GLOBAL ANDROLOGY FORUM
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Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation
Article #41: “Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation”.
Authors: Sulagna Dutta, Ralf Henkel & Ashok Agarwal
Andrologia, Published: 28 May 2020, DOI: 10.1111/and.13718
CAPSULE
Contributors: Sulagna Dutta (UAE), and Ralf Henkel, PhD (UK)
Preamble:
Traditional semen analysis, which evaluates parameters like sperm concentration, motility, and morphology, often falls short in pinpointing fertility issues, as these aspects can overlap between fertile and infertile men and fail to highlight deeper cellular or molecular dysfunctions in sperm. One crucial aspect of understanding male infertility is assessing sperm chromatin integrity, as it reveals subtle sperm defects that routine semen analysis might miss. Issues in chromatin condensation increase the susceptibility of sperm DNA to damage, making the evaluation of sperm chromatin integrity, through various methods like the SCSA, TUNEL, and SCD assays extremely important. These tests directly assess DNA fragmentation, while others like aniline blue and chromomycin A3 evaluate chromatin condensation quality.
The significance of chromatin integrity tests lies in their ability to identify specific causes of sperm dysfunction, thus enhancing the diagnosis and management of male infertility. They provide a comprehensive understanding of male fertility, crucial given the limitations of conventional semen analysis. Clinically, sperm DNA integrity is acknowledged as vital for human reproduction, and recent guidelines recommend Sperm DNA Fragmentation (SDF) testing in cases like infertile patients with varicocele, unexplained infertility, recurrent IVF failures, and those exposed to certain infertility inducers. Despite this, SDF tests are not universally endorsed in male infertility assessments.
Impaired sperm DNA integrity can result from factors like oxidative stress during spermatogenesis, leading to adverse outcomes like altered semen parameters, recurrent miscarriages, and reduced fertility. Various factors, including lifestyle, environmental exposures, and genetics, contribute to SDF. For assessing this damage, techniques like the TUNEL assay (the gold standard), SCD, Comet, SCSA assays, and the determination of 8-hydroxy-2-deoxyguanosine are used. These tests correlate the extent of sperm DNA fragmentation with other sperm characteristics and reproductive outcomes.
Commentary:
In this article, the authors emphasized the complexity and necessity of evaluating sperm chromatin integrity and sperm DNA fragmentation (SDF) in understanding male infertility and advocate for more standardized, comprehensive diagnostic approaches to enhance the accuracy and efficacy of treatments in male reproductive health.
SDF testing is facing challenges due to the usage of different assays, lack of standardization and significant variability between individuals and laboratories. This necessitates method revalidation and strict quality control. Differentiating between tests for SDF and chromatin condensation is vital, as these tests assess different, distinct sperm functions and aspects. The variety of DNA damages, each needing specific assays, complicates the correlation of results from different tests.
Concluding Remarks:
This article is another significant contribution from the Global Androgen Forum (GAF), emphasizing the importance of analyzing sperm chromatin integrity and DNA fragmentation as distinct functional features in understanding male infertility. It highlights that a differentiated analysis is not only pivotal in unraveling the mechanisms behind male infertility but also serves as a predictor of fertility outcomes in natural reproduction and assisted reproductive technologies (ART). The article points out that high SDF is a clinically recognized paternal-derived defect that can lead to repeated ART failures and miscarriages. It discusses the availability of various assays to evaluate sperm chromatin integrity and SDF, noting that each assay has its own set of advantages and limitations. Therefore, it's crucial to compare these tests carefully before selecting one, ensuring the most relevant information is obtained. Since there are different types of DNA damage (e.g. mismatched bases, abasic sites, base modifications) with sperm DNA fragmentation (DNA strand breaks) being the end point of one type of damage, the article also stresses the importance of identifying the actual causes of sperm DNA damage to provide appropriate therapeutic strategies. While cut-off values for SDF tests have been proposed, the authors suggest that they require further validation and a broader consensus within the scientific community. Overall, the article strongly supports including sperm DNA fragmentation assessments in the evaluation of infertile men, underscoring its critical role in the diagnosis and treatment of male infertility.
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Take Home Message: (Contributor: Ashok Agarwal)
- Sperm DNA integrity is crucial for male fertility: Evaluation of sperm chromatin integrity and DNA fragmentation is vital for diagnosing and managing male infertility, given its significant impact on reproductive outcomes.
- Conventional semen analysis is insufficient: Routine semen analysis does not provide information on sperm DNA integrity, necessitating the use of specialized tests to uncover subtle defects affecting fertility.
