fragmentation
Journal of Andrology, 2017Co-Authors: Sofia C Ribeiro, Rakesh Sharma, Sajal Gupta, Zeynep Cakar, C De Geyter, Ashok AgarwalAbstract:Summary 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.
Rakesh Sharma - One of the best experts on this subject based on the ideXlab platform.
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TUNEL Assay-Standardized method for testing sperm DNA fragmentation.
Andrologia, 2020Co-Authors: Rakesh Sharma, Ashok Agarwal, Concetta Iovine, Ralf HenkelAbstract:Sperm DNA integrity is important for normal functions such as fertilization, implantation, pregnancy and fetal development. Sperm DNA fragmentation (SDF) is more common in infertile men and may be responsible for poor reproductive function. Although there are a number of tests available to measure SDF, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling TUNEL) Assay using flow cytometry is becoming more popular to measure the sperm DNA fragmentation. It is a direct test that measures both single- and double- DNA strand breaks. In this review, we describe the protocol, quality control and measurement of sperm DNA fragmentation using a benchtop flow cytometer. We also briefly discuss the factors that can affect the results, challenges and clinical implications of TUNEL in assessing male infertility.
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TUNEL Assay by Benchtop Flow Cytometer in Clinical Laboratories
A Clinician's Guide to Sperm DNA and Chromatin Damage, 2018Co-Authors: Rakesh Sharma, Zeynep Cakar, Ashok AgarwalAbstract:A high level of sperm DNA damage is associated with impaired infertility and increased risk of miscarriages. Evaluating semen samples utilizing the advanced sperm markers such as sperm DNA fragmentation (SDF) is important to identify its role in male infertility and the success of subsequent treatment outcome such as in ART. Sperm DNA fragmentation tests are increasingly being used to understand the molecular mechanism of DNA fragmentation and subsequent male infertility. Of these tests, sperm chromatin structure Assay or SCSA has been the most studied and is considered the gold standard. While other tests such as comet and sperm DNA dispersion test are also being increasingly used, their utility has not been widespread. In recent years, the TUNEL or the terminal deoxynucleotidyl transferase dUTP nick end labeling is gaining increasing recognition in the literature as the marker of sperm DNA fragmentation that provides the most relevant information for the clinicians and the patients alike. TUNEL test is fast becoming the Assay of choice. In this chapter, we provide details on the measurement of sperm DNA fragmentation using a simple bench flow cytometer.
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Inter-and Intra-Laboratory Standardization of TUNEL Assay for Assessment of Sperm DNA Fragmentation.
Current protocols in toxicology, 2017Co-Authors: Sajal Gupta, Rakesh Sharma, Ashok AgarwalAbstract:The functional aspects of sperm activity such as sperm chromatin integrity and ability to fertilize cannot be characterized by routine semen parameters. Men with unexplained infertility and idiopathic infertility, as well as men with normozoospermic semen profiles, show high DNA fragmentation. Molecular anomalies in the sperm can be detected by a sperm DNA fragmentation (SDF) Assay which can be used in adjunct to conventional semen analysis. While the sperm chromatin structure Assay (SCSA) remains the "gold standard," the TUNEL Assay using flow cytometry is becoming popular among the different tests that are currently available to measure sperm DNA fragmentation. In this unit, we describe the inter-laboratory and intra-laboratory standardization of the TUNEL Assay using a benchtop cytometer. The article also provides a step-by-step protocol for measuring sperm DNA fragmentation using the TUNEL Assay and a bench-top flow cytometer, and also points out the inherent challenges with this test. © 2017 by John Wiley & Sons, Inc.
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inter and intra laboratory standardization of TUNEL Assay for assessment of sperm dna fragmentation
Journal of Andrology, 2017Co-Authors: Sofia C Ribeiro, Rakesh Sharma, Sajal Gupta, Zeynep Cakar, C De Geyter, Ashok AgarwalAbstract:Summary 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.
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Current Protocols in Toxicology - Inter- and intra-laboratory standardization of TUNEL Assay for assessment of sperm DNA fragmentation
Andrology, 2017Co-Authors: Sofia C Ribeiro, Rakesh Sharma, Sajal Gupta, Zeynep Cakar, C De Geyter, Ashok AgarwalAbstract:Summary 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.
Gilles Bleau - One of the best experts on this subject based on the ideXlab platform.
