Sperm DNA fragmentation and infertility: a narrative review

Commentary:
The study of Sperm DNA fragmentation and infertility represents a significant revolutionary factor in addressing a new possible etiology of male factor infertility (MFI) (1). As the complexity of MFI diagnosis continues to grow, and the need for multidimensional tools to uncover the etiology of MFI, the importance of this timely paper which provides a practical understanding of the role of sperm DNA fragmentation (SDF) in the management of male infertility is mandated (1).

SDF is increasingly recognized as a significant biomarker for assessing male infertility. High levels of DNA fragmentation in sperm are associated with lower fertilization rates, poor embryo quality, and increased risk of miscarriage. (2)

Three main mechanisms are involved in the process of SDF which include defective maturation, abortive apoptosis, and elevated OS from intrinsic or extrinsic factors. Defective maturation and abortive apoptosis occur during spermatogenesis in the testes and OS from ROS usually affects sperm in the post-testicular male excurrent ductal system (3). Various causes of ROS with its consequences include infection, inflammation, leucocytes, smoking, alcohol, radiation, toxic chemicals, diseases of the male reproductive accessory gland, genital tract inflammation, varicocele, testicular torsion, or cryptorchidism. (4)

The exact mechanism of SDF during spermatogenesis is not fully known. However, apoptosis, protamination failure, and the excess of reactive oxygen species (ROS) which results in lipid peroxidation, with its impact on sperm motility are the main causes of SDF (5). The chromatin is highly compacted and tightly packed in mature sperm, primarily through the replacement of histones with protamines. Elevated SDF represents its impact with poor histone-chromatin transition, affecting the protection of sperm DNA integrity (6).

The WHO has acknowledged the significance of SDF testing in evaluating male fertility by including it in their latest semen analysis guidelines. While organizations such as ASRM and ESHRE have not fully endorsed routine SDF testing, they recommend it in cases of unexplained infertility, recurrent pregnancy loss, repeated IVF failures, and in men with risk factors like varicocele or smoking (7).

Several methods are available to measure SDF, such as the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling assay), SCSA (Sperm Chromatin Structure Assay), SCD (Sperm Chromatin Dispersion or Halo Test), and Comet assay. Each method has its advantages and limitations, and the choice of test may impact clinical decision-making (8). Standardization of SDF testing across laboratories is crucial for improving the reliability, comparability, and clinical utility of SDF assessments in male infertility. This can be achieved by developing uniform testing protocols, establishing consensus on thresholds, and quality control measures, and integrating standardized practices into clinical guidelines. (9)

Therapeutic interventions for sperm DNA fragmentation include lifestyle modifications, antioxidant supplementation, hormone therapy, varicocele repair, and assistedreproductive technologies. All these measures could alleviate DNA fragmentation and improve male fertility potential. The choice of treatment depends on the underlying causes of DNA damage and the severity of the condition (9). Future research on SDF in male infertility should focus on developing more accurate and sensitive SDF assays, investigating the underlying molecular mechanisms of SDF, the role of SDF in emerging fields like epigenetics and intergenerational effects, exploring novel pharmacological and therapeutic interventions to reduce SDF levels and improve fertility outcome (10).

Take Home Message:

SDF testing is increasingly required in the evaluation and treatment of MFI, which is why more research is necessary to standardize SDF testing and its reliability, and clear guidelines for its use in clinical practice are urgently needed. Although, most of the guidelines and the published studies agreed that the level of evidence and grade of recommendations for indications and therapeutic interventions for sperm DNA fragmentation relied upon good quality and well-designed studies; some of the recommendations are based on poorer quality studies (retrospective, case series, expert opinion). Finally, this narrative review highlighted the importance of SDF and emphasized its role in achieving successful reproductive outcomes.

References:
1. Ziouziou I, Rambhatla A, Shah R, Agarwal A. Sperm DNA fragmentation and infertility: a narrative review. World J Urol. 2024 Jul 11;42(1):408.
2. Agarwal A, Rana M, Qiu E, AlBunni H, Bui AD, Henkel R: (2018) Role of oxidative stress, infection and inflammation in male infertility. Andrologia 50(11):e13126. https://doi.org/10.1111/and.13126
3. Agarwal A, Majzoub A, Baskaran et al (2020) Sperm DNA fragmentation: a new guideline for clinicians. World J men’s Health 38(4):412–471.

