Best urological practices on testing and management of infertile men with abnormal sperm DNA fragmentation levels: the SFRAG guidelines

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 (1), 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 (2). 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 (3). 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 (4). Infertile men often have abnormal levels of sperm DNA fragmentation (SDF), which is a marker of damaged chromatin (5). 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 (6, 7). 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.


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 (1), 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 (2).
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 (3). 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 (4). Infertile men often have abnormal levels of sperm DNA fragmentation (SDF), which is a marker of damaged chromatin (5).
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 (6,7). 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) (8). 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 Best urological practices on testing and management of infertile men with abnormal sperm DNA fragmentation levels: the SFRAG guidelines _______________________________________________ _______________________________________________ Sandro C. Esteves 1, 2, 3 , Armand Zini 4 , Robert Matthew Coward 5 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).
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.
The primary goals of the SFRAG guideline are to provide clinicians -urologists, andrologists, gynecologists, and reproductive endocrinologistswith 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. Any of the four SDF tests (SCSA, alkaline Comet, SCD, and TUNEL) may provide valid information concerning the probability of reproductive success for couples embarking on IUI, IVF, and ICSI.

Conditional Grade B
A standardized protocol with strict quality control is essential for a reliable SDF testing result. Tests should be validated by the laboratory, with thresholds established based on the evaluation of fertile and infertile populations.

Strong Grade A-B
A neat semen sample should be used for SDF testing, collected after ejaculatory abstinence of 2-5 days. Strong Grade B Patients should be asked not to have prolonged abstinence periods before the ejaculation that precedes the one used for semen collection and testing.

Conditional
Grade D A fixed ejaculatory abstinence length should be used for SDF testing when monitoring the effects of medical and surgical interventions aimed at decreasing SDF levels.

Conditional
Grade B Fresh or frozen-thawed specimens can be used for testing, but the analysis should start as quickly as possible after liquefaction (e.g., 30-60 minutes) or thawing.

Strong Grade C-D
If a frozen specimen is to be used for SDF testing, freezing should be immediately done after liquefaction is achieved.

Conditional Grade B
Overall, thresholds exceeding 20-30% (SCSA, alkaline Comet, and SCD) indicate a statistical probability of increased time to achieve natural pregnancy, increased miscarriage risk (after both natural and assisted conception), and low odds of reproductive success by IUI, IVF, and ICSI.

Conditional
Grade B SDF results -in combination with the current tools for infertility diagnosis-provide useful information concerning the probability of reproductive success.

Conditional
Grade B SDF tests cannot perfectly discriminate fertile from infertile men or couples that will have a successful IUI, IVF, or ICSI cycle from those that will not.

Strong
Grade B The usefulness of any test for one partner is also dependent on the fertility of the other partner. Before testing, clinicians should have some understanding of the characteristics of SDF assays (e.g., sensitivity and specificity, positive and negative predictive value).  Among couples with ICSI failure and elevated SDF, testicular rather than ejaculated sperm may be considered for sperm injection in subsequent treatment cycles.

Conditional
Grade B The use of testicular sperm in preference over ejaculated sperm for ICSI, when both are available, may be particularly relevant for couples with no apparent reasons for a failed ICSI (e.g., no relevant female factors). This advice implies that a reproductive urologist has evaluated the male partner and all possible corrective measures taken to improve overall reproductive health and sperm chromatin integrity.

Conditional
Grade D

Fertility Counseling for Individuals with Infertility Risk Factors
SDF testing may be considered to provide laboratory evidence of defective sperm chromatin to couples who seek fertility counseling and family planning, particularly when the male partner has an infertility risk factor.

Conditional
Grade C Men with infertility risk factors (e.g., tobacco smoking, obesity, metabolic syndrome, exposure to environmental or occupational toxicants, use of licit or illicit drugs with gonadotoxic effects, and advanced paternal age) should be informed that these factors may cause SDF and that lifestyle changes may alleviate SDF, potentially increasing the likelihood of reproductive success.

Conditional Grade C
An abnormal SDF test result should prompt a complete male evaluation by a reproductive urologist to help identify and possibly treat conditions associated with poor sperm DNA quality.

Strong Grade D
An abnormal SDF test result may be used for counseling, reinforcing the importance of lifestyle changes and avoiding exposure to toxins.

Conditional
Grade C Early ICSI may be considered for individuals with persistently high SDF levels despite corrective interventions, mainly when the reproductive window is limited.

Conditional
Grade D The information provided by SDF testing may guide the choice of assisted conception modality, IUI, IVF, or ICSI, in infertile couples with a male partner of advanced age.

Conditional
Grade D SDF testing may be used to monitor the effects of lifestyle interventions.

Conditional
Grade D

Sperm Cryopreservation
SDF testing can be considered before sperm cryopreservation to provide additional information about semen quality.

Conditional
Grade D The information provided by SDF testing may guide the decision to use IUI or IVF/ICSI for future conception with cryopreserved sperm -in case both options are available-, and the choice of the optimal sperm freezing method.