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    World J Mens Health. 2020 Oct;38(4):412-471. English.
    Published online Aug 06, 2020.
    https://doi.org/10.5534/wjmh.200128
    Copyright © 2020 Korean Society for Sexual Medicine and Andrology
    Review

    Sperm DNA Fragmentation: A New Guideline for Clinicians

    Ashok Agarwal,1 Ahmad Majzoub,2,3 Saradha Baskaran,1 Manesh Kumar Panner Selvam,1 Chak Lam Cho,4,5 Ralf Henkel,6,7 Renata Finelli,1 Kristian Leisegang,8 Pallav Sengupta,9 Catalina Barbarosie,10 Neel Parekh,11 Marco G. Alves,12 Edmund Ko,13 Mohamed Arafa,2,14 Nicholas Tadros,15 Ranjith Ramasamy,16 Parviz Kavoussi,17 Rafael Ambar,18 Manish Kuchakulla,16 Kathy Amy Robert,1 Concetta Iovine,19 Damayanthi Durairajanayagam,20 Sunil Jindal,21 and Rupin Shah22
      • 1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA.
      • 2Department of Urology, Hamad Medical Corporation, Doha, Qatar.
      • 3Department of Urology, Weill Cornell Medicine - Qatar, Doha, Qatar.
      • 4Department of Surgery, Union Hospital, Hong Kong.
      • 5S. H. Ho Urology Centre, Department of Surgery, The Chinese University of Hong Kong, Hong Kong.
      • 6Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK.
      • 7Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa.
      • 8School of Natural Medicine, Faculty of Community and Health Sciences, University of the Western Cape, Bellville, South Africa.
      • 9Department of Physiology, Faculty of Medicine, Bioscience and Nursing, MAHSA University, Jenjarom, Malaysia.
      • 10Department of Genetics, Faculty of Biology, University of Bucharest, Bucharest, Romania.
      • 11Department of Urology, Cleveland Clinic Foundation, Cleveland, OH, USA.
      • 12Department of Microscopy, Laboratory of Cell Biology & Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal.
      • 13Department of Urology, Loma Linda University, Loma Linda, CA, USA.
      • 14Andrology Department, Cairo University, Giza, Egypt.
      • 15Division of Urology, Southern Illinois University School of Medicine, Springfield, IL, USA.
      • 16Department of Urology, University of Miami, Miami, FL, USA.
      • 17Austin Fertility & Reproductive Medicine/Westlake IVF, Austin, TX, USA.
      • 18Urology Department of Centro Universitario em Saude do ABC, Santo André, Brazil.
      • 19Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Caserta, Italy.
      • 20Department of Physiology, Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia.
      • 21Department of Andrology and Reproductive Medicine, Jindal Hospital, Meerut, India.
      • 22Department of Urology, Lilavati Hospital and Research Centre, Mumbai, India.
    • Correspondence to: Ashok Agarwal. Andrology Center and American Center for Reproductive Medicine, Cleveland Clinic, Mail Code X-11, 10681 Carnegie Avenue, Cleveland, OH 44195, USA. Tel: +1-216-444-9485, Fax: +1-216-445-6049, Email: agarwaa@ccf.org , Website: www.Clevelandclinic.org/ReproductiveResearchCenter
    Received July 11, 2020; Revised July 13, 2020; Accepted July 13, 2020.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Sperm DNA integrity is crucial for fertilization and development of healthy offspring. The spermatozoon undergoes extensive molecular remodeling of its nucleus during later phases of spermatogenesis, which imparts compaction and protects the genetic content. Testicular (defective maturation and abortive apoptosis) and post-testicular (oxidative stress) mechanisms are implicated in the etiology of sperm DNA fragmentation (SDF), which affects both natural and assisted reproduction. Several clinical and environmental factors are known to negatively impact sperm DNA integrity. An increasing number of reports emphasizes the direct relationship between sperm DNA damage and male infertility. Currently, several assays are available to assess sperm DNA damage, however, routine assessment of SDF in clinical practice is not recommended by professional organizations. This article provides an overview of SDF types, origin and comparative analysis of various SDF assays while primarily focusing on the clinical indications of SDF testing. Importantly, we report four clinical cases where SDF testing had played a significant role in improving fertility outcome. In light of these clinical case reports and recent scientific evidence, this review provides expert recommendations on SDF testing and examines the advantages and drawbacks of the clinical utility of SDF testing using Strength-Weaknesses-Opportunities-Threats (SWOT) analysis.

    Keywords
    Assisted reproductive techniques outcome; Clinical guidelines; Infertility, male; Oxidative stress; Sperm DNA fragmentation

    INTRODUCTION

    Infertility is defined as the failure of a couple to achieve a clinical pregnancy after one year of regular, unprotected sexual intercourse [1]. Infertility affects more than 15% of couples globally with male factors alone or in combination with female factors, contributing to 50% of the cases [2]. Evaluation of infertile men still relies on conventional semen analysis, though it alone does not accurately predict male fertility potential and success of assisted reproductive technology (ART) [3]. In fact, about 15% of infertile patients have a normal semen analysis [4]. However, assessment of sperm concentration, motility and morphology may not fully reflect impaired sperm DNA integrity [5], which is detrimental for normal fertilization, embryo development and success of ART [6].

    Sperm DNA fragmentation (SDF) can be caused by extrinsic factors (i.e., heat exposure, smoking, environmental pollutants, and chemotherapeutics) as well as intrinsic factors (i.e., defective germ cell maturation, abortive apoptosis, and oxidative stress [OS]) [7]. Compelling evidence demonstrates that OS is a major contributor to male infertility [8]. Reactive oxygen species (ROS) are vital for physiological processes such as apoptosis and capacitation, but an overproduction leads to various deleterious consequences including SDF [9]. Types of DNA damage include mismatch of bases, loss of base (abasic site), base modifications, DNA adducts and crosslink, pyrimidine dimers and single strand breaks (SSB) and double strand breaks (DSB) (Fig. 1). Any of these alterations can induce SDF and compromise natural conception or ART outcomes.

    Fig. 1
    Different types of DNA damage that can occur at DNA level: mismatched bases, abasic sites, base modifications (oxidation, alkylation, deamination), adducts and intrastrand crosslinks, pyrimidine dimers, and single and double strand fragmentation. ROS: reactive oxygen species, UV: ultraviolet.

    Since 1999, there has been a significant increase in the number of studies reporting an association between SDF and male infertility. According to a recent scientometric analysis, the primary focus of SDF research in the past 20 years has emphasized lifestyle factors, varicocele, and asthenozoospermia [10]. Increased SDF levels have been implicated in male infertility while being associated with conditions such as varicocele, male accessory gland infection, advanced paternal age, cancer, chronic illness, exposure to environmental toxins and lifestyle factors [11].

    Moreover, numerous studies have found that increased SDF adversely impacts conception rates [12, 13, 14]. Evidence shows that DNA damage in spermatozoa can affect the health and well-being of offspring [15]. Consequently, the negative impact of SDF on the male fertility potential may encourage more clinicians to utilize SDF testing in the clinical setting [16]. Interventions have also been explored to improve fertility outcomes and promote healthy offspring.

