INTRODUCTION
The prevention and management of male infertility is an integral component of sexual and reproductive health services. Male factors, alone or combined with female factors, explain up to 50% of infertility cases, and when present, an evaluation by a urologist experienced in diagnosing and treating male factor infertility is highly recommended. In Brazil, like the United States and Canada (11. Samplaski MK, Smith JF, Lo KC, Hotaling JM, Lau S, Grober ED, et al. Reproductive endocrinologists are the gatekeepers for male infertility care in North America: results of a North American survey on the referral patterns and characteristics of men presenting to male infertility specialists for infertility investigations. Fertil Steril. 2019; 112:657-62.), most patients are referred to urologists by (reproductive) gynecologists based on an abnormal semen analysis result. The work-up involves a detailed medical history and physical examination and, when indicated, hormone, genetic, and imaging tests, all of which are used to guide clinical management (22. Esteves SC, Miyaoka R, Agarwal A. An update on the clinical assessment of the infertile male. Clinics (Sao Paulo). 2011; 66:691-700. Erratum in: Clinics (Sao Paulo). 2012; 67:203.).
The semen analysis is one of the earliest tests in the infertility work-up. The standard assessment of semen characteristics includes ejaculate volume, sperm count, sperm motility, and sperm morphology. Although informative, they provide limited discriminatory information about the male fertility potential, unless at extremely low levels (33. Esteves SC. Clinical relevance of routine semen analysis and controversies surrounding the 2010 World Health Organization criteria for semen examination. Int Braz J Urol. 2014; 40:443-53.). Recently, increased attention has been given to the evaluation of sperm DNA, whose integrity is indispensable for post-fertilization events and the birth of healthy offspring (44. Krawetz SA. Paternal contribution: new insights and future challenges. Nat Rev Genet. 2005; 6:633-42.). Infertile men often have abnormal levels of sperm DNA fragmentation (SDF), which is a marker of damaged chromatin (55. Esteves SC. Are specialized sperm function tests clinically useful in planning assisted reproductive technology? Int Braz J Urol. 2020; 46:116-123.).
Measurement of SDF in the ejaculated semen is used to obtain information about sperm DNA quality at the molecular level. Sperm DNA breaks can be detected using probes or dyes under fluorescence or optical microscopy or flow cytometry examination. Several interventions have been proposed to mitigate the potential deleterious effect of SDF on reproduction (66. Esteves SC, Santi D, Simoni M. An update on clinical and surgical interventions to reduce sperm DNA fragmentation in infertile men. Andrology. 2020; 8:53-81., 77. Esteves SC. Testicular versus ejaculated sperm should be used for intracytoplasmic sperm injection (ICSI) in cases of infertility associated with sperm DNA fragmentation | Opinion: Yes. Int Braz J Urol. 2018; 44:667-675.). Despite robust evidence relating SDF with infertility, clear guidance on how testing should be performed and to whom it should be offered has been lacking. Moreover, the general belief that high SDF is untreatable has hampered testing in routine clinical practice.
The sperm DNA fragmentation study group (SFRAG) guidelines
An evidence-based guideline for the investigation and treatment of SDF was published in late 2020 on behalf of the Sperm DNA Fragmentation Study Group (SFRAG) (88. Esteves SC, Zini A, Coward RM, Evenson DP, Gosálvez J, Lewis SEM, et al. Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations. Andrologia. 2020:e13874.). This consensus guideline provides a comprehensive evidence summary about the role of SDF on infertility and offers best practice advice on testing and care of couples confronted with elevated SDF. Furthermore, the guideline provides an overview of the treatments currently available for mitigating elevated SDF, and which ones may be recommended. Recommendations are also formulated on what test should be used and how testing should be conducted to select patients for possible therapeutic interventions.
The guideline was developed in three main sections. In the first part, it outlines the SDF pathophysiology and explains each SDF test. This section provides thirteen recommendations on how testing should be carried out and results analyzed (Table-1). Also, a new nomenclature is proposed to classify the sperm chromatin damage tests into two groups, that is, one for the tests that measure SDF (TUNEL, SCSA, SCS, and Comet; Figure-1), and another related to tests that assess chromatin compaction (e.g., chromomycin A3, acridine orange staining, toluidine blue staining, and aniline blue staining).
