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© 2021 The Authors. Société Internationale d'Urologie Journal, published by the Société Internationale d'Urologie, Canada.

Increasing Awareness About Male Infertility:  
An Overview of the Sperm DNA Fragmentation 
Study Group (SFRAG) Guidelines
Sandro C. Esteves 

Sandro C. Esteves: https://orcid.org/0000-0002-1313-9680  
ANDROFERT, Andrology and Human Reproduction Clinic, Referral Center for Male Reproduction, Campinas, Brazil 
Department of Surgery (Division of Urology), University of Campinas (UNICAMP), Campinas, Brazil. Faculty of Health, Aarhus University, Aarhus, Denmark

Sperm DNA integrity is indispensable for the birth of healthy offspring[1]. Increasing evidence indicates that sperm 
DNA fragmentation (SDF), a marker of damaged chromatin, has an independent and critical role in male infertility 
and reproductive success[2]. The reasons relate to the often higher SDF levels in ejaculated semen of infertile men 
(versus their fertile counterparts) and the adverse impact of SDF on the sperm’s ability to fertilize the egg and promote 
healthy embryo development[2,3]. Consequently, couples in whom the male partner exhibits high SDF on neat semen 
may have longer time-to-pregnancy, increased risk of pregnancy loss, and decreased success in medically assisted 
reproductive (MAR) modalities (eg, intrauterine insemination [IUI] and in vitro fertilization/intracytoplasmic sperm 
injection [IVF/ICSI])[4]. The adverse effect of SDF on reproductive success is modulated by the oocyte’s DNA repair 
capacity intrinsically related to female age[5]. Sperm DNA damage exceeding the oocyte’s repair capacity–or the 
oocyte’s failure to repair DNA damage–negatively influences the embryo’s development potential and the offspring’s 
health[6].

Routine semen analysis–the laboratory backbone of infertility investigation–has shown little progress over the last 
century. Semen analysis results have low discriminatory power (except at extremely low levels) as there is considerable 
overlap between semen characteristics (eg, sperm count, motility, and morphology) of fertile and infertile men[7]. The 
need for more robust male infertility diagnosis methods has been the driving force of the ongoing efforts to develop 
and implement SDF testing in clinical practice. Indeed, SDF tests represent one of the best examples of translational 
medicine in andrology.

While it is not a replacement for the current tools for infertility diagnosis, SDF testing may add independent 
information about sperm quality at the molecular level, and its integration into practice may provide better 
counseling, diagnosis, and treatment planning. Despite that, societies like the American Urological Association and 
the American Society for Reproductive Medicine have not recommended using SDF testing routinely during the 
infertility evaluation, albeit acknowledging that the tests’ results might be informative. Insufficient clinical data, 
tests’ technical limitations and lack of effective treatment options to overcome SDF-related infertility have been the 
common grounds for the reluctance to endorse the clinical application of SDF tests[8]. However, evidence on these 
areas has increased steadily, justifying the development of clinical practice guidelines to refine efficiency in diagnosing 
and treating clinical conditions associated with SDF.

Key Words Competing Interests Article Information

Male infertility, practice guideline, sperm 
DNA fragmentation, semen analysis, assisted 
reproductive technology

SCE declares the receipt of unrestricted 
research grants and lecture fees from Merck 
outside the submitted work. The author is a 
member of the Sperm DNA Fragmentation 
Group and lead author of the SFRAG 
guidelines.

Received on November 2, 2020 
Accepted on December 13, 2020

Soc Int Urol J.2021;2(2):129–132

DOI: https://doi.10.48083/ WNAU8209

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https://doi.10.48083/WNAU8209
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This commentary highlights the recently published 
ev idence-based guideline from the Sperm DNA 
Fragmentation Study Group (SFRAG), which provides 
a comprehensive summary about the role of SDF in 
infertility and offers best practice advice on testing 
and care of couples with SDF[9]. 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. The SFRAG recommendations were based on the 
best available evidence, which ranged from moderate 
to low quality. The guideline may be used to help 
standardize care while securing physician autonomy, 
making it an invaluable resource for a broad range of 
professionals providing infertility care, particularly 
urologists.

The first part of the SFRAG guideline outlines the 
SDF pathophysiology and explains each SDF test. In 
brief, many conditions, including varicocele, chronic 
illness, accessory gland infections, advanced paternal 
age, lifestyle, obesity, occupational and environmental 
factors, medications, ionizing and non-ionizing 
radiation, and heat exposure, have been associated with 
elevated SDF levels. These conditions can promote SDF 
by causing defective spermatogenesis, evoking abortive 
apoptosis, or increasing the generation of reactive 
oxygen species (ROS). Excessive ROS causes oxidative 
stress, a significant causative factor of SDF in live sperm. 
The SFRAG guideline states there are 4 reliable tests to 
measure SDF. These tests are grouped in methods that 
use (a) enzymatic reactions to label the DNA breaks 
(eg, terminal deoxynucleotidyl transferase‐mediated 
dUTP‐biotin nick end labeling [TUNEL] assay) and (b) 
controlled DNA denaturation combined with protein 
depletion as intermediates to reveal the DNA breaks 
(eg, sperm chromatin structure assay [SCSA], sperm 
chromatin dispersion test [SCD], and the Comet assay). 
This guideline includes 13 recommendations on how 
testing should be carried out and results analyzed. 
Notably, it states that a standardized protocol with strict 
quality control is essential for a reliable test result and 
that a neat semen sample should be used for SDF testing, 
collected after ejaculatory abstinence of 2 to 5 days. It 
notes that although the results provided by the most 
common SDF tests do not necessarily line up, there 
is a good correlation between SDF rates reported by 
TUNEL, SCSA, SCD, and alkaline Comet. Thresholds 
of about 20% by TUNEL, SCSA, SCD, and alkaline 
Comet, assessed on neat semen, best discriminate fertile 
from infertile men. Additionally, thresholds of 20% 
to 30% evaluated by SCSA, alkaline Comet, and SCD 
are clinically useful for classifying infertile couples 
into those with a statistical probability of longer time 
to achieve natural pregnancy, a decreased chance of 

pregnancy with MAR, and an increased miscarriage 
risk. Lastly, it highlights that 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.