- Various methods are available for assessment: Techniques such as the TUNEL assay, SCD (sperm chromatin dispersion) test, and SCSA (sperm chromatin structure assay) offer different degrees of diagnostic and prognostic capabilities for evaluating sperm DNA damage and chromatin condensation.
- Choice of method matters: Each method has its specific advantages, limitations, and applications, making it important to select the most appropriate test based on clinical needs, personal experience and laboratory capabilities.
- Clinical implications of sperm DNA damage: High levels of SDF are associated with decreased semen quality, reduced reproductive potential, and adverse ART outcomes, underscoring the need for inclusion of these assessments in male infertility workups.
My Personal Viewpoint on “Diagnostic Value of Advanced Semen Analysis”
Dr. Sulagna Dutta responds to the questions by Ashok Agarwal
Q1. Regarding our current emphasis on Sperm DNA Fragmentation (SDF) testing for male infertility, do you believe it is justified, exaggerated, or understated in the existing literature and scientific discussions?
Dr. Dutta: In current scientific literature and discussions, Sperm DNA Fragmentation (SDF) testing is understated. Considering the limited understanding of the molecular mechanisms behind idiopathic male infertility, increased knowledge about SDF and its various testing methods could significantly improve the diagnosis and assessment of male infertility.
Q2. Can you identify Sperm DNA Fragmentation (SDF) tests that are straightforward, provide accurate cutoff values, demonstrate reproducibility, have validated results in clinical trials, are extensively published, and are cost-effective?
Dr. Dutta: Tests like the Sperm Chromatin Structure Assay (SCSA), the TUNEL assay, and the Comet assay are noted for their reliability. These tests offer straightforward procedures, reproducibility, and have been validated in various clinical settings. However, cost-effectiveness and clear cutoff values vary, and more extensive publishing in clinical trials is needed for some.
Q3. What, in your opinion, are the three primary reasons behind the absence of a gold standard test for assessing sperm chromatin integrity? Additionally, could you share your preference for a specific SDF test and the reasons behind your choice?
Dr. Dutta: The three primary reasons behind the absence of a gold standard test for assessing sperm chromatin integrity can be:
- Variability in Test Methodologies: Different SDF tests use varied techniques, leading to inconsistencies in results.
- Lack of Universal Cutoff Values: There's no consensus on the threshold values for SDF levels that indicate fertility issues.
- Complex Nature of Sperm DNA Damage: Sperm DNA damage is multifaceted, making it challenging to standardize a single test.
Preference for SDF Test: The TUNEL assay is favored for its balance of sensitivity and specificity. However, the choice often depends on the specific clinical context and available resources.
Q4. I'm curious about your perspective on the utilization of AI-based devices to interpret the results of SDF testing. How do you see this technology influencing the accuracy and efficiency of such assessments?
Dr. Dutta: With further improvisation of the AI technology, AI-based devices hold promise for enhancing the accuracy and efficiency of SDF test interpretations. These technologies can potentially standardize readings, reduce human error, and provide quicker, more reliable assessments.
Q5. Looking ahead, do you envision SDF testing becoming commonplace in the future, recognized by major professional societies in the field of male infertility? Your insights into the potential trajectory of SDF testing would be valuable.
Dr. Dutta: It is likely that SDF testing will become more mainstream in male infertility assessment, as understanding and technology improve. This likelihood increases if major professional societies acknowledge its significance, especially if future research and publications further establish the link between SDF levels, fertility outcomes, and testing methods, similar to our article under discussion.
Sulagna Dutta, PhD: Short Biography
Sulagna Dutta, PhD
Assistant Professor
School of Life Sciences
Manipal Academy of Higher Education
Dubai, UAE
sulagna_dutta11@yahoo.com
https://orcid.org/0000-0002-7893-5282
Dr. Sulagna Dutta is a Physiologist with >150 research publications and an h-index of 30 on Scopus. She is a Faculty at the School of Medicine, Manipal Academy of Higher Education, Dubai, UAE. She earned her PhD in Physiology, with specialization in Immunology from the University of Calcutta, India, and later pursued a Research Internship in Reproductive Medicine at the Cleveland Clinic, USA. Sulagna has over 12 years of experience in teaching and research in India, Malaysia, and UAE. Her research interests include immunology, reproductive physiology, and infertility. She is being ranked among the Top 2% Scientists in the world by Stanford University since 2020
My Personal Viewpoint on “Diagnostic Value of Advanced Semen Analysis”
Prof. Ralf Henkel responds to the questions by Ashok Agarwal
Q1. Regarding our current emphasis on Sperm DNA Fragmentation (SDF) testing for male infertility, do you believe it is justified, exaggerated, or understated in the existing literature and scientific discussions?