4. Szabó A, Váncsa S, Hegyi et al (2023) Lifestyle-, environmental-, and additional health factors associated with an increased sperm DNA fragmentation: a systematic review and meta-analysis. Reproductive Biology Endocrinology: RB&E 21(1):5.
5. Finelli R, Leisegang K, Kandil H, Agarwal A (2022) Oxidative stress: a comprehensive review of biochemical, molecular, and genetic aspects in the Pathogenesis and management of Varicocele. World J men’s Health 40(1):87–103.
6. González-Marín C, Gosálvez J, Roy R. Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells. Int J Mol Sci. 2012 Oct 31;13(11):14026-52. doi:10.3390/ijms131114026.
7. Chua, S. C., Yovich, S. J., Hinchliffe, P. M., & Yovich, J. L. (2023). The sperm DNA fragmentation assay with SDF level less than 15% provides a useful prediction for clinical pregnancy and live birth for women aged under 40 years. Journal of Personalized Medicine, 13(7).

8. Agarwal, A., Cho, C., Majzoub, A., & Esteves, S. (2017). The Society for Translational Medicine: clinical practice guidelines for sperm DNA fragmentation testing in male infertility. Translational Andrology And Urology, 6(Suppl 4), S720-S733. doi:10.21037/tau.2017.08.06
9. Agarwal, A., Farkouh, A., Saleh, R., Hamoda, T. A. A. A. M., Harraz, A. M., Kavoussi, P., Arafa, M., Salvio, G., Rambhatla, A., Toprak, T., Gül, M., Phuoc, N. H. V., Boitrelle, F., Birowo, P., Ghayda, R. A., Cannarella, R., Kuroda, S., Durairajanayagam, D., Zini, A., … Le, T. V. (2023). Controversy and consensus on indications for sperm DNA fragmentation testing in male infertility: A global survey, current guidelines, and expert recommendations. World Journal of Men’s Health, 41.https://doi.org/10.5534/wjmh.22028
10. Farkouh, A., Saleh, R., Shah, R., & Agarwal, A. (2023). Sperm DNA fragmentation in male infertility: From bench to bedside. In Arab Journal of Urology (Vol. 21, Issue 4).https://doi.org/10.1080/20905998.2023.2278200

Ahmed I. El-Sakka, MBChB, MSc, MD: Short Biography

Q1. What are the primary mechanisms leading to sperm DNA fragmentation (SDF) during spermatogenesis?

Dr. El-Sakka: The exact mechanism of SDF during spermatogenesis is not fully known. However, apoptosis, protamination failure, and the excess of reactive oxygen species (ROS) which results in lipid peroxidation, with its impact on sperm motility are considered to be the main causes of SDF. Post-testicular mechanisms that result in the elevation of OS leading to the elevation of SDF. When the level of ROS is high then OS occurs. This results in lipid peroxidation which is toxic to DNA and ultimately damaging to cellular components that negatively affect fertility.

Q2. What are the clinical implications of elevated sperm DNA fragmentation in male infertility?

Dr. El-Sakka: Several interesting studies have reported that high SDF can act in a way as a barrier to male fertility. SDF levels are also affected by many factors like varicocele, obesity, unexplained infertility, idiopathic infertility, testicular cancer, and aging in men. SDF was categorized into viable and non-viable depending upon its impact on natural fertility in

normozoospermic males, in viable SDF males, spermatozoa showed the ability to fertilize but later failed in good embryo development, whereas non-viable SDF males are not able to initiate fertilization.

Q3. How does sperm DNA fragmentation affect natural pregnancy rates and ART outcomes?

Dr. El-Sakka: Basically, the elevated SDF contributes to male infertility, and its association with reduced natural conception and ART outcomes is almost the same. The rate of natural pregnancy is affected by DNA damage. The correlation between the SDF index and Pregnancy outcome was investigated by several studies, and was concluded that 20-30%, decreases the chance of natural pregnancy. A negative correlation was found between SDF and sperm motility in couples with recurrent spontaneous abortion. However, there is controversy on the clinical implications of SDF with contradictory findings reported amongst the SRMAs conducted on the effect of DNA fragmentation on natural pregnancy rates and ART outcomes.

Q4. What are the indications for SDF testing in the evaluation of male infertility?

 Dr. El-Sakka: Several indications for SDF testing in the evaluation of male infertility were reported by different societies i.e. AUA, EAU, and others in addition to different published studies.
• Men with unexplained or idiopathic male infertility.
• Couples experiencing recurrent pregnancy loss.
• Infertile couples before initiating or after failure of IUI or IVF.
• Couples with recurrent miscarriage following ICSI
• Other indications such as Men with clinical varicocele or modifiable lifestyle risk factors were also reported.

All the guidelines and the published studies agreed that the level of evidence and grade of recommendations for such indications relied upon good quality and well-designed studies.
However, some of the recommendations are based on poorer quality studies (retrospective, case series, expert opinion).