    The present article aims to highlight the clinical utility of SDF testing by providing current evidence for its use in the management of the infertile male. This review begins by examining the underlying mechanisms and risk factors of SDF. It then describes the clinical tests associated with different types of DNA fragmentation, followed by the clinical indications for SDF testing. Finally, male infertility case scenarios with high SDF are presented along with expert recommendations on its management and an illustration of strengths, weaknesses, opportunities and threats (SWOT) of SDF testing.

    ORIGIN OF SPERM DNA FRAGMENTATION

    1. Primary mechanisms underlying sperm DNA fragmentation

    SDF is primarily induced by defective maturation and abortive apoptosis occurring within the testis, or by OS throughout the male reproductive tract [17]. During spermatogenesis, chromatin is compacted through histone exchange with transitional proteins and protamines [18]. This is facilitated by the endogenous nuclease topoisomerase II, creating DNA breaks to reduce torsional stress for histone disassembly and chromatin packaging [19, 20, 21]. If these breaks are not repaired, impairment of chromatin packaging may result in defective maturation and the appearance of sperm with increased SDF in the ejaculate [22, 23, 24, 25, 26, 27]. SDF can also be induced by abortive apoptosis during spermatogenesis. Apoptosis ensures that no defective germ cells differentiate into spermatozoa, however failure of this process may result in the accumulation of spermatozoa expressing apoptotic markers in the ejaculated semen (Fig. 2) [28, 29, 30]. Extrinsic apoptosis is mediated through Fas-ligand binding to a death receptors, such as Fas, activating caspase-8 or 10 [31]. Indeed, the expression of Fas in the ejaculated sperm is an indicator of increased abortive apoptosis [32]. Excessive ROS can induce DNA damage [33] and also activate apoptotic pathways in spermatozoa [34]. Moreover, SDF can be indirectly induced by OS through by-products of lipid peroxidation, particularly malondialdehyde (MDA) and 4-hydroxynonenal (4HNE) which can introduce DNA adducts, such as 8-hydroxy-2′-deoxyguanosine (8-OHdG), 1,N6-ethenoadenosine, and 1,N6-ethenoguanosine, resulting in DNA damage [33, 35, 36, 37]. On the other hand, direct oxidative damage to DNA bases results in formation of adducts such as 8-hydroxy-20-deoxyguanosine (8OHdG), particularly at sites with poor protamine shielding [24, 25]. OS further activates the MAPK pathway, increasing p53 and caspase 3 expression and reducing bcl-2, thereby impairing maturation and promoting apoptosis [38]. OS activates intrinsic apoptotic pathways in spermatozoa, where externalization of phosphatidylserine is an early marker and SDF is a late marker of apoptosis [34]. This process is initiated through a mitochondrial-mediated pathway, where cytochrome c is released into the cytosol resulting in proteolytic activation of caspase 3, 6, and 7 [39, 40].

    Fig. 2
    Overview of the origins of sperm DNA fragmentation (SDF). SDF result from underlying mechanisms such as defective maturation, abortive apoptosis, and oxidative stress. Moreover, clinical (age, infection, cancer, hormonal imbalances, obesity, diabetes) and environmental (heat exposure, environmental toxins, radiation, smoking, drug abuse, diet) risk factors lead to SDF. MAPK: mitogen-activated protein kinase, ERK: extracellular signal-regulated kinase, JNK: c-JUN N-terminal kinase, ROS: reactive oxygen species, ART: assisted reproductive techniques.

    2. Clinical and environmental risk factors of sperm DNA fragmentation

    SDF increases with age, starting in reproductive years and doubling between the ages of 20 and 60 years [41, 42, 43]. This association has been attributed to higher exposure to OS, defective sperm chromatin packaging, and disordered apoptosis that occur with aging [44]. Clinical associations with increased SDF include varicocele, which induces testicular damage and SDF through increased intratesticular temperature and retrograde flow of renal and adrenal metabolites resulting in OS and apoptosis [45, 46]. Genitourinary infections and subsequent leukocytospermia increases ROS production, increasing SDF [47, 48, 49, 50, 51]. Increase in SDF has also been reported in men with testicular cancer and other malignancies, which is suggested to be secondary to the associated endocrine alterations or OS in these pathologies [52, 53, 54, 55].

    Lifestyle and environmental factors induce SDF. Importantly, obese men have higher levels of OS and SDF compared to normal weight or overweight men [56, 57, 58]. Increased scrotal temperature, endocrine imbalance and chronic systemic inflammation are believed to be the mechanisms linking obesity with altered sperm function and reduced fertility potential. Indeed, studies have shown significant improvement in SDF and overall fertility with weight loss [59, 60]. Men with diabetes demonstrate higher levels of SDF due to OS, in association with the generation of advanced glycation end products [61, 62].

    SDF and chromatin decondensation is observed with a subtle 2℃–3℃ increase in physiologic scrotal temperature [63, 64, 65, 66], partly mediated through OS induced apoptosis and elevated stress-inducible protein expression [67, 68, 69]. Increased scrotal temperature is induced by physical abnormalities such as cryptorchidism, retractile testes and varicocele, as well as in acute febrile illnesses and sedentary lifestyles [68, 70, 71, 72].

    Some studies demonstrate increased SDF with air pollution [73, 74, 75]; while others have found no difference [76, 77, 78]. Exposure to heavy metals such as lead, cadmium [79, 80], fenvalerate (synthetic insecticide) [81] and organophosphorus pesticides [82] can cause DNA damage. The effect of occupational toxins depends on proximity and duration of exposure [83]. Bisphenol A and styrene found in synthetic rubber or polyesters, also alters sperm DNA integrity [84, 85, 86, 87].

    Cigarette smoking negatively impacts DNA integrity [88, 89, 90, 91] due to tobacco metabolites [92] such as nicotine [93], cadmium [79, 94], lead [79, 80, 95] and benzopyrene [96]. Alcohol consumption can also increase SDF and cause apoptosis [97, 98, 99].

    Electromagnetic waves, particularly from cell phones, increase mitochondrial ROS production and DNA adduct formation causing DNA damage [100, 101, 102]. Furthermore, radiation therapy for cancer can cause SDF [103].

    These clinical and environmental risk factors increase the production of ROS by different mechanisms, leading to OS and ultimately result in SDF [104, 105, 106, 107, 108].