Recommendations on technical aspects of Sperm DNA Fragmentation testing, clinical thresholds, and interpretation of results.
Sperm DNA fragmentation tests.
A) Sperm Chromatin Dispersion test (SCD): Sperm sample of a patient with varicocele presenting with elevated SDF. Open arrowheads indicate sperm with halos of dispersed chromatin representing a normal DNA molecule with no fragmented DNA. Black arrowheads indicate sperm with small or absent halos of dispersed chromatin, representing sperm with fragmented DNA. Arrows in indicate sperm with no halos at all, fragmented-degraded DNA. B) Alkaline Comet assay under fluorescence microscopy: Sperm sample of a patient exhibiting elevated sperm DNA fragmentation (SDF). Several comets are shown, which represent sperm with DNA fragmentation. The longer and brighter the ‘Comet’ tail, the more fragmentation is present. Open arrow: spermatozoon with DNA fragmentation. White arrow: spermatozoon with a hardly visible ‘Comet’ tail, representing a cell with minimal DNA fragmentation. As the Comet test measures the amount of damage in each cell, it is rare to find a perfect spermatozoon with 0% damage, even from fertile donors. C) TUNEL Assay: Visualization of sperm DNA damage using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Digoxigenin-dUTP was incorporated into DNA breaks using a terminal transferase that was detected using anti-digoxigenin-FITC (green color). TUNEL+ represents sperm presenting DNA damage. Slides were counterstained with propidium iodide (red color). TUNEL- represents sperm free of DNA breaks. D) Sperm Chromatin Structure Assay (SCSA): Test data (SCSA Diagnostics, Brookings, USA). Left panel (top box): raw data from a flow cytometer showing each of 5.000 sperm as a single dot on a scattergram. Y-axis = green fluorescence with 1.024 gradations (channels) of DNA stainability (intact double-stranded DNA). X-axis = red fluorescence with 1.024 gradations of red fluorescence (single-strand DNA). Axes shown are 1.24/10. The line at Y = 75 marks the upper boundary of DNA staining of normal sperm chromatin; above that line are sperm (dots) with partially uncondensed chromatin allowing more DNA stainability. The bottom left corner shows gating out of seminal debris. Middle panel: Raw data from the left panel are converted by SCSAsoft software (or equivalent) to red/red+green fluorescence. This transforms the angled sperm display in the left panel to a vertical pattern that is often critical for accurately delineating the percentage of sperm with fragmented DNA.
Y-axis = total DNA stainability vs. X-axis = red/red+green fluorescence (DFI). Right panel: Frequency histogram of data from middle panel showing computer gating into %DFI and Mean DFI. Bottom box: SCSAsoft software calculations of the mean of two independent measures of mean and standard deviation (std dev) of median DFI, %DFI, and %HDS (high DNA stainability).
Modified from: Esteves SC, Zini A, Coward RM, Evenson DP, Gosálvez J, Lewis SEM, Sharma R, Humaidan P. Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations. Andrologia. 2020 Oct 27:e13874. Epub ahead of print. This is an open-access article distributed under the Creative Commons Attribution License. The license permits unrestricted use, distribution, reproduction in any medium, remixing, transformation, and building upon the material for any purpose provided the original work is properly cited.
The second part details seven clinical situations that may benefit from SDF testing, including i. Varicocele, ii. Unexplained/idiopathic infertility, iii. Recurrent pregnancy loss, iv. Intrauterine insemination, v. In vitro fertilization/intracytoplasmic sperm injection, vi. Infertility risk factors, and vii. Sperm cryopreservation. The guideline provides specific recommendations for each condition-twenty-eight in total (Table-2)- and best practices for treatment. Lastly, the third part lists the main gaps in knowledge and provides recommendations for future research.
Why and how to use the SFRAG guideline
The SGRAG guideline is unique as it unites reproductive urologists with vast clinical experience in diagnosing and treating male factor infertility. Moreover, for the first time, a group of scientists pivotal in developing the four major SDF assays used nowadays worked together. They deciphered each test’s technical aspects, making it easier to interpret the results and understand the intrinsic limitations of these assays. Furthermore, the SFRAG guideline includes an experienced reproductive endocrinologist with vast clinical experience, who added unique insights concerning the application of SDF testing in couples undergoing assisted reproduction.