Q5. How does varicocele repair influence sperm DNA fragmentation levels?

Dr. El-Sakka: Varicoceles have been consistently associated with increased SDF and immature chromatin, leading to infertility. One possible cause for poor semen quality and quantity is scrotal hyperthermia or heat stress, causing oxidative stress in  varicocele males. Varicocele repair can improve OS, decrease ROS, increase antioxidants, and ultimately better natural pregnancy as well as ART outcomes. Varicocelectomy reduced SDF, improved sperm concentration, progressive motility, and morphology difference thus was considered as a treatment during abnormal DNA fragmentation index.

Ayman S. Rashed, MBChB, MSc, MD, PhD: Short Biography

Q1. What is the role of lifestyle and environmental factors in increasing sperm DNA fragmentation?

Dr. Rashed: Lifestyle and environmental factors play significant roles in increasing sperm DNA fragmentation (SDF), contributing to male infertility. Many lifestyle factors, such as smoking, excessive alcohol consumption, poor diet, heat exposure, lack of exercise, and chronic mental stress, lead to oxidative stress. This results in an imbalance between reactive oxygen species (ROS) and antioxidants in the body, causing damage to sperm DNA. Mitigating these lifestyle and environmental factors by adopting a healthy diet, regular exercise, avoiding smoking and alcohol consumption, reducing exposure to environmental toxins, and managing stress can help lower sperm DNA fragmentation and improve fertility.

Q2. How do different SDF measurement techniques compare in terms of reliability and clinical applicability?

Dr. Rashed: Different sperm DNA fragmentation (SDF) measurement techniques vary in reliability, sensitivity, and clinical applicability. Here's a comparison of the most commonly used methods:

1. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) Assay:
• Reliability: The TUNEL assay is highly reliable and sensitive. It directly labels DNA breaks as either single- or double-strand breaks. It can also be used for both fresh and frozen samples.
• Clinical applicability: Due to its accuracy, TUNEL is widely used in research and clinical settings.

2. SCSA (Sperm Chromatin Structure Assay):
• Reliability: SCSA is also considered reliable, particularly for its ability to measure DNA denaturation, a sign of chromatin instability. It has a standardized protocol, and a large number can be tested. It can be used for fresh and frozen samples.
• Clinical Applicability: SCSA is one of the most standardized tests frequently used in clinical practice. It is highly reproducible and provides quick results.

3. Comet assay:
• Reliability: The comet assay is sensitive and allows for directly detecting single- and double-strand DNA breaks. It can also detect specific types of DNA damage and be used in cases with marked oligozoospermia.
• Clinical Applicability: Though very sensitive, it is less commonly used in routine clinical settings due to its complexity, less standardization across laboratories, only done on fresh samples, Inter-observer variability, time-consuming, and technically demanding.

4. SCD (Sperm Chromatin Dispersion) Test:
• Reliability: Although the SCD test is simpler, easier to perform, and requires minimal equipment, commercial kits are available and less expensive. However, it is somewhat less reliable than TUNEL and SCSA.
• Clinical Applicability: Commonly used in clinical practice due to its simplicity and low cost.

In Summary:

❖ Best for Research: TUNEL and Comet assays are preferred in research due to their high sensitivity and detailed insights into DNA damage.
❖ Best for Clinical Use: SCSA and SCD tests are more commonly used in clinical practice because they balance accuracy with ease of use and costeffectiveness.
❖ Each method has strengths and limitations, and the choice of test often depends on the clinical context, available resources, and patient-specific needs. Multiple tests may be used together for a comprehensive assessment.

Q3. What are the recommended SDF thresholds for clinical decision-making in ART?

Dr. Rashed: Sperm DNA fragmentation (SDF) thresholds are crucial for clinical decisionmaking in assisted reproductive technologies (ART) such as IVF and ICSI. These thresholds help determine the likelihood of successful fertilization, embryo development, and pregnancy outcomes. While certain SDF thresholds have been suggested for clinical decision-making, the exact cut-off points are still debated, and their application can vary depending on the clinical context (e.g., natural conception vs. ART)

Recommended SDF Thresholds:

A. General ART thresholds:
• 20-30%: An SDF level within this range is generally considered a critical threshold.
Above 30% are associated with significantly reduced pregnancy rates and increased miscarriage rates. This threshold is widely used in clinical practice to guide decisions regarding ART techniques.
• 15-20%: Values below 15-20% are typically associated with better ART outcomes, including higher successful fertilization and pregnancy rates.