    SPERM DNA FRAGMENTATION: SINGLE-VERSUS DOUBLE-STRAND BREAKS

    DNA fragmentation is characterized by both SSBs and DSBs. In DNA with SSBs, the other strand can act as a template for replication. SSBs are caused by the action of abortive topoisomerase or DNA ligase activity adjacent to a lesion, which can covalently bind to phosphate and can thereby be fixed. The most commonly occurring lesions are base and sugar modifications and SSBs following oxidation, alkylation, deamination, and spontaneous hydrolysis [109]. When these lesions are not repaired, they can compromise the integrity of the genome [110]. Moreover, OS, lipid peroxidation and protein alteration may also lead to SSBs [111] (Fig. 3A). In general, DSBs are considered harmful to the genomic DNA as they result in genetic rearrangements. DSBs are produced from endogenous sources as a consequence of SSBs during the DNA replication process [112], collapsed replication forks [113], or increased levels of free radicals [112] (Fig. 3B). Furthermore, exogenous causes such as ionizing radiation, genotoxic chemicals, radiomimetic drugs can also lead to DSBs [112, 114, 115].

    Fig. 3
    (A) Main insults that result in DNA single strand breaks are abortive topoisomerase, free radicals, and DNA ligase activity adjacent to lesion. (B) Main insults that result in DNA double strand breaks are free radicals, collapsed replication forks, replication in DNA strand with single-stranded breaks, ionizing radiation, genotoxic chemicals, and radiomimetic drugs.

    Both SSBs and DSBs present in sperm DNA can affect the overall fertility and reproductive outcomes. DSBs negatively affect embryo kinetics and implantation rates, and have been associated with recurrent miscarriages in couples without a female factor [116, 117]. In contrast, SSBs do not significantly impact embryo development or implantation rates [117]. Nonetheless, higher levels of SSBs are inversely related to the natural pregnancy outcome [118]. Thus, evaluation of SSB and DSB may provide important information during fertility evaluation of men [119]. Sperm DNA integrity can be determined using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and other direct tests, such as sperm chromatin structure assay (SCSA) and sperm chromatin dispersion (SCD). However, these assays cannot distinguish between SSBs and DSBs present in the DNA [117]. Depending on the methodology, i.e., either neutral (DSBs) or alkaline (SSBs and DSBs), this distinction can be made only by the Comet assay [120]. The two-tailed Comet assay can directly differentiate between the SSBs and DSBs [121]. On the other hand, the newest SDF test introduced for the immunodetection of gamma histone 2AX (γH2AX), can only assess DSBs [115]. The γH2AX is the phosphorylated form of γH2AX from the histone 2A family and is a highly specific and sensitive molecular marker of DSBs [122].

    WHAT TEST SHOULD I ORDER?

    Several assays are used for SDF evaluation in clinical practice (Table 1). The TUNEL assay is based on labelling free 3′-OH nicks with dUTP [123]. While Comet assay identifies SDF based on electrophoretic separation of DNA where damaged DNA forms a comet-like profile. In the SCD test, a distinct “halo” (dispersed DNA loops) is observed after removal of DNA-linked proteins, while no or small halos indicate DNA damage [124]. The SCSA uses metachromatic acridine orange, which fluoresces green and red after binding to double- (native) or single-stranded (damaged) DNA, respectively [125].

    Table 1
    Published SDF cut-off values for the prediction of pregnancy outcomes using different laboratory assays

    As summarized in Table 1, several studies have attempted to identify clinical SDF cut-offs for the prediction of natural or ART-related pregnancy [126, 127, 128, 129, 130, 131]. Although there remains no unanimous consensus on a specific cut-off value, a recent meta-analysis suggests that a cut-off of 20% can potentially differentiate between fertile and infertile men [108]. Different SDF values are reported for prediction of pregnancy in natural conception or ART (in vitro fertilization [IVF], intracytoplasmic sperm injection [ICSI], or both) settings by analyzing native semen, sperm processed by swimup or density gradient centrifugation (DGC) as well as in cases where own or donor oocytes are used. Despite the heterogeneity of the published studies, the challenges related to the identification of unbiased cut-off values for the prediction of pregnancy and the use of SDF assays in clinical practice, it appears that the TUNEL assay is most commonly used [10], as it is accurate and reliable [132]. While it would be highly desirable if a globally accepted assay with strong predictive value would be performed by all clinics, in reality the choice of the SDF assay in individual clinics often depends on instrumentation availability, trained personnel and the cost of the assay to be performed in terms of reagents and run-time.

    The diagnostic value of these tests for assisted reproduction can be increased by the evaluation of OS. A moderate correlation between ROS and SDF has been reported [133, 134, 135]. Decreased total antioxidant capacity, reflecting the amount of seminal antioxidants [136], has been associated with elevated SDF and male infertility [137] and an increased risk of spontaneous miscarriage [138]. By using oxidation reduction potential (ORP) as a measure of redox balance and SDF (by TUNEL) with cut-off values of 1.36 mV/106 sperm/mL and 32%, respectively, fertilization has been predicted with high sensitivity and specificity [139]. However, few studies report weak [140, 141] or no correlation between ORP and SDF [134], suggesting that ORP and SDF testing might reflect the general impact of seminal OS on sperm functions and specifically on DNA, respectively. Therefore, ORP cannot be recommended as a standalone test in substitution of SDF evaluation, considering that other factors, such as abortive apoptosis or defects in DNA protamination, can render spermatozoa more susceptible to OS and DNA damage, even at relatively low ROS levels [142]. Moreover, defects in the protamination and in DNA condensation can leave unligated nicks [143] while a faulty DNA rearrangement may lead to severe DNA damage [144].

    WHICH PATIENTS ARE SUITABLE FOR SPERM DNA FRAGMENTATION TESTING?

    The considerable research conducted in recent years has improved our understanding of the clinical scenarios where SDF testing is most beneficial. We recently published clinical practice guidelines endorsed by the Society of Translational Medicine recommending SDF testing in patients with unexplained infertility, recurrent pregnancy loss (RPL), and clinical varicocele, prior to undergoing ART and in patients exposed to lifestyle risk factors and environmental toxicants [145]. An updated evidence supporting these recommendations are presented in Table 2.

    Table 2
    Correlation between clinical outcomes and SDF testing: evidence-based report

    For evidence-based reporting on SDF, the PubMed database was searched from the time of inception to December 2019. The search was limited to human studies published in English. The term ‘sperm DNA fragmentation’ was searched in combination with the following keywords using the Boolean expression “AND”: ‘intracytoplasmic sperm injection’, ‘fertilization in vitro’, ‘intrauterine insemination, ‘recurrent pregnancy loss’, ‘varicocele’, ‘idiopathic male infertility’, ‘unexplained male infertility’, ‘genital tract infections’, ‘male age’, ‘obesity’, ‘alcohol’, ‘smoking’, ‘air pollution’, ‘lifestyle’, ‘plastics’, ‘industrial’.

    The inclusion criteria for the evidence-based reporting on SDF were (a) studies with male patients having primary or secondary infertility as target population and (b) studies reporting clinical outcome parameters including changes in semen parameters or SDF levels, fertilization, pregnancy, birth, and miscarriage rates.

    The search yielded a total of 1,584 publications. The title and abstracts were cross-checked by three independent researchers and 251 articles were considered in this review. Relevant information was extracted from the studies that fulfilled the selection criteria and presented in Table 2.