The second part describes 7 clinical situations that may 
benefit from SDF testing:

1. Varicocele
2. Unexplained/idiopathic infertility
3. Recurrent pregnancy loss
4. Intrauterine insemination
5. In vitro fertilization/intracytoplasmic sperm 

injection
6. Infertility risk factors
7. Sperm cryopreservation.
• Varicocele: Repairing a clinical varicocele may 

alleviate SDF, potentially increasing the likelihood of 
reproductive success. SDF testing may help identify 
patients with a profile that would not fit the standard 
indication of varicocele repair (eg, clinical varicocele 
of any grade and normal/borderline routine semen 
analysis) but would benefit from varicocele repair. 
SDF testing may be used to monitor treatment 
outcomes. However, SDF testing in subfertile 
men with subclinical varicocele is not currently 
recommended.

• Unexplained/idiopathic infertility: Couples with 
unexplained/idiopathic male infertility should be 
informed that abnormal SDF levels may adversely 
impact their chances of achieving a live birth. An 
abnormal test result should prompt a complete male 
fertility evaluation by a reproductive urologist to help 
identify and possibly treat conditions associated with 
poor sperm DNA quality. ICSI may be considered if 
no correctable male factor is identified, or if abnormal 
SDF levels persist after treatment, particularly among 
couples with a limited reproductive time window.

• Medically assisted reproduction: Infertile couples 
eligible for MAR treatment should be informed that 
abnormal SDF levels may adversely impact their 
chances of achieving a live birth. As in idiopathic/
unexplained infertility, a reproductive urologist’s 
evaluation is recommended to help identify and 
possibly treat conditions associated with SDF. 
Among couples with ICSI failure and elevated SDF, 
sperm retrieved from the testis may be considered 
for sperm injection in subsequent treatment cycles 
because of the lower SDF rates in testicular than in 
epididymal and ejaculated sperm and the higher ICSI 
success rates with use of testicular sperm rather than 
ejaculated sperm.

130 SIUJ  •  Volume 2, Number 2  •  March 2021 SIUJ.ORG

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• Risk factors: SDF testing is recommended in 
men with infertility risk factors (eg, tobacco 
smoking, obesity, metabolic syndrome, exposure 
to environmental or occupational toxicants, use 
of drugs with gonadotoxic effects, and advanced 
paternal age). An abnormal SDF test result may be 
used for counseling, reinforcing the importance of 
lifestyle changes and avoiding exposure to toxins, 
and monitoring the effect of lifestyle changes. It 
should also prompt a urologist’s evaluation to help 
identify other hidden and potentially correctable 
conditions linked to SDF.

The SFRAG guideline was developed by reproductive 
urologists with clinical experience in diagnosing and 
treating male factor infertility and a reproductive 

endocrinologist with insight into the application of 
SDF testing in couples undergoing assisted conception. 
Additionally, a group of scientists pivotal in developing 
the main SDF assays worked together, making the 
interpretation of tests' results and their limitations easy 
to understand. For each recommendation, a strength 
rating based on both expert judgment and evidence 
levels is provided. The guideline emphasizes the central 
role of urologists in the evaluation of the male partner 
and highlights the importance of corrective measures to 
improve the male reproductive health overall and SDF 
in particular (Figure 1). Lastly, the SFRAG guideline 
discusses the main gaps in knowledge and provides 
recommendations for future research.

FIGURE 1

A pictorial summary of the recommendations for SDF testing and possible management in couples with high SDF

Reprinted with permission. Esteves SC, et al.[9]

IUI: intrauterine insemination; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; RPL: recurrent pregnancy loss.

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References
1. Krawet z SA. Paternal contribution: new insights and future 

challenges. Nat Rev Genet. 2005;6:633–642.

2. 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.

3. Champroux A, Torres-Carreira J, Gharagozloo P, Drevet JR, Kocer 
A . Mammalian sperm nuclear organization: resiliencies and 
vulnerabilities. Basic Clin Androl. 2016;26:17.

4. 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.

5. Jin J, Pan C, Fei Q, Ni W, Yang X, Zhang L, et al. Effect of sperm 
DNA fragmentation on the clinical outcomes for in vitro fertilization 
and intracy toplasmic sperm injection in women with dif ferent 
ovarian reserves. Fertil Steril. 2015;103:910-916.

6. Aitken R J. DN A damage in human spermatozoa; impor tant 
contributor to mutagenesis in the offspring. Transl Androl Urol. 
2017;6:S761-S764.

7. 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.

8. Esteves SC, Agar wal A , Cho CL , Majzoub A . A strengths-
weaknesses-opportunities-threats (SWOT) analysis on the clinical 
utilit y of sperm DNA fragmentation testing in specific male 
infertility scenarios. Transl Androl Urol.2017;6(Suppl 4):S734-S760.

9. 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;Oct 
27:e13874. Epub ahead of print.

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