Sperm DNA Fragmentation: A New Guideline for Clinicians

Commentary:
This narrative review represents a state-of-the-art guide for physicians on sperm DNA fragmentation (SDF), including its mechanism, types of SDF tests, indications of testing, and management of high SDF levels. Herein, we provide a comprehensive summary of the article to enhance the understanding of SDF and its implications on male fertility.

Mechanisms of SDF: Sperm DNA fragmentation (SDF) primarily arises from underlying mechanisms such as defective maturation, abortive apoptosis (testicular), and oxidative stress (post-testicular) (1). In addition, clinical factors such as age (2), varicocele (3), genitourinary infection (4), obesity (5), diabetes (6), and environmental factors such as heat exposure, environmental toxins, radiation, smoking, and diet can all lead to raised SDF (7).

Types of SDF tests: Sperm DNA fragmentation (SDF) can be assessed by various tests, including sperm chromatin structure assay (SCSA) (8), Comet assay (9), TUNEL (10), and sperm chromatin dispersion (SCD) (11). However, the TUNEL assay is the most widely utilized due to its accuracy and reliability. The lack of consensus on specific cut-off values for predicting fertility outcomes underscores the complexity of integrating these tests into clinical practice (12). Additionally, the interplay between oxidative stress and SDF highlights the need for a comprehensive approach to male fertility assessment, as no single test can fully capture the multifaceted nature of sperm health (13).

Indications of SDF testing: Sperm DNA fragmentation (SDF) testing is recommended for patients experiencing unexplained infertility (14), recurrent pregnancy loss (15), recurrent ART failure (16), and clinical varicocele (17) with normal conventional semen analysis, particularly before embarking on assisted reproductive technology (ART) trials, as well as for those exposed to lifestyle and environmental risk factors (18). This approach is supported by extensive research and clinical guidelines aimed at improving reproductive outcomes.

1. Natural conception
The integrity of sperm DNA is essential for successful fertilization and early embryonic development. High SDF significantly reduces the natural pregnancy rate, as evidenced by many studies (19-21).
2. Assisted reproductive technology outcomes

The evidence suggests that sperm DNA integrity plays a significant role in ART
outcomes (19, 20). The current review analyzed the available evidence and concluded that elevated SDF is associated with a decreased pregnancy rate with intrauterine insemination (IUI) and in vitro fertilization (IVF) and an increased miscarriage rate
following both IVF and intracytoplasmic sperm injection (ICSI).
3. Varicocele
Varicocele is a significant correctable cause of male infertility. The available literature suggests that varicocele repair significantly improves SDF and reproductive outcomes, regardless of the type of surgical technique, SDF test type, or the baseline SDF value. However, more studies are needed to confirm these findings and to determine the optimal management for these men. Men with palpable varicocele and normal conventional semen analysis are a clear indication for SDF testing, as the sperm analysis is normal and a functional test should be imposed, especially before varicocele repair or commencing ART treatment (22, 23).
4. Recurrent pregnancy loss
There is strong evidence that links recurrent pregnancy loss to elevated SDF. This has been proven irrespective of the type of SDF test used (24).
5. Idiopathic and unexplained male infertility
Unexplained infertility means infertility of the couple despite an unremarkable fertility assessment. Of them, 20% proved to have significantly high SDF. Idiopathic infertility means abnormality in one or more semen parameters but without identifiable cause. Those also proved to have significantly higher SDF as compared to normal controls (14, 25).
6. High-risk patients
Lifestyle and environmental factors (physical, chemical, and biological) proved to correlate with significantly high oxidative stress levels, eventually leading to SDF and poor reproductive outcomes. Understanding these associations is crucial for developing strategies to improve the fertility potential of these patients (18, 26, 27).

Management of elevated SDF: This review uniquely describes a road map for the management of high SDF. Firstly, treatment should be directed to identifiable causes such as varicocele (28), genital tract infection (29), and risk factor modification such as cessation of smoking (30) and weight reduction (31). Another way is recurrent ejaculation (32), which may reduce SDF and improve pregnancy rates in ICSI. Moreover,  antioxidants can alleviate OS (33) but should be used cautiously to avoid reductive
stress. Another approach would be using testicular sperm for ICSI (34), which may reduce SDF despite surgical risks and concerns about aneuploidy. Lastly, advanced sperm selection techniques like MACS (35) and IMSI (36) can be used to super-select sperm with lower SDF levels.

Pros and cons of SDF testing: Sperm DNA fragmentation (SDF) testing has the advantage of exploring and assessing male fertility on a molecular and functional basis in contrast to conventional semen analysis. This is particularly helpful in difficult cases such as unexplained infertility (14), recurrent pregnancy loss (15), clinical varicocele (37) with normal sperm analysis, and patients with recurrent ART failure (16). On the other hand, SDF testing faces a lot of limitations, such as the absence of universally accepted cut-off values, its reliability in clinical practice needs more research, test-to-test, and interpersonal variability are also limiting factors, and lastly, the cost of the test can be a burden, especially in developing countries (12,26).