    1. Natural conception

    As stated previously, sperm DNA integrity plays a crucial part in the fertilization process and in early embryo development, thereby directly influencing the likelihood of natural conception. Reports have linked SDF to low cleavage rates [146, 147] and to the arrest of embryonic development after the second cleavage state [148]. Using the SCSA on 215 Danish first pregnancy planners, Spanò et al [149] reported an inverse relationship between the level of SDF and probability of natural pregnancy in a menstrual cycle. Moreover, evidence linking SDF to natural pregnancy rates can be drawn from the meta-analysis by Zini [146] which included three studies and 616 couples and revealed that high SDF, determined by the SCSA test, was associated with failure to achieve natural pregnancy with an odds ratio (OR) of 7.01 (95% confidence interval [CI]=3.68–13.36).

    2. Assisted reproductive technology outcomes

    Numerous reports investigating the predictive role of SDF on ART outcomes have reported contradictory results [41, 88, 146, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164]. This controversy can be attributed to a number of factors, such as the study selection methods used by these reviews, heterogeneity of the conducted studies, differences in the SDF testing methods and female age and fertility status to name a few. With regards to intrauterine insemination (IUI), Chen et al [150] analyzed the results of 10 articles and demonstrated that high SDF levels were associated with significantly lower pregnancy (relative risk [RR]=0.34, 95% CI=0.22–0.52; p < 0.001) and delivery rates (RR=0.14, 95% CI=0.04–0.56; p < 0.001). This result was also echoed by two other meta-analyses reporting that patients with low SDF had an OR for clinical pregnancy ranging between 7.01 and 16 [41, 146]. However, a recent meta-analysis by Sugihara et al [151] analyzed the results of 3 studies and reported that while low SDF was associated with better pregnancy rates with a RR of 3.30 (95% CI=1.16–9.39), the test performance was low as it had a low positive predictive value (17%).

    As for SDF impact on IVF/ICSI, various meta-analyses have been published assessing the rates of pregnancy, miscarriage and live birth. Four meta-analyses [146, 152, 154, 157] reported that high SDF was associated with lower pregnancy rates with conventional IVF with an OR ranging between 0.68 and 1.7. With regards to ICSI, only Simon et al [157] reported significantly lower pregnancy rates with high SDF, while the remaining three meta-analyses failed to find a significant association [146, 152, 154].

    Live birth rate was examined by one meta-analysis and was found to be significantly lower in men with high SDF following both IVF and ICSI with a combined OR of 1.17 (95% CI=1.07–1.28; p=0.0005) [158].

    Three meta-analyses examined the miscarriage rate following ART in relation to SDF [146, 153, 154]. Overall, high SDF was associated with greater risk for miscarriage following both IVF and ICSI with a combined OR ranging between 2.28 and 2.48.

    Contrary to the abovementioned studies, two metaanalyses of slightly different design reported rather discouraging results. Cissen et al [155] analyzed 30 studies to assess the value of SDF in predicting the chance of ongoing pregnancy with IVF or ICSI. Overall, SDF testing had fair to good sensitivity with poor specificity. The authors constructed a hierarchical summary receiver operating characteristic curve, which reported fair predictive performance for TUNEL and Comet assays, while the predictive power for SCSA and SCD was poor. The authors concluded that the current SDF testing methods had a limited ability in predicting the chance of pregnancy in the context of ART. Furthermore, Collins et al [156] analyzed 13 studies having an extensive study heterogeneity and reported random effect model of the diagnostic OR rather than sensitivities and specificities. While the authors detected that sperm DNA integrity was significantly associated with pregnancy following IVF and ICSI with a diagnostic OR of 1.44, 95% CI=1.03–2.03; p=0.04 the likelihood ratios (LR(+)=1.23, LR(−)=0.81) were in a range suggesting that testing did not alter the outcome and was hence not clinically relevant. Subgroup analyses showed that the test accuracy was not materially affected by the testing method (TUNEL or SCSA) or the ART modality (IVF or ICSI).

    Taking the abovementioned results all together, there is reasonable evidence to state that SDF is relevant in the context of ART. A high SDF value is associated with decreased pregnancy rate with IUI and IVF and with increased miscarriage rate following both IVF and ICSI.

    3. Varicocele

    Varicocele is the most common correctable cause of male infertility, prevalent in about 40% of men with primary infertility and up to 80% of men with secondary infertility [165]. Improper patient selection for varicocele ligation was an important reason for the controversy regarding the effect of treatment on pregnancy outcome. This has been resolved by indicating surgery only for patients with clinically evident disease and abnormal semen parameters [166]. However, even when proper patient selection is practiced, pregnancy is observed in only, 40% to 50% of patients following surgery [167]. Hence efforts have been made to refine the indications of surgery in varicocele patients and interest in SDF has emerged after finding a significant positive association with varicocele [168].

    Studies have shown that men with varicocele have significantly higher levels of SDF than controls regardless of their fertility status, suggesting that varicocele is independently associated with impaired DNA integrity [168, 169, 170, 171, 172, 173, 174, 175]. A recent cross-sectional study was carried out on 2,399 men attending a fertility clinic, 16.3% (391/2,399) of whom were diagnosed with varicocele [171]. Men with varicocele had a significantly increased percentage of seminal SDF (p=0.03), abnormal chromatin packaging (p=0.001), and abnormal mitochondrial membrane potential (p=0.03) in comparison to men without varicocele. It is important to note that varicocele treatment is associated with a reduction in the SDF level. A meta-analysis conducted by Wang et al [176] invloving 12 studies (7 studies assessed SDF in patients with varicocele, while 6 studies determined the outcome of surgery) revealed that varicocele was associated with significantly higher levels of SDF compared with controls with a mean difference (MD) of 9.84% (95% CI=9.19–10.49; p<0.00001). Varicocele treatment resulted in significant reduction of SDF levels when compared to control group (MD of −3.37%; 95% CI −4.09 to −2.65; p<0.00001).

    A recent review by Roque and Esteves [177] investigated 21 studies, including >1,200 subjects in whom the effect of varicocele ligation on SDF was assessed. The authors observed that all studies reported a significant decrease in SDF following varicocele ligation during a follow-up period ranging from 3 to 12 months. Few studies reported the pregnancy outcome following treatment and generally identified lower SDF values in couples who conceived compared with those who did not. Smit et al [178] utilized the SCSA in 49 patients before and after varicocelectomy and reported significant reduction in SDF values following surgery (MD=5%; p=0.019). Out of the 49 subjects, 18 (37%) conceived spontaneously and 11 (22%) conceived with ART. The SDF levels were significantly lower in patients who conceived spontaneously or with ART (26.6%±13.7%) in comparison to patients who did not conceive at all (37.3%±13.9%) (p=0.013). Another study by Ni et al [179] assessed the ratio between protamine 1 and 2 mRNAs (P1/P2) as well as SDF levels using PCR and SCSA, respectively, in 42 infertile men with varicocele and 10 fertile donors with normal semen parameters. The study group underwent varicocelectomy and pregnancy was achieved by 23.81% of patients 6 months after surgery. Compared with couples who failed to conceive following varicocelectomy, pregnant couples had significantly lower mean P1/P2 mRNA ratio and SDF levels.