The guideline summarizes and critically appraises the most relevant studies published to date. Thus, for each recommendation, a strength rating based on both expert judgment and evidence levels is provided. The clinical scenarios warranting SDF testing are dissected, and the best evidence-based treatment practices are provided. Notably, the guideline emphasizes the central role of urologists in the evaluation of the infertile male partner and highlights the importance of corrective measures to improve the male reproductive health overall, and SDF in particular. Figure-2 summarizes the SFRAG guideline in a snapshot.
A Pictorial summary of the recommendations for sperm DNA fragmentation testing and possible management in couples with elevated sperm DNA fragmentation.
IUI: intrauterine insemination; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; RPL: recurrent pregnancy loss.
Reprinted from: Esteves SC, Zini A, Coward RM, Evenson DP, Gosálvez J, Lewis SEM, Sharma R, Humaidan P. Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations. Andrologia. 2020 Oct 27:e13874. Epub ahead of print. This is an open-access article distributed under the Creative Commons Attribution License. The license permits unrestricted use, distribution, reproduction in any medium, provided the original work is properly cited.
The primary goals of the SFRAG guideline are to provide clinicians -urologists, andrologists, gynecologists, and reproductive endocrinologists - with clear advice on best practices in SDF testing and treatment. Besides treating conditions known to impair fertility and SDF, like varicocele, the reproductive urologist may identify other factors associated with the SDF, including subclinical infections, systemic diseases, and unhealthy lifestyle factors. For couples who need assisted reproductive technology, the reduction in SDF rates may help improve success rates, and downgrade the complexity and cost of the method potentially, or even help achieve natural conception.
The SFRAG guideline statements were developed based on the best available evidence, with the grade of recommendation ranging from low to moderate. This thematic area still lacks high-quality studies, thus offering ample research opportunities. Such a guideline should be used as a tool to help standardize care, however, it does not mandate clinical care pathways. The SFRAG guideline is a clear, concise summary of best practices in SDF testing and treatment that represents an invaluable resource for a broad range of professionals providing infertility care.
Data availability statement
This paper provides an abridged version of SFRAG guidelines, an open-access article distributed under the Creative Commons Attribution License. The license permits unrestricted use, distribution, reproduction in any medium, remixing, transformation, and building upon the material for any purpose provided the original work is properly cited. The full version can be found at https://onlinelibrary.wiley.com/doi/10.1111/and.13874.
REFERENCES
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1Samplaski MK, Smith JF, Lo KC, Hotaling JM, Lau S, Grober ED, et al. Reproductive endocrinologists are the gatekeepers for male infertility care in North America: results of a North American survey on the referral patterns and characteristics of men presenting to male infertility specialists for infertility investigations. Fertil Steril. 2019; 112:657-62.
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2Esteves SC, Miyaoka R, Agarwal A. An update on the clinical assessment of the infertile male. Clinics (Sao Paulo). 2011; 66:691-700. Erratum in: Clinics (Sao Paulo). 2012; 67:203.
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3Esteves SC. Clinical relevance of routine semen analysis and controversies surrounding the 2010 World Health Organization criteria for semen examination. Int Braz J Urol. 2014; 40:443-53.
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4Krawetz SA. Paternal contribution: new insights and future challenges. Nat Rev Genet. 2005; 6:633-42.
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5Esteves SC. Are specialized sperm function tests clinically useful in planning assisted reproductive technology? Int Braz J Urol. 2020; 46:116-123.
-
6Esteves SC, Santi D, Simoni M. An update on clinical and surgical interventions to reduce sperm DNA fragmentation in infertile men. Andrology. 2020; 8:53-81.
-
7Esteves SC. Testicular versus ejaculated sperm should be used for intracytoplasmic sperm injection (ICSI) in cases of infertility associated with sperm DNA fragmentation | Opinion: Yes. Int Braz J Urol. 2018; 44:667-675.
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8Esteves SC, Zini A, Coward RM, Evenson DP, Gosálvez J, Lewis SEM, et al. Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations. Andrologia. 2020:e13874.
Publication Dates
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Publication in this collection
01 Oct 2021 -
Date of issue
Nov-Dec 2021
History
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Received
08 Nov 2020 -
Accepted
09 Nov 2020 -
Published
20 Dec 2020