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sperm dna fragmentation threshold value in male fertility
Human Reproduction, 2005Co-Authors: Martin Sergerie, G Laforest, Louis Bujan, Francois Bissonnette, Gilles BleauAbstract:BACKGROUND: The extent of sperm DNA fragmentation, which can be measured by the TUNEL Assay, is one of the determinants of male fertility. However, the clinical application of this test to in-vivo situations is difficult owing to the absence of a statistically validated threshold value. METHODS: The aim of this study was to compare the results of TUNEL Assay applied to semen samples from men of proven fertility (n = 47) and patients from an infertile population (n = 66), in order to establish a discriminating threshold value. RESULTS: Infertile patients had a higher mean level of DNA fragmentation than men of proven fertility (40.9 +/- 14.3% versus 13.1 +/- 7.3%, respectively; P < 0.001). The area under the receiver operating characteristics curve was 0.93 for 20% sperm DNA fragmentation. The calculated threshold value for TUNEL Assay to distinguish between fertile controls and infertile men was 20%. At this threshold, specificity was 89.4 [95% confidence interval (CI) 83.7-95.1] and sensitivity was 96.9% (95% CI 93.8-100). The positive and negative predictive values of the 20% sperm DNA fragmentation threshold were high: 92.8% (95% CI 87.9-97.5) and 95.5% (95% CI 91.6-99.3), respectively. CONCLUSION: This study demonstrates that sperm DNA fragmentation, as measured by TUNEL Assay, is a highly valuable indicator of male fertility.
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Innocuity of tetradecyl-dimethyl-benzyl ammonium fluoride on the DNA of human spermatozoa.
Contraception, 2004Co-Authors: G Laforest, Martin Sergerie, Gilles BleauAbstract:Tetradecyl-dimethyl-benzyl-ammonium fluoride (TDBAF) is a powerful new spermicide. In cases of failure of local contraception, there is a theoretical risk that a spermatozoon exposed to a sublethal concentration of spermicide might suffer DNA strand breaks, with transmission of damaged DNA to the oocyte. The present study aims to ascertain the innocuity of TDBAF in this regard. Spermatozoa from 32 fertile men were exposed to a sublethal concentration of TDBAF. The degree of DNA fragmentation was measured by modified terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin end labeling (TUNEL) Assay, pulsed field gel electrophoresis of purified sperm DNA and radioenzymatic labeling. With the TUNEL Assay, a statistically significant increase in sperm labeling was observed after TDBAF treatment compared to the controls. However, pulsed field gel electrophoresis and enzymatic labeling of purified DNA showed no evidence of augmented DNA fragmentation in TDBAF-treated sperm. Under electron microscopy, TDBAF caused sperm chromatin decondensation. In the TUNEL Assay, this phenomenon would allow easier access of terminal transferase to its DNA substrate, explaining the apparent increase in DNA fragmentation in the presence of TDBAF. In conclusion, the use of TDBAF as a spermicide does not place progeny at heightened risk of abnormalities should pregnancy occur. In regard to the TUNEL Assay, we suggest that pretreatment of spermatozoa with TDBAF promotes a more accurate measurement of sperm DNA fragmentation.
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Original research article Innocuity of tetradecyl-dimethyl-benzyl ammonium fluoride on the DNA of human spermatozoa
2004Co-Authors: G Laforest, Martin Sergerie, Gilles BleauAbstract:Tetradecyl-dimethyl-benzyl-ammonium fluoride (TDBAF) is a powerful new spermicide. In cases of failure of local contraception, there is a theoretical risk that a spermatozoon exposed to a sublethal concentration of spermicide might suffer DNA strand breaks, with transmission of damaged DNA to the oocyte. The present study aims to ascertain the innocuity of TDBAF in this regard. Spermatozoa from 32 fertile men were exposed to a sublethal concentration of TDBAF. The degree of DNA fragmentation was measured by modified terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin end labeling (TUNEL) Assay, pulsed field gel electrophoresis of purified sperm DNA and radioenzymatic labeling. With the TUNEL Assay, a statistically significant increase in sperm labeling was observed after TDBAF treatment compared to the controls. However, pulsed field gel electrophoresis and enzymatic labeling of purified DNA showed no evidence of augmented DNA fragmentation in TDBAF-treated sperm. Under electron microscopy, TDBAF caused sperm chromatin decondensation. In the TUNEL Assay, this phenomenon would allow easier access of terminal transferase to its DNA substrate, explaining the apparent increase in DNA fragmentation in the presence of TDBAF. In conclusion, the use of TDBAF as a spermicide does not place progeny at heightened risk of abnormalities should pregnancy occur. In regard to the TUNEL Assay, we suggest that pretreatment of spermatozoa with TDBAF promotes a more accurate measurement of sperm DNA fragmentation. © 2004 Elsevier Inc. All rights reserved.