    4. Recurrent pregnancy loss

    RPL, defined as spontaneous loss of 2 or more pregnancies. Prior to 20 weeks of gestation, has been linked to elevated levels of SDF in several investigations. Studies performed using different SDF testing methods such as SCD [180], TUNEL assay [181] and SCSA [182] reported significantly higher SDF levels among patients with RPL in comparison to normal controls [183]. Aiming to understand the male contribution to RPL, Tan et al [184] recently conducted a meta-analysis on 12 prospective and 2 retrospective studies including 530 men with RPL and 639 fertile controls. The study revealed a significant association between RPL and SDF with an average MD of 11.98, 95% CI 6.64–17.32; p<0.001 indicating that men with RPL had significantly higher SDF values than the control group. Similar result was reported during subgroup analysis according to the SDF testing method.

    5. Idiopathic and unexplained male infertility

    Unexplained male infertility (UMI) is a term given to couples who otherwise have a completely normal fertility evaluation. Studies have revealed that men with normal semen parameters may still have elevated levels of SDF. Oleszczuk et al [185] have shown that about 1 out of 5 men with unexplained infertility had a SDF level above 30%. Another prospective study conducted on 25 men with unexplained infertility revealed that a SDF level above 30%, measured by SCD, was detected in 29% of the subjects [185]. Similarly, idiopathic male infertility, a term given to describe men with one or more abnormality in semen parameters without an identifiable etiology, has been associated with high SDF. Studies have confirmed a significant inverse correlation between the SDF level and sperm count, motility and normal morphology [140, 186, 187]. A few comparative studies also have revealed that men with idiopathic male infertility tend to have significantly higher SDF than normal fertile controls [188] (Table 2).

    6. High risk patients

    Various studies have been conducted linking several lifestyle factors/environmental exposures to elevated levels of SDF [78, 94, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198]. These factors include (i) Physical agents such as radiation and heat; (ii) Chemical agents such as cigarette smoke, airborne pollutants, and chemotherapeutic drugs; and (iii) Biological factors including sexually transmitted infections, increasing male age, elevated body mass index (BMI) and medical conditions such as insulin dependent diabetes [78, 94, 189, 190, 191, 192, 193, 194, 195, 196, 197, 199, 200, 201]. Elevated OS levels is believed to be the main mechanism resulting in SDF with these exposures. Moreover, occupational exposures have been considerably linked to SDF and altered fertility potential. Examples of such exposures include lead and cadmium [83], organochlorine pollutants found in pesticides [82], and bisphenol A, a compound widely utilized in plastic containers used in food and drink industries [86].

    MANAGEMENT OF HIGH SPERM DNA FRAGMENTATION

    1. Oral antioxidant therapy

    Antioxidants play an important role in general health by scavenging excess free radicals and thus, preventing oxidative damage to macromolecules. However, the benefits of exogenous antioxidant therapy are less clear [202]. Clinicians commonly utilize antioxidant therapy to maintain redox balance by scavenging ROS [203]. Several clinical trials have demonstrated the positive effects of antioxidants on SDF in infertile men (Table 3) [204, 205, 206, 207, 208]. However, with no validated guidelines on antioxidant supplementation, they are frequently used empirically. Antioxidants can be easily purchased over the counter and are commonly considered safe. However, excess antioxidant supplementation may have a paradoxical effect on OS and SDF, a condition referred to as reductive stress [209]. As a result, the indiscriminate use of oral antioxidants in men without elevated OS should be avoided [210].

    Table 3
    Therapeutic interventions for SDF: evidence based report

    2. Control of infections/inflammation/leukocytospermia

    Among infertile men, the incidence of infection ranges from 2% to 18% [211]. Sexually transmitted infections or prostatitis are associated with elevated OS and leukocytospermia, which may result in elevated SDF and impaired fertility [212, 213]. Antibiotic therapy has been reported to be effective in treating infection-induced elevated SDF levels (Table 3) [47]. Moreover, empirical antibiotic therapy for leukocytospermia may improve natural pregnancy rates [214].

    3. Varicocelectomy

    Varicocele has been consistently associated with increased SDF values. It has been established that varicocele repair can improve OS markers and reduce SDF indices [11]. Current data supports the value of varicocele repair in reducing SDF and improving fertility (Table 3). In a systematic review of 21 studies evaluating the effect of varicocelectomy on SDF, all studies reported a significant decrease in SDF by an average of approximately 8% [177]. Moreover, varicocele repair has demonstrated improvements in pregnancy success in both natural conception and assisted reproduction by way of improved SDF indices [178]. Given these observations, the association between palpable varicocele and SDF should be considered, and varicocelectomy discussed with patients as a potential solution to improving fertility.

    4. Lifestyle modifications

    Exposure to environmental and lifestyle factors have far-reaching implications on male fertility. Current data has consistently associated smoking with higher SDF values when compared to non-smokers [91, 99, 191, 215], however no study has yet evaluated the impact of smoking cessation on SDF. There have also been numerous environmental factors such as airborne pollutants, ionizing radiation, and pesticides linked with increased SDF values [74, 82, 193, 216, 217]. Several studies have demonstrated higher SDF in obese men, yet a recent meta-analysis found no robust association between BMI and SDF [218]. No concrete evidence in lifestyle modification impact on SDF exists [219], however, weight loss and dietary changes have been proposed to benefit SDF indices in patients [220, 221] (Table 3).

    5. Short ejaculatory abstinence

    The negative impact of prolonged ejaculatory abstinence (EA) on SDF has been reported without significant detrimental effect on conventional semen parameters [222, 223]. Therefore, short-term recurrent ejaculation may be a simple noninvasive maneuver to improve SDF. Although the beneficial effect of short EA on natural conception is unclear, application of the technique to assisted reproduction may have its value [224, 225, 226]. In addition to higher pregnancy rates in ICSI, recurrent ejaculation has been associated with a significantly lower SDF [227] (Table 3).

    6. Sperm processing and preparation

    Laboratory conditions (i.e., prolonged incubation, centrifugation, cryopreservation and use of different media) can significantly impact SDF by increasing OSmediated DNA damage [228, 229, 230, 231]. Conventional (swimup, DGC) and advanced techniques can select sperm with low levels of SDF [232, 233, 234, 235, 236] (Table 3). Magneticactivated cell sorting (MACS), based on the detection of phosphatidylserine [237], shows a better selection alone [238, 239] or in combination with DGC [240, 241, 242, 243]. Intracytoplasmic morphologically selected sperm injection (IMSI) uses high magnification to select the most morphologically normal sperm, as the presence of vacuoles in the nuclear region has been associated with high SDF [244, 245]. Other approaches include the physiological intracytoplasmic sperm injection (PICSI), based on sperm binding to hyaluronic acid, and microfluidic devices, allowing sperm migration along microchannels [246, 247, 248, 249].