References cited in the commentary:
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Q1. What are the primary mechanisms underlying sperm DNA fragmentation (SDF)?

Dr. Ragab: This entails endogenous and exogenous mechanisms. Endogenous mechanisms include defective sperm maturation and abortive apoptosis. Exogenous sources include varicocele, infection, hormonal imbalances, lifestyle risk factors (e.g., smoking, alcoholism), and environmental factors such as pollution and ionizing radiation. All these factors may cause sperm DNA damage via increased OS.

Q2. How does oxidative stress contribute to sperm DNA fragmentation?

Dr. Ragab: OS can lead to sperm DNA damage by altering the chemical structure of the DNA and inducing sperm apoptosis. Exploring more details on a molecular basis, OS can lead to lipid peroxidation by-products, particularly malondialdehyde (MDA) and 4-hydroxynonenal (4HNE), which can introduce DNA adducts such as 8-hydroxy-2’-deoxyguanosine (8-OHdG) and 1, N2-thioguanine. On the other hand, direct oxidative damage to DNA bases results in the formation of adducts, particularly at sites with poor protamine shielding. OS further activates the mitogen-activated protein kinase (MAPK) pathway, thereby impairing maturation and promoting apoptosis.

Q3. What are the clinical implications of sperm DNA fragmentation in male infertility?

Dr. Ragab: The integrity of sperm DNA is essential for successful fertilization, embryonic development, natural and assisted pregnancy, and live birth. High SDF proved to significantly reduce fertilization rate, natural pregnancy rate, IUI, and IVF outcomes. Moreover, high SDF proved to increase the incidence of recurrent pregnancy loss (RPL). However, it seems that the ICSI outcome is not significantly affected by SDF. However, these conclusions need more well-designed studies for confirmation.

Q4. How does age influence the levels of sperm DNA fragmentation??

Dr. Ragab: Previously, reproductive outcomes were thought to be affected by maternal, not paternal, age. Recently, after extensive research on SDF, it seems that SDF increases with age, starting in reproductive years and doubling between 20 and 60 years of age. This association may be due to higher exposure to OS, defective sperm chromatin packaging, and disordered apoptosis that occurs with aging.

Q5. What role does varicocele play in increasing sperm DNA fragmentation?

Dr. Ragab: Clinical varicocele was reported to significantly elevate SDF levels through high OS in the seminal plasma. Some studies found that men with varicocele have significantly higher levels of SDF as compared to healthy controls. Moreover, varicocele repair (VR) is associated with a reduction in SDF. Many studies and meta-analyses proved the positive effect of VR on the level of SDF and reproductive outcomes after surgery.

Q6 How do genitourinary infections contribute to increased sperm DNA fragmentation?

Dr. Ragab: Genitourinary infections and leukocytospermia can elevate SDF via OS-mediated mechanisms and consequently negatively affect fertility. Antibiotic therapy has been reported to be effective in treating infection-induced elevated SDF levels. Moreover, empirical antibiotic therapy for leukocytospermia may improve natural pregnancy rates.

Q7. What lifestyle factors are associated with increased sperm DNA fragmentation?

Dr. Ragab: Lifestyle and environmental factors that can induce SDF include obesity, sedentary life, cigarette smoking, alcohol consumption, electromagnetic waves, particularly from cell phones, environmental pollution, etc.

Q8. How does obesity influence sperm DNA fragmentation?

Dr. Ragab: Obese men have higher levels of OS and SDF as compared to normal controls. The proposed mechanisms include increased scrotal temperature, hormonal imbalance (aromatization of testosterone to estradiol leading to T/E2 imbalance), and lastly, chronic systemic inflammation. Indeed, studies have shown significant improvement in SDF and overall fertility with weight loss.

Q9. What are the common extrinsic factors that cause sperm DNA fragmentation?

Dr. Ragab: Sperm DNA fragmentation (SDF) can be caused by common extrinsic factors such as heat exposure, smoking, environmental pollutants, and chemotherapeutics.

Q10. How is sperm DNA fragmentation linked to assisted reproductive techniques (ART) outcomes?

Dr. Ragab. The evidence suggests that high SDF levels are linked to reduced pregnancy rates and increased miscarriage rates in both intrauterine insemination (IUI) and in vitro fertilization (IVF) procedures. Despite some conflicting findings regarding the predictive value of SDF testing methods, the overall consensus indicates that elevated SDF negatively impacts ART outcomes.