Prof. Henkel: I believe that sperm DNA fragmentation is an essential diagnostic parameter that should be tested in order to have a more complete analysis of the male fertilizing ability. My analyses show that even in the group of patients which are supposedly to be normal, the normozoospermic men, 27% of these men show elevated sperm DNA fragmentation. In the other patient groups, the percentage of patients with high sperm DNA fragmentation is even much higher (oligozoospermia: 24%; teratozoospermia: 53%; asthenozoospermia: 98%). Therefore, considering that this parameter is providing significant additional information for the patient and for the physician on how to treat and manage the patient, I would even recommend testing routinely for sperm DNA fragmentation. In addition, in order to shorten the time period from diagnosing the man until possible assisted reproduction, sperm DNA fragmentation testing should be done together with a test for seminal redox stress. The aim should be to diagnose and treat the man properly, rather than treating the sperm or using potentially defective sperm for ICSI attempts.
In summary, the scientometric analysis offers a thorough overview of the research in male infertility and ART, providing valuable insights for private andrologists to improve their practice and patient care.
Q2. Can you identify Sperm DNA Fragmentation (SDF) tests that are straightforward, provide accurate cutoff values, demonstrate reproducibility, have validated results in clinical trials, are extensively published, and are cost-effective?
Prof. Henkel: The SDF tests with the highest accuracy and reproducibility are tests that employ flow cytometry, namely the TUNEL assay or the sperm chromatin structure assay (SCSA). The other test systems such as COMET assay or Halosperm test have a higher inter-rater variability than the TUNEL or SCSA because they are analyzing a much smaller number of sperm and are manually evaluated. The advantage of the Halosperm test or similar tests, however, is that they are cheaper.
Q3. What, in your opinion, are the three primary reasons behind the absence of a gold standard test for assessing sperm chromatin integrity? Additionally, could you share your preference for a specific SDF test and the reasons behind your choice??
Prof. Henkel: In my opinion, reasons why there is still no generally accepted “gold standard test” for sperm DNA fragmentation are the lack of standardization and as a result of that different cut-off values. In addition, the cost factor seems to play a big role as not all laboratories can afford a flow cytometer and therefore rather perform a cheaper one which has a higher inter-observer variability.
Q4. I'm curious about your perspective on the utilization of AI-based devices to interpret the results of SDF testing. How do you see this technology influencing the accuracy and efficiency of such assessments?
Prof. Henkel: At the moment, AI is tested for tests such as the Halosperm test in order to improve reliability and reduce inter-observer variation. I would also see opportunities for the implementation of AI in novel test systems that could perhaps analyze the number of DNA strand breaks as a measure of the extend of the sperm DNA damage. Important is however, that such systems are getting cheaper, more standardized, and more accurate and reproducible.
Q5. Looking ahead, do you envision SDF testing becoming commonplace in the future, recognized by major professional societies in the field of male infertility? Your insights into the potential trajectory of SDF testing would be invaluable.
Prof. Henkel: Yes, definitely. However, it will still take some time for medical societies to fully adopt sperm DNA fragmentation as a routine test.
Ralf Henkel, PhD: Short Biography
Ralf Henkel, PhD, Habil
Chief Scientific Advisor: LogixX Pharma, Reading, UK
Visiting Reader, Department of Metabolism, Digestion and Reproduction, Imperial College London, UK
ralf.henkel@logixxpharma.com
https://orcid.org/0000-0003-1128-2982
Prof. Ralf Henkel, a distinguished scientist, pursued his studies in Biology and Chemistry at Marburg, Germany. Following the completion of his PhD, he contributed to the fields of Dermatology and Andrology in Giessen, Germany. In 2004, he assumed the role of Professor at the Urology department in Jena, Germany and later served as the Head of the Department of Medical Bioscience at the University of the Western Cape in Cape Town, South Africa He currently holds the position of Extraordinary Professor at the same institution. Since June 2020, Ralf has been engaged with LogixX Pharma, UK. Prof. Henkel is also the Editor-in-Chief of Andrologia. Throughout his illustrious career, he has supervised 76 students, published over 300 articles, chapters, and books, and has an h-index of 46 on Scopus. His collaboration with Ashok Agarwal dates back to their time at the Cleveland Clinic, and since 2022, he has been a senior member of the Global Andrology Forum.