    7. Use of testicular sperm for intracytoplasmic sperm injection

    Testicular sperm has been explored as a treatment option for high SDF based on the finding of lower SDF in testicular sperm than ejaculated sperm [250, 251], and better ICSI outcome [12, 252, 253]. However, surgical sperm retrieval [254] carries risk of anesthetic and surgical complications. Furthermore, possible higher aneuploidy rate in testicular sperm is a concern [255] despite a recent report of opposing view [256]. Therefore, the use of testicular sperm in clinical management of nonazoospermic patients with high SDF is still debated.

    CLINICAL CASE REPORTS

    1. Case 1

    A 37-year-old male, presented to the male infertility unit complaining of primary infertility of 3 years duration. He is a navy lieutenant and is physically fit. He does not have a history of recent febrile illness, genitourinary infections or trauma. He does not have a significant past medical or surgical history. He smokes half a pack of cigarettes a day for the past 15 years. There is no family history of infertility. His wife is 26 years old with regular menses and normal fertility evaluation. There is no consanguinity between the couple. On physical examination, he was of normal BMI (26 kg/m2). Genital examination revealed normal phallus, normal testis size, palpable vasa bilaterally and no palpable varicocele. An outside semen analysis demonstrated a volume of 3 mL, sperm concentration of 11 million/mL, total motility of 40% (progressive motility 20%) and normal morphology of 10%.

    Repeat semen analysis with SDF testing performed at our center demonstrated a volume of 4.5 mL, sperm concentration 9 million/mL, total motility 30% (progressive motility 10%), normal morphology of 3% (WHO, 2010) and SDF of 45% (using the SCD test [Halosperm], normal<30%). His serum hormone levels were as follows: testosterone 17.5 nmol/L (normal=10.4–30.86 nmol/L), follicle stimulating hormone (FSH) 2.5 IU/L (normal=1.5–12.4 IU/L), luteinizing hormone (LH) 2 IU/L (normal=1.7–8.6 IU/L), estradiol 122 pmol/L (normal=94.8–223 pmol/L), and prolactin 245 mIU/L (normal=85–323 mIU/L). Scrotal ultrasound demonstrated normal testicular volume echogenicity, and vascularity and absence of epididymal cysts and varicoceles.

    The patient was diagnosed with idiopathic oligoasthenoteratozoospermia and high SDF. He had a lifestyle risk factor and was counselled about the importance of smoking cessation on his overall health and fertility potential.

    He was prescribed the following antioxidants: vitamin C 500 mg twice daily, L-carnitine+zinc 1,000 mg twice daily, and folic acid 0.5 mg once daily for 3 months.

    On the 3-month follow-up visit, a repeat semen testing showed a volume of 3 mL, sperm concentration of 17 million/mL, total motility 45% (progressive motility 25%), normal morphology 5%, and SDF 26%. There was still no pregnancy and hence, the couple were advised to undergo IUI. The patient was kept on vitamin C and L-carnitine+zinc regimen.

    The procedure was performed 5 weeks following the last patient visit. The prewash total motile sperm count was 22.5 million and the post-wash total motile sperm count was 13 million. The patient was seen in the clinic 6 weeks following his IUI and reported that his wife was pregnant.

    2. Case 2

    A 41-year-old male, presented for fertility evaluation after failure of conception for 3 years. His past medical and surgical history were unremarkable. He did not smoke or consume alcohol. On physical examination, the patient had normal built (height 176 cm, weight 82 kg, BMI 26.5 kg/m2). On genital examination, both testes were normally descended with normal size and consistency, both epididymes were normal, vasa deferentia could be felt bilaterally, and no varicocele could be appreciated either side. The spouse was 32 years old with regular menses, no gynecological problems and normal ovarian reserve (anti-mullerian hormone: 18.9 pmol/L, normal=0.071–52.4 pmol/L).

    Semen analysis demonstrated a volume of 2.5 mL, sperm concentration of 34 million/mL, total motility of 8%, 0% progressive motility and 5% normal morphology. SDF testing was performed using the Halosperm Kit and was found to be high (90%). Hormonal profile assessment showed normal levels of testosterone, FSH, LH, prolactin and estradiol. The patient was given antioxidants in the form of L-carnitine 1,000 mg+zinc twice daily, vitamin C 1,000 mg once daily, co-enzyme Q10 and selenium for 3 months and on repetition of SDF, it was still high (85%). The couple were counseled and decided to go for a trial of ICSI using ejaculated sperm. The female was started with standard long protocol. On the day of ICSI, 16 cumulus-oophorus complexes were collected, 13 of which were in metaphase II (MII) and were used in ICSI. After 24 hours only one oocyte was fertilized and at day 3, it showed no division therefore no embryo transfer was done.

    After 4 months, SDF was still elevated (85%) and the couple were scheduled for a second ICSI trial using testicular sperm which was retrieved by testicular sperm aspiration on day of ICSI. In total, 22 oocytes were collected, 15 of which were MII. At 24 hours, 9 oocytes were fertilized. Two embryos were transferred on day 5. Pregnancy test was positive after 2 weeks and the spouse delivered a healthy girl.

    3. Case 3

    A 44-year-old male, presented for fertility evaluation after failure of conception for 6 years. He had an unremarkable past medical and surgical history. He was a non-smoker and non-drinker. On physical examination, the patient has normal built (height 177 cm, weight 77 kg, BMI 24.6 kg/m2). On genital examination, both testes were normally descended with normal size and consistency, both epididymis were normal, vasa deferentia could be felt bilaterally, and varicocele could be appreciated on both sides clinically (left grade III and right grade I). The spouse was 30 years old with regular menses and no gynecological problems. The couple performed one IUI trial 3 years ago but it was unsuccessful.

    Semen analysis showed oligoasthenoteratozoospermia with a sperm concentration of 6 million/mL, total motility of 34%, 4% progressive motility, and 2% normal morphology. SDF testing with the Halosperm Kit was high (45%). Hormonal profile assessment showed normal levels of testosterone, FSH, LH, prolactin and estradiol. Scrotal ultrasound confirmed bilateral varicocele with vein diameters of 4.8 mm and 3.2 mm on left and right sides, respectively. The couple were counseled on treatment options and consented to proceed with surgical varicocelectomy. Bilateral microsurgical subinguinal varicocelectomy was performed without any complications.

    After 3 months, the patient repeated the semen analysis which demonstrated improvement but with continued oligoasthenoteratozoospermia with a sperm concentration of 9 million/mL, total motility of 65%, 8% progressive motility, and 3% normal morphology. SDF was normalized (25%). The couple were counseled for assisted conception, but they opted to try for natural conception for another 3 months. At 6 months following surgery, they achieved a spontaneous pregnancy and subsequently delivered a healthy girl.