Ahmed Ragab, M.B.B.Ch, MSc, MD: Short Biography

Q1. What are the different types of DNA damage that can occur in sperm?

Dr. Almekaty: Sperm DNA damage can be single-strand or double-strand beaks. The type of damage can be classified on a molecular basis into mismatched bases, abasic sites, base modifications (oxidation, alkylation, deamination), adducts and intrastrand crosslinks, and pyrimidine dimmers.

Q2. What are the clinical indications for testing sperm DNA fragmentation?

Dr. Almekaty: SDF testing is indicated in patients with unexplained infertility, recurrent pregnancy loss (RPL), repeated ART failure, palpable varicocele with apparently normal semen analysis, and in patients exposed to lifestyle risk factors and pollution.

Q3. How does oxidative stress (OS) cause DNA damage in spermatozoa?

Dr. Almekaty: OS can occur in the seminal tract and can contribute to SDF through the following possible mechanisms: (i) activation of the mitogen-activated protein kinase (MAPK) pathway, thereby impairing maturation and promoting apoptosis; (ii) activation of caspases and endonucleases leading to defective chromatin packaging and sperm maturation; (iii) also, OS can indirectly enhance SDF through lipid peroxidation by-products, which can introduce DNA adducts; (iv) Moreover, OS can directly damage DNA bases, resulting in the formation of adducts, particularly at sites with poor protamine shielding.

Q4. What are the consequences of increased sperm DNA fragmentation on embryo development?

Dr. Almekaty: SDF can negatively affect natural pregnancy rates. Moreover, it has a detrimental effect on ART outcomes. The literature suggests a negative impact of high SDF on IUI and IVF but not ICSI outcomes. Some studies also mentioned a “delayed effect” of high SDF in the form of recurrent pregnancy loss (RPL) despite initial successful fertilization and embryo formation. Of note, DSBs negatively impact fertility outcomes, affecting embryo kinetics, implantation rates, and recurrent miscarriages in couples without a female factor, while SSBs do not significantly impact these aspects. Nonetheless, higher levels of SSBs are inversely related to the natural pregnancy outcome.

Q5. How does oxidative stress activate apoptotic pathways in spermatozoa?

Dr. Almekaty: This can occur by 2 possible mechanisms; first, OS can activate the MAPK pathway. Second, activation of caspases and endonucleases leads to defective chromatin condensation. Both mechanisms eventually lead to abortive apoptosis and defective maturation.

Q6. What are the advantages and drawbacks of sperm DNA fragmentation testing?

Dr. Almekaty: SDF testing has the advantage of exploring and assessing male fertility on a molecular and functional basis, in contrast to conventional semen analysis, which lacks such analysis. It can help in the assessment of unexplained infertility, recurrent pregnancy loss (RPL), clinical varicocele with normal semen analysis, and recurrent ART failure. On the other hand, SDF testing faces a lot of limitations, such as the absence of universally accepted cut-off values, its reliability in clinical practice needs more research, test-to-test, and interpersonal variability are also limiting factors, and lastly, the cost of the test can be a burden, especially in developing countries.

Q7. How do environmental toxins contribute to sperm DNA fragmentation?

Dr. Almekaty: Environmental toxins in the air, soil, and water can lead to high SDF. For example, air pollution, heavy metals, occupational toxins, and even electromagnetic waves,  particularly from cell phones, all can lead to OS and high SDF. Some studies suggested that the level of SDF depends on proximity and duration of exposure to environmental factors, which makes sense.

Q8. What is the impact of smoking on sperm DNA fragmentation?

Dr. Almekaty: Cigarette smoking enhances SDF due to the toxic effects of many tobacco metabolites such as nicotine, cadmium, lead, and benzopyrene.

Q9. How does defective germ cell maturation lead to sperm DNA fragmentation?

Dr. Almekaty: This can occur through several pathways: 1) abnormal activity of the topoisomerase II enzyme, inadequate protamination, and DNA damage inflicted by endonucleases and mutagens during spermiogenesis. 2) OS, during sperm transport through the epididymal passage. The latter is the rationale upon which some authors recommend the use of testicular rather than ejaculated in patients with high SDF and repeated ART failure.

Q10. What roles do reactive oxygen species (ROS) play in sperm DNA fragmentation?

Dr. Almekaty: Maintaining the balance of ROS in the seminal environment is of crucial importance for sperm vitality and for “within normal” physiological processes such as apoptosis and capacitation. An overproduction of ROS can lead to deleterious effects on sperm DNA integrity. ROS can alter DNA integrity in the sperm nucleus by inducing singleor double-strand DNA breaks, base modifications, chromatin cross-linking, etc. Moreover, spermatozoa have limited defense mechanisms against ROS-induced DNA damage in contrast to the ova, which can correct itself.