Darius A. Paduch - One of the best experts on this subject based on the ideXlab platform.
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Concordance among sperm deoxyribonucleic acid integrity Assays and semen parameters.
Fertility and sterility, 2015Co-Authors: Peter J. Stahl, Darius A. Paduch, Akanksha Mehta, Chava Cogan, Alex Bolyakov, Marc GoldsteinAbstract:Objective To assess the concordance of sperm chromatin structure Assay (SCSA) results, epifluorescence TUNEL Assay results, and standard semen parameters. Design Prospective, observational study. Setting Tertiary referral andrology clinic. Patient(s) A total of 212 men evaluated for subfertility by a single physician. Intervention(s) Clinical history, physical examination, semen analysis, SCSA, and TUNEL Assay. Main Outcome Measure(s) Spearman's rank correlation coefficients ( r ) between SCSA DNA fragmentation index (DFI), percentage TUNEL-positive sperm, and semen analysis parameters. Result(s) There was a positive correlation between SCSA DFI and TUNEL ( r = 0.31), but the strength of this correlation was weaker than has previously been reported. The discordance rate between SCSA and TUNEL in classifying patients as normal or abnormal was 86 of 212 (40.6%). The SCSA DFI was moderately negatively correlated with sperm concentration and motility. The TUNEL results were unrelated to standard semen parameters. Conclusion(s) The SCSA DFI and percentage TUNEL-positive sperm are moderately correlated measures of sperm DNA integrity but yield different results in a large percentage of patients. The DFI is well-correlated with semen analysis parameters, whereas TUNEL is not. These data indicate that the SCSA and TUNEL Assay measure different aspects of sperm DNA integrity and should not be used interchangeably.
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rejection treshholds and seasonal variability of epifluorescent sperm TUNEL Assay
Fertility and Sterility, 2012Co-Authors: Darius A. Paduch, Alexander Bolyakov, J. Kiper, J. Bazarnik, Akanksha Mehta, M. GoldsteinAbstract:OBJECTIVE: To determine: seasonal and batch effects on positive and negative controls results of TUNEL Assay. To analyze cut-off points for rejection of Assay. DESIGN: Retrospective review. MATERIALS AND METHODS: TUNEL Assay was performed using 10 ul sperm spread on each of 5 microscope slides. Two slides for negative and 2 for positive controls were analyzed. 200 sperm per slides were counted. All positive controls were incubated with 500 ul of DNAse type I for 10 min at 37C incubator prior to adding enzyme solution and labeling solution. For negative controls only labeling solution was added. All slides were then incubated for 1 h at 37C, counter stained with DAPI and analyzed by the same observer. Results were reported as percentage of TUNEL positive (FITC) sperm per total number of sperm (DAPI). RESULTS: 125 positive and 126 negative Assays were scored between April of 2009 and 2012 by the same observer. Mean % TUNEL and 95% CI for negative control was 1.02 (0.94-1.13) and 98.6% (98.5-98.8) for positive controls. Distribution curves fitting showed Weilbull as most appropriate model. Normal Q-Q plot and weighted percentiles analysis revealed that Assay should be rejected if negative TUNEL is> 2% or positive TUNEL1⁄4 2% or positive control is <1⁄497%. TUNEL Assays is extremely reproducible as evident by very close clustering of values. Seasonal variability although surprising may be explained by variability in temperatures in the culture rooms or other factors like DNAse stability. Seasonal variability does not increase Assay rejection rate. Supported by: Robert Dow.