    4. Case 4

    A 34-year-old male, presented for fertility evaluation. His wife was 33 years old with regular menses and no gynecological problems. They were married for 8 years and had a 6.5-year-old boy who was conceived spontaneously. The husband had an unremarkable past medical history. He underwent left orchidectomy following failed orchiopexy for an intra-abdominal left undescended testis at the age of 6 years. He was a nonsmoker and did not consume alcohol. On physical examination, the patient had normal built (height 182 cm, weight 98 kg, BMI 29.6 kg/m2). On genital examination, the left scrotal sac was empty and underdeveloped. The right testis, epididymis, and vas deferens were normal, and there was no palpable varicocele.

    Semen analysis showed a sperm concentration of 14 million/mL, total motility of 45%, 15% progressive motility and 4% normal morphology. SDF was tested twice using Halosperm Kit and was high (47% and 49%). ORP was assessed using the MiOXSYS system and was high (2.9 mV/106 sperm/mL, normal=1.34 mV/106 sperm/mL). Hormonal profile assessment showed normal levels of testosterone, FSH, LH, prolactin and estradiol. The patient was given antioxidants (containing mainly selenium, L-carnitine, L-arginine, Coenzyme Q10, Lycopene, N acetyle l-cysteine, vitamin C, and E) for 3 months. On repetition of semen analysis, it showed 31 million/mL, total motility of 50%, 25% progressive motility and 8% normal morphology, while SDF (25%) and ORP (1.2 mV/106 sperm/mL) normalized. One month later, his wife achieved a spontaneous pregnancy and she delivered a healthy boy.

    EXPERT RECOMMENDATIONS ON SPERM DNA FRAGMENTATION TESTING

    The extensive literature search conducted in this review reveals that SDF significantly impacts male fertility and its testing in specific clinical circumstances may augment the treatment strategy resulting in better outcomes. Accordingly, a clinical algorithm is set forth by this expert panel to elucidate the application of SDF testing in clinical practice (Fig. 4). Patients presenting with infertility should be evaluated with a complete medical and reproductive history, undergo physical examination by a reproductive specialist or urologist and provide at least two semen specimens for conventional analysis [257, 258, 259].

    Fig. 4
    Clinical algorithm to elucidate the applications of sperm DNA fragmentation (SDF) testing in clinical practice. ICSI: intracytoplasmic sperm injection.

    Men with idiopathic and UMI, RPL, and modifiable lifestyle risk factors should undergo SDF testing (grade C recommendation). This recommendation is based on the evidence linking high SDF levels in the abovementioned conditions. It is also aimed at providing pertinent treatment strategies directed at lowering SDF levels. Oral antioxidant therapy may be considered in these regards (grade C recommendation). While its benefit in alleviating SDF and improving live birth rates in infertile men has been reported by a Cochrane meta-analysis [260], further research is needed to refine the ideal candidates and treatment regimen.

    Diet modification and weight reduction may help in reducing SDF (grade C recommendation). However, further research is needed to confirm the role of lifestyle modifications in improving sperm DNA integrity and possibly translate into better reproductive results. Nonetheless, the information provided by SDF testing might help to monitor patient compliance and treatment prognosis.

    Another indication for SDF testing is in patients who are diagnosed with clinical varicocele (grade C recommendation). The findings of higher SDF in both fertile and infertile men with varicocele than controls [168] and significant decrease in SDF levels after varicocele repair [261] provide the rationale of SDF testing in refining the selection of varicocelectomy candidates. In addition, reduction in SDF seems to translate into better reproductive outcomes [262, 263, 264]. Although the association between SDF and high-grade varicocele is much stronger, patients with low-grade varicocele had achieved improvement in natural pregnancy rate that were similar to those with high-grade varicocele after surgery [265].

    SDF testing should also be offered to infertile couples prior to initiating or after failure of IUI/IVF (grade C recommendation). The relationship between SDF and ART outcomes has been extensively investigated. Controversies persist in view of heterogeneous nature of the studies [16, 266]. In general, high SDF is one of the etiologies in patients with recurrent IUI or IVF failure [267]. In contrast to the association between SDF and IUI/IVF outcomes, there is compelling evidence suggesting that SDF has a negligible effect on ICSI outcome measures [154, 158, 268]. These results signify the potential role of ICSI in the treatment of men with high SDF. Patients with persistently high SDF result should be directed towards ICSI, such recommendation will avoid unnecessary delay in definitive treatment which is particularly important in couples with limited reproductive window (grade C recommendation).

    Finally, SDF testing is indicated in couples with recurrent miscarriage following ICSI (grade C recommendation). While high levels of SDF appear not have a significant impact on ICSI pregnancy rates [146, 152, 154, 157], a greater risk of miscarriage following ICSI has been reported by several meta-analyses [146, 153, 154]. A number of interventions have been explored in the context of ICSI to reduce SDF levels and consequently achieve a better outcome. Various sperm selection methods (swim-up, DGC, MACS, IMSI, PICSI) are able to identify sperm with intact DNA integrity for injection [237, 240, 241, 242, 243, 244, 246]. The significantly lower SDF levels in testicular compared to ejaculated sperm supports the use sperm harvested from testis as a plausible maneuver to bypass sperm DNA damage which occurs during the epididymal transit [250]. A metaanalysis of five studies favored the use of testicular sperm by demonstrating better clinical pregnancy and live birth rates [12]. The utilization of testicular sperm is further supported by recent reports and better reproductive outcomes that have been reported in both oligozoospermic and normozoospermic men with prior ICSI failure [269, 270]. Nonetheless, the invasive nature of sperm retrieval procedures and the higher rates of sperm aneuploidy with testicular sperm can be considered as potential disadvantages for this treatment approach which certainly warrants further investigation [271, 272].

    STRENGTHS-WEAKNESSES-OPPORTUNITIES-THREATS (SWOT) ANALYSIS ON THE CLINICAL UTILITY OF SPERM DNA FRAGMENTATION TESTING IN SPECIFIC MALE INFERTILITY SCENARIOS

    SWOT analysis, a system that was originally developed for financial studies, has been recently applied to health sciences. It explores the strengths and weaknesses of a given method in an attempt to identify the threats and opportunities accessible to overcome certain gaps hindering its broad application. Studies included in this review (Table 2) were analyzed using the SWOT method to understand the perceived advantages and drawbacks for the clinical utility of SDF in specific clinical scenarios (Fig. 5).

    Fig. 5
    Strengths-Weaknesses-Opportunities-Threats (SWOT) analysis on the clinical utility of sperm DNA fragmentation (SDF) testing in specific male infertility scenario. ART: assisted reproductive techniques.

    1. Strengths

    SDF testing can serve as an ancillary test to conventional semen analysis in specific clinical scenarios. Evidence indicates that higher levels of SDF are observed in patients who are unable to conceive naturally [149, 273, 274, 275], who present with UMI/idiopathic infertility [185, 188, 276, 277, 278, 279, 280, 281, 282], have RPL [116, 138, 180, 181, 182, 183, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295], are diagnosed with varicocele [169, 170, 171, 172, 173, 174, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305], have a negative ART outcome [13, 41, 88, 117, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330] and who are found to have lifestyle/environmental risk factors [42, 47, 56, 65, 74, 82, 84, 88, 190, 191, 192, 193, 195, 196, 197, 243, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349].