B. IVF/ICSI-specific thresholds:
• ICSI: An SDF level above 30% is often considered for ICSI. However, due to the direct injection of a single sperm into the egg, ICSI can sometimes overcome the negative impact of high SDF. Still, a higher SDF may lead to poorer embryo quality and lower pregnancy rates even with ICSI.
• IVF: A lower SDF is preferred for standard IVF. If SDF exceeds 20-25%, couples may be counseled to consider ICSI instead of IVF to improve their chances of success.

C. Male infertility cases:
• In cases of unexplained infertility or recurrent pregnancy loss, a more conservative threshold of around 15-20% may be applied. Higher SDF levels in these cases may prompt more aggressive interventions, such as the use of testicular sperm retrieval (which often has lower SDF) combined with ICSI. Also, surgical treatment of clinical varicocele should be considered even with ICSI intention.

D. Testicular vs. ejaculated sperm:
• In cases where SDF is high in ejaculated sperm, clinicians might consider using testicular sperm for ART since testicular sperm typically exhibit lower DNA fragmentation levels. This approach is advantageous in severe male factor infertility or recurrent ART failures.

Q4. How does sperm DNA fragmentation correlate with recurrent pregnancy loss?

Dr. Rashed: Sperm DNA fragmentation (SDF) has been increasingly recognized as a significant factor in recurrent pregnancy loss (RPL). RPL is generally defined as two or more consecutive miscarriages, and while various factors can contribute to RPL, SDF has emerged as a crucial male-related factor.
Correlation Between SDF and RPL:

A. Increased miscarriage rates:
• High SDF can lead to impaired embryo development, resulting in pregnancy loss. The DNA integrity in sperm is vital for successful fertilization and the subsequent development of a healthy embryo.

B. Impact on embryo quality:
• High SDF can result in poor-quality embryos that fail to implant properly or have abnormal development, leading to early pregnancy loss. Embryos with fragmented sperm DNA are more likely to experience cell division errors and chromosomal abnormalities, which can result in miscarriage.
• Clinical studies have consistently shown that men whose partners experience RPL tend to have higher SDF levels compared to men whose partners have successful pregnancies. For instance, a meta-analysis found that men with high SDF are nearly twice as likely to be involved in cases of RPL.

Q5. What is the significance of SDF in unexplained and idiopathic male infertility?

Dr. Rashed:
A. Unexplained male infertility:
• In many cases of unexplained infertility, standard semen parameters appear normal.
However, these parameters do not assess the genetic integrity of the sperm. Elevated SDF can be present despite normal semen analysis, indicating underlying DNA damage that could impair fertility.
• Men with unexplained infertility often have higher SDF levels compared to fertile men, suggesting that sperm DNA damage may be a hidden factor contributing to infertility.
• High levels of SDF can negatively impact fertilization rates, embryo development, and ultimately, pregnancy outcomes. Sperm with fragmented DNA may fertilize an egg, but the resulting embryo might fail to develop properly, leading to failed implantation or early miscarriage.

B. Idiopathic male infertility:
• In cases of idiopathic infertility, where no clear cause is identified after thorough evaluation, SDF testing provides valuable diagnostic information. It can help identify men at risk of poor reproductive outcomes due to sperm DNA damage.
• Elevated SDF levels in idiopathic infertility cases may prompt the consideration of advanced reproductive techniques, such as ICSI or the use of testicular sperm, which often have lower DNA fragmentation levels.

Q6. What are the limitations of current SDF testing methodologies?

Dr. Rashed: While SDF testing provides valuable insights into sperm DNA integrity, its clinical application is hindered by limitations such as lack of standardization, technical complexity, and challenges in interpretation. Ongoing research and efforts to standardize testing protocols are essential to improve the reliability and utility of SDF tests in clinical practice. Here are some of the key limitations:

A. Lack of standardization:
• Variation in Protocols: Different laboratories often use varying protocols and thresholds for SDF testing, leading to inconsistent results and comparability of SDF test results across different clinical settings.
• Different Assay Techniques: There are multiple SDF assays, each with its methodology, sensitivity, and specificity. The results obtained from one method may not correlate perfectly with those from another, complicating the interpretation and clinical decisionmaking.

B. Technical complexity:
• Requirement for Specialized Equipment: This limits accessibility and increases the cost of testing.
• Need for Technical Expertise: If the tests are not conducted under optimal conditions, the results can be variable.

C. Biological variability:
• Sperm Heterogeneity: Sperm samples are inherently heterogeneous, with varying levels of DNA fragmentation among different sperm cells within the same ejaculate. This variability can lead to sampling bias and may affect the accuracy of the test results.
• Temporal Variability: SDF levels can fluctuate over time due to factors such as illness, stress, or environmental exposures. A single test may not accurately reflect a man’s longterm sperm DNA integrity.