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Rejection treshholds and seasonal variability of epifluorescent sperm TUNEL Assay
Fertility and Sterility, 2012Co-Authors: Darius A. Paduch, Alexander Bolyakov, J. Kiper, J. Bazarnik, Akanksha Mehta, M. GoldsteinAbstract:OBJECTIVE: To determine: seasonal and batch effects on positive and negative controls results of TUNEL Assay. To analyze cut-off points for rejection of Assay. DESIGN: Retrospective review. MATERIALS AND METHODS: TUNEL Assay was performed using 10 ul sperm spread on each of 5 microscope slides. Two slides for negative and 2 for positive controls were analyzed. 200 sperm per slides were counted. All positive controls were incubated with 500 ul of DNAse type I for 10 min at 37C incubator prior to adding enzyme solution and labeling solution. For negative controls only labeling solution was added. All slides were then incubated for 1 h at 37C, counter stained with DAPI and analyzed by the same observer. Results were reported as percentage of TUNEL positive (FITC) sperm per total number of sperm (DAPI). RESULTS: 125 positive and 126 negative Assays were scored between April of 2009 and 2012 by the same observer. Mean % TUNEL and 95% CI for negative control was 1.02 (0.94-1.13) and 98.6% (98.5-98.8) for positive controls. Distribution curves fitting showed Weilbull as most appropriate model. Normal Q-Q plot and weighted percentiles analysis revealed that Assay should be rejected if negative TUNEL is> 2% or positive TUNEL1⁄4 2% or positive control is
M. Goldstein - One of the best experts on this subject based on the ideXlab platform.
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rejection treshholds and seasonal variability of epifluorescent sperm TUNEL Assay
Fertility and Sterility, 2012Co-Authors: Darius A. Paduch, Alexander Bolyakov, J. Kiper, J. Bazarnik, Akanksha Mehta, M. GoldsteinAbstract:OBJECTIVE: To determine: seasonal and batch effects on positive and negative controls results of TUNEL Assay. To analyze cut-off points for rejection of Assay. DESIGN: Retrospective review. MATERIALS AND METHODS: TUNEL Assay was performed using 10 ul sperm spread on each of 5 microscope slides. Two slides for negative and 2 for positive controls were analyzed. 200 sperm per slides were counted. All positive controls were incubated with 500 ul of DNAse type I for 10 min at 37C incubator prior to adding enzyme solution and labeling solution. For negative controls only labeling solution was added. All slides were then incubated for 1 h at 37C, counter stained with DAPI and analyzed by the same observer. Results were reported as percentage of TUNEL positive (FITC) sperm per total number of sperm (DAPI). RESULTS: 125 positive and 126 negative Assays were scored between April of 2009 and 2012 by the same observer. Mean % TUNEL and 95% CI for negative control was 1.02 (0.94-1.13) and 98.6% (98.5-98.8) for positive controls. Distribution curves fitting showed Weilbull as most appropriate model. Normal Q-Q plot and weighted percentiles analysis revealed that Assay should be rejected if negative TUNEL is> 2% or positive TUNEL1⁄4 2% or positive control is <1⁄497%. TUNEL Assays is extremely reproducible as evident by very close clustering of values. Seasonal variability although surprising may be explained by variability in temperatures in the culture rooms or other factors like DNAse stability. Seasonal variability does not increase Assay rejection rate. Supported by: Robert Dow.
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Rejection treshholds and seasonal variability of epifluorescent sperm TUNEL Assay
Fertility and Sterility, 2012Co-Authors: Darius A. Paduch, Alexander Bolyakov, J. Kiper, J. Bazarnik, Akanksha Mehta, M. GoldsteinAbstract:OBJECTIVE: To determine: seasonal and batch effects on positive and negative controls results of TUNEL Assay. To analyze cut-off points for rejection of Assay. DESIGN: Retrospective review. MATERIALS AND METHODS: TUNEL Assay was performed using 10 ul sperm spread on each of 5 microscope slides. Two slides for negative and 2 for positive controls were analyzed. 200 sperm per slides were counted. All positive controls were incubated with 500 ul of DNAse type I for 10 min at 37C incubator prior to adding enzyme solution and labeling solution. For negative controls only labeling solution was added. All slides were then incubated for 1 h at 37C, counter stained with DAPI and analyzed by the same observer. Results were reported as percentage of TUNEL positive (FITC) sperm per total number of sperm (DAPI). RESULTS: 125 positive and 126 negative Assays were scored between April of 2009 and 2012 by the same observer. Mean % TUNEL and 95% CI for negative control was 1.02 (0.94-1.13) and 98.6% (98.5-98.8) for positive controls. Distribution curves fitting showed Weilbull as most appropriate model. Normal Q-Q plot and weighted percentiles analysis revealed that Assay should be rejected if negative TUNEL is> 2% or positive TUNEL1⁄4 2% or positive control is