    The widespread use of SDF testing has been hampered by the belief that no effective treatment exists to alleviate high SDF in clinical practice. On the contrary, studies have shown that a number of interventions can be utilized in this regard. Examples of such interventions include recurrent ejaculation to shorten the abstinence time [222, 227, 350, 351, 352], oral antioxidant therapy [205, 207, 208, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362], performing varicocelectomy for patients with clinical varicocele [168, 176, 177, 179, 263, 264, 360, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382], treating genitourinary infections when diagnosed [47], and utilizing advanced sperm selection techniques for ICSI such as PICSI/IMSI [383, 384, 385] or using testicular sperm instead of ejaculated sperm [251, 252, 269, 386, 387].

    2. Weaknesses

    Perhaps the main limitation of SDF testing is the lack of a definitive cut-off value above which a sample is considered anomalous. It is worth mentioning that various SDF thresholds may be determined based on the predicted outcome measure (fertility/infertility, ART success/failure, etc.). Indeed, several cut-off values were reported having a fair to good overall accuracy in predicting various outcome measures (Table 1). Despite the differences in the reported cut-offs, a recent meta-analysis by Santi et al. compared the SDF results of four different assays (TUNEL, SCD, SCSA, and Comet) between 2,883 infertile men and 1,294 fertile men. The authors identified a SDF cut-off of 20% which had a good predictive power in differentiating between fertile and infertile men with a sensitivity of 79% and a specificity of 86% (area under the curve=0.844) [108].

    Another weakness for the utility of SDF testing is the existing moderate to low evidence in support of its use in the above-mentioned clinical scenarios. The heterogenous nature of the conducted studies and the scarcity of randomized clinical trials are possibly the main reasons behind the obtained level of evidence. Furthermore, few contradictory studies have been reported in almost every clinical scenario. A number of studies failed to find a significant association between high levels of SDF and UMI/idiopathic infertility [190, 388], RPL [389, 390, 391, 392], and likelihood of conception whether natural [381], or following ART [314, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410]. While 50%–60% of patients with varicocele have elevated SDF levels, it is not uncommon to find a normal SDF result in infertile men with varicocele who might have a conventional semen parameter abnormality. As for lifestyle/environmental risk factors, no solid evidence exists to support the benefit of lifestyle modification on the SDF level [199, 411, 412].

    3. Threats

    The lack of sufficient high-quality evidence supporting the utility of SDF testing resulted in international societies (American Society for Reproductive Medicine, American Urologic Association, European Association of Urology [EAU]) not to recommend its routine use for the evaluation of male infertility. However, since many confounding factors can impact the likelihood of conception, it is not uncommon in the field of reproduction to provide recommendations for diagnostic tests based on lower quality of evidence. Nonetheless, the increasing number of publications exploring the utility of SDF testing in recent years should provide enough fuel for an update to reproductive society guidelines. This is recently witnessed in the latest update of the EAU guidelines on male infertility which recommends SDF testing for the assessment of couples with RPL following natural or ART conceptions and in men with unexplained infertility [259].

    Case scenarios are commonly used in medical literature to describe a certain clinical condition. However, they may not accurately represent all the possible presentations that might be seen in the clinic. Despite this, we have utilized this method to personalize the message giving it a clinical perspective.

    Finally, the cost of SDF testing ranges between $150–300 (Table 4) which is another important factor limiting its routine use in clinical practice. However, cost is also a major drawback to several fertility related therapeutic interventions that are usually not covered by medical insurance [413]. While SDF testing may be considered an additional cost for patients undergoing fertility treatments, such as ART or varicocelectomy, understanding the circumstances where this assay is most beneficial should help in improving the outcome of treatment and may possibly impact the overall treatment cost.

    Table 4
    Comparison of different SDF tests

    4. Opportunities

    Further studies of adequate power and controlled design are necessary to enhance our understanding of the clinical utility of SDF. This review inspected the available literature with regards to various applications for SDF testing in clinical practice. However, a number of gaps remain and are considered potential areas of research. These areas are particularly involved with demonstrating the impact of interventions on SDF reduction and more importantly on fecundity.

    CONCLUSIONS

    SDF is detrimental for normal fertilization, embryo development and success of ART and therefore, SDF testing is increasingly being utilized in the evaluation of male infertility. SDF can be induced endogenously by defective maturation and abortive apoptosis occurring within the testis, or by OS throughout the male reproductive tract. It can also result from exogenous sources including clinical disease states (varicocele, cancer, diabetes), lifestyle risk factors (smoking, alcoholism, obesity), and environmental exposures (air pollution, pesticides, industrial chemicals). Various SDF testing methods are available; while a single specific cut-off value has not been unanimously identified, a threshold of 20% is believed to be hold a good discriminative accuracy between fertile and infertile men. The thorough literature review presented in this manuscript identifies specific clinical scenarios where SDF testing is most beneficial. These include patients with unexplained and idiopathic infertility, RPL, varicocele, opting for ART and in those with lifestyle/environmental risk factors. A number of therapeutic interventions can be undertaken in patients with high SDF result to improve their likelihood of conception. Recurrent ejaculation, antioxidant therapy, lifestyle modification, varicocelectomy, and the use of advanced sperm selection techniques or testicular sperm for ICSI are examples of treatment methods that can be utilized in such patients.

    Key points

    1) Sperm DNA integrity is crucial for fertilization and development of healthy offspring.

    2) SDF results from defective maturation, abortive apoptosis and OS and can be induced by a number of disease states and lifestyle/environmental exposures.

    3) There are several assays available to assess sperm DNA damage and most commonly utilized tests include TUNEL, SCD, SCSA and single cell gel electrophoresis assay.

    4) Evidence indicates that SDF testing is most beneficial in patients with unexplained and idiopathic infertility, RPL, varicocele, opting for ART and in those with lifestyle/environmental risk factors.

    5) High SDF fragmentation can be treated by recurrent ejaculation, antioxidant therapy, lifestyle modification, varicocelectomy, and the use of advanced sperm selection techniques or testicular sperm for ICSI.

    Notes

    Conflict of Interest:The authors have nothing to disclose.

    Author Contribution:

    • Conceptualization: AA.

    • Writing — original draft: all the authors.

    • Writing — review & editing: all the authors.

    ACKNOWLEDGEMENTS

    Authors are thankful to the artists from the Cleveland Clinic's Center for Medical Art & Photography for their help with the illustrations. The study was supported by the American Center for Reproductive Medicine (Andrology Research Fund #500000105879).

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    MeSH Terms
    DNA Damage
    DNA Fragmentation*
    DNA*
    Fertility
    Fertilization
    Infertility, Male
    Male
    Oxidative Stress
    Reproduction
    Societies
    Spermatozoa*
    Metrics
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