Khaled Mohamed Hafez Almekaty, M.B.B.Ch, MSc, MD: Short Biography

Q1. How can interventions to reduce sperm DNA fragmentation improve fertility outcomes?

Dr. Alghobary: Elevated SDF can be treated by many strategies: 1. Recurrent ejaculation, which can reduce SDF and improve pregnancy rates in ICSI. 2. Antioxidant therapy, which may reduce OS in infertile men but should be used cautiously as its overuse can lead to reductive stress. 3. Lifestyle modification, such as weight loss dietary control, and cessation of smoking may improve SDF, but evidence is scarce. 4. VR significantly reduces SDF and boosts fertility. 5. Advanced sperm selection, e.g., MACS and IMSI, can select sperm with lower SDF. 6. Lastly, using testicular sperm for ICSI can improve outcomes for high SDF, despite surgical risks and concerns about aneuploidy.

Q2. What are the limitations of conventional semen analysis in predicting male fertility potential?

Dr. Alghobary: In fact, about 15% of infertile patients have an apparently normal semen analysis. This means that having a normal semen analysis cannot exclude subfertility. So, think that societies and communities dealing with and treating fertility should consider introducing alternative tests that can assess sperm function, such as SDF testing, for a better diagnosis of male subfertility and improved decision-making on management plans.

Q3. How does sperm DNA fragmentation affect natural conception rates?

Dr. Alghobary: Sperm DNA integrity is essential for successful fertilization and early embryo development. High SDF significantly reduces the likelihood of natural conception, as evidenced by various studies. Research indicates that SDF is associated with reduced cleavage rates and can lead to the halting of embryonic development after the second cleavage stage. Furthermore, DNA damage in spermatozoa can affect the health and wellbeing of offspring.

Q4. What is the relationship between sperm DNA fragmentation and male accessory gland infections?

Dr. Alghobary: Genitourinary infections, leading to leukocytospermia, can elevate ROS production and SDF, negatively affecting fertility. Antibiotic therapy has been reported to be effective in treating infection-induced elevated SDF levels. Moreover, empirical antibiotic therapy for leukocytospermia may improve natural pregnancy rates.

Q5. How do chemotherapeutic agents influence sperm DNA fragmentation?

Dr. Alghobary: Onco-fertility is a very important and interesting field that has been extensively explored in recent years. The use of chemotherapy is based on the suppression of rapidly dividing cells, and as everyone knows, spermatogonia is a very good example of these cells. Thus, it is strongly recommended to do sperm banking before embarking on chemotherapy and tumor treatment. I think the mechanism by which chemotherapeutic agents lead to sperm DNA damage is by their direct antimitotic properties and also by enhancing OS in the seminal plasma, eventually leading to sperm DNA damage and defective maturation.

Q6. What is the role of the MAPK pathway in sperm DNA fragmentation?

Dr. Alghobary: The MAPK pathway has a critical role in regulating cell survival and death mechanisms. The activation of the MAPK pathway by OS leads to an increase in p53 and caspase 3 expression while decreasing bcl-2 levels, eventually resulting in impaired maturation and promotion of apoptosis.

Q7. How does advanced paternal age contribute to sperm DNA fragmentation?

Dr. Alghobary: SDF increases with age, starting in reproductive years and doubling between the ages of 20 and 60 years. This association has been attributed to higher exposure to OS, defective sperm chromatin packaging, and disordered apoptosis that occurs with aging.

Q8. What are the strengths, weaknesses, opportunities, and threats (SWOT) associated with sperm DNA fragmentation testing?

Dr. Alghobary:

A) Strengths: SDF testing can be more beneficial than conventional semen analysis in particular clinical situations, such as unexplained infertility, recurrent pregnancy loss (RPL), and clinical varicocele with normal semen analysis;

B) Weaknesses: the absence of universally accepted cut-off values and only moderate evidence is available to support its use;

C) Threats: the absence of strong evidence to support its use and the relatively high cost of the test;

D) Opportunities: further studies are essential to clarify the clinical implications of SDF testing and to explore effective treatment of high SDF.

Q9. How does oxidative stress-mediated apoptosis result in sperm DNA fragmentation?

Dr. Alghobary: OS activates the mitogen-activated protein kinase (MAPK) pathway, increasing p53 and caspase 3 expression and reducing bcl-2, thereby impairing maturation and promoting apoptosis.

Q10. What are the recommended clinical scenarios for performing sperm DNA fragmentation testing?

Dr. Alghobary: The recommended clinical scenarios for performing SDF include patients with unexplained infertility, recurrent pregnancy loss (RPL), clinical varicocele with apparently normal semen analysis, a negative ART outcome, and patients exposed to lifestyle risk factors and environmental toxicants.

Moheiddin Fakhry Alghobary, M.B.B.Ch, MSc, MD: Short Biography