D. Challenges in clinical interpretation:
• Uncertain Clinical Thresholds: While certain SDF thresholds have been suggested for clinical decision-making, the exact cut-off points are still debated.
• Limited Predictive Value: Although high SDF is associated with poorer reproductive outcomes, the predictive value of SDF testing is still limited.

E. Cost and accessibility
• Expense: SDF testing can be expensive, especially when using more advanced methods like TUNEL or SCSA, making it less accessible to some patients.
• Availability: Not all fertility clinics or labs offer SDF testing, limiting access to this diagnostic tool for many patients.

F. Limited evidence in specific populations

• Understudied Populations: Most SDF research has focused on men with known infertility issues, leaving gaps in knowledge regarding the role of SDF in fertile men or populations with specific reproductive challenges, such as older men or those with chronic illnesses.

Q7. How does sperm DNA fragmentation relate to sperm chromatin structure abnormalities?

Dr. Rashed: Sperm DNA fragmentation (SDF) and sperm chromatin structure abnormalities are closely related aspects of sperm quality that contribute to male infertility. Both involve issues with the integrity and packaging of genetic material within sperm, and their interplay can significantly affect reproductive outcomes.

Relationship Between SDF and Sperm Chromatin Structure Abnormalities:
A. Chromatin packaging and SDF:
• Chromatin Structure: In mature sperm, chromatin is highly compacted and tightly packed, primarily through the replacement of histones with protamines. This tight packing protects the DNA from damage and ensures its integrity during fertilization.
• Abnormal Chromatin Packaging: When chromatin is not properly compacted, it becomes more vulnerable to damage from various factors, such as oxidative stress. Poor chromatin packaging is often associated with incomplete histone-to-protamine transition, leading to areas of the sperm DNA being less protected and more prone to fragmentation.

B. Oxidative stress and DNA damage:
• Role of Oxidative Stress: ROS can cause breaks in the DNA strands, particularly when chromatin is loosely packed, and the DNA is exposed.
• Correlation with SDF: Studies have shown a strong correlation between poor chromatin structure and increased levels of SDF. Sperm with abnormal chromatin structure often exhibit higher levels of DNA fragmentation.

C. Implications for fertility:
• Impact on Sperm Function: Abnormal chromatin structure and elevated SDF can impair sperm function, including its ability to fertilize an egg and support embryo development.

Maged M. Rageb, MBChB, MSc, MD: Short Biography

Q1. What are the recommendations for SDF testing by various fertility societies?

Dr. Ragab: The WHO has acknowledged the significance of SDF testing in evaluating male fertility by including it in their latest semen analysis guidelines. While organizations such as ASRM and ESHRE have not fully endorsed routine SDF testing, they recommend it in cases of unexplained infertility, recurrent pregnancy loss, repeated IVF failures, and in men with risk factors like varicocele or smoking. (Chua et al., 2023).

Q2. How do single-strand and double-strand DNA breaks differ in their impact on fertility?

Dr. Ragab: Sperm DNA fragmentation can occur as either single-strand breaks (SSBs) or double-strand breaks (DSBs). DSBs are more severe and high levels are associated with reduced pregnancy rates and increased risk of miscarriage. (Agarwal, Barbăroșie, et al.,2020).

Q3. What are the potential therapeutic interventions to reduce sperm DNA fragmentation?

Dr. Ragab: Therapeutic interventions for sperm DNA fragmentation include lifestyle modifications, antioxidant supplementation, hormone therapy, varicocele repair, and assisted reproductive technologies. The choice of treatment depends on the underlying causes of DNA damage and the severity of the condition. (Agarwal et al., 2023).

Q4. What is the role of apoptosis in the development of sperm DNA fragmentation?

Dr. Ragab: Apoptosis may play a role in sperm DNA fragmentation. Apoptosis plays a critical role in sperm DNA fragmentation (SDF) by eliminating damaged sperm cells. Some germ cells escape this programmed elimination process and undergo maturation. These are then identified as defective spermatozoa, associated with high levels of DNA damage and contributing to male infertility. Understanding and targeting these abortive apoptotic pathways may offer therapeutic potential for improving male reproductive health.(Sakkas et al., 1999).

Q5. What are the future research directions for understanding the role of SDF in male infertility?

Dr. Ragab: Future research on SDF in male infertility should focus on developing more accurate and sensitive SDF assays, investigating the underlying molecular mechanisms of SDF, the role of SDF in emerging fields like epigenetics and intergenerational effects, exploring novel pharmacological and therapeutic interventions to reduce SDF levels and improve fertility outcome. (Farkouh et al., 2023).