What is Sperm DNA fragmentation, how do single- and double-stranded DNA breaks occur, and how do they affect fertilization, embryo development, and reproductive outcomes?
Sperm DNA fragmentation (SDF) refers to damage within the genetic material carried by sperm. This DNA is essential because it combines with the oocyte’s DNA to form an embryo. In some sperm, the DNA can develop breaks, which may affect either one strand (single-strand breaks) or both strands (double-strand breaks). Since the introduction of the Sperm Chromatin Structure Assay in the 1980s (Rex et al., 2017), interest in SDF has increased, as standard semen analysis does not assess DNA quality.
Higher levels of sperm DNA fragmentation are more commonly seen in men with fertility problems, although it can also be present even when routine semen test results appear normal. There are several reasons why this damage can occur. During sperm development, the DNA is normally tightly packed and protected, but if this process is faulty, the DNA can become more vulnerable. In addition, the body usually removes damaged sperm cells through a natural process, but sometimes this process does not work properly, allowing abnormal sperm to be released. One of the main causes is oxidative stress, which occurs when harmful molecules, called reactive oxygen species (ROS), overwhelm the body’s natural defences and damage DNA (Sakkas & Alvarez, 2010; Muratori et al., 2019).
A range of health and lifestyle factors have been linked to increased sperm DNA damage. These include increasing paternal age, varicocele (enlarged veins in the testicles), infections in the reproductive tract, chronic illnesses, obesity, and diabetes. Environmental exposures such as toxins and pollution can also play a role, as can smoking, alcohol use, and long periods without ejaculation (Ribas-Maynou & Benet, 2019; Agarwal et al., 2014; Gosálvez et al., 2024; Pfeiffer et al., 2000).
Single-strand DNA breaks are most often caused by oxidative stress. Harmful molecules can come from within the body, for example due to inflammation or conditions such as varicocele (Ribas-Maynou & Benet, 2019), as well as from external sources like pollution, smoking, and poor nutrition, which can damage the DNA (Agarwal et al., 2014). These types of breaks can affect how well sperm move and are often linked to reduced chances of natural conception (Ribas-Maynou & Benet, 2019).
Double-strand DNA breaks are generally more serious because both sides of the DNA are damaged. These are often linked to problems in sperm development, how DNA is packaged, or failures in the normal process that removes damaged cells (Gosálvez et al., 2024; Pardiñas et al., 2022; Lange et al., 2011). They may also result from environmental exposures such as radiation or toxins (Pfeiffer et al., 2000). After fertilization, the oocyte can repair some DNA damage using natural repair processes, but this ability is limited and influenced by maternal age. If the damage is too severe or not properly repaired, it may affect the stability of the embryo (Ribas-Maynou et al., 2022).
In terms of fertility outcomes, higher levels of sperm DNA fragmentation have been associated with delayed embryo development, reduced implantation (when the embryo attaches to the womb), and a higher risk of early miscarriage, particularly in couples where no female factor has been identified (Casanovas et al., 2019). Overall, increased SDF is linked to lower chances of natural pregnancy and reduced success in fertility treatments.
In summary, sperm DNA fragmentation is an important aspect of male fertility that is not detected in routine semen analysis. Single-strand damage is mainly caused by oxidative stress and tends to affect sperm function and natural conception, while double-strand damage is more severe and is linked to embryo development and pregnancy loss. Because higher levels of SDF are associated with poorer outcomes in both natural and assisted conception, testing for SDF can provide valuable additional information when assessing and managing male infertility.
Rex, A. S., Aagaard, J., & Fedder, J. (2017). DNA fragmentation in spermatozoa: A historical review. Andrology, 5(4), 622–630. https://doi.org/10.1111/andr.12381
Sakkas, D., & Alvarez, J. G. (2010). Sperm DNA fragmentation: Mechanisms of origin, impact on reproductive outcome, and analysis. Fertility and Sterility, 93(4), 1027–1036. https://doi.org/10.1016/j.fertnstert.2009.10.046
Muratori, M., Tarozzi, N., Carpentiero, F., Danti, S., Perrone, F. M., Cambi, M., Casini, A., Azzari, C., Boni, L., Maggi, M., Borini, A., & Baldi, E. (2019). Sperm selection with density gradient centrifugation and swim up: Effect on DNA fragmentation in viable spermatozoa. Scientific Reports, 9(1), 7492. https://doi.org/10.1038/s41598-019-43981-2
Ribas-Maynou, J., & Benet, J. (2019). Single and Double Strand Sperm DNA Damage: Different Reproductive Effects on Male Fertility. Genes, 10(2), 105. https://doi.org/10.3390/genes10020105
Agarwal, A., Virk, G., Ong, C., & du Plessis, S. S. (2014). Effect of Oxidative Stress on Male Reproduction. The World Journal of Men’s Health, 32(1), 1–17. https://doi.org/10.5534/wjmh.2014.32.1.1
Gosálvez, J., Johnston, S. D., Prado, A., López-Fernández, C., Contreras, P., Bartolomé-Nebreda, J., González-Martínez, M., Fernández, J. L., De La Vega, C. G., & Góngora, A. (2024). Strong Correlation Between Double-Strand DNA Breaks and Total Sperm DNA Fragmentation in the Human Ejaculate. Archives of Medical Research, 55(8), 103122. https://doi.org/10.1016/j.arcmed.2024.103122
Pfeiffer, P., Goedecke, W., & Obe, G. (2000). Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis, 15(4), 289–302. https://doi.org/10.1093/mutage/15.4.289
Pardiñas, M. L., Martin, A., Ortega-Jaén, D., De los Santos, J. M., Viloria, T., Gamiz, P., & De los Santos, M. J. (2022). Sperm DNA fragmentation and microfluidics: A new era in human sperm selection. Medicina Reproductiva y Embriología Clínica, 9(3), 100121. https://doi.org/10.1016/j.medre.2022.100121
Lange, J., Pan, J., Cole, F., Thelen, M. P., Jasin, M., & Keeney, S. (2011). ATM controls meiotic double-strand break formation. Nature, 479(7372), 237–240. https://doi.org/10.1038/nature10508
Ribas-Maynou, J., Novo, S., Torres, M., Salas-Huetos, A., Rovira, S., Antich, M., & Yeste, M. (2022).
Sperm DNA integrity does play a crucial role for embryo development after ICSI, notably when good-quality oocytes from young donors are used. Biological Research, 55(1), 41. https://doi.org/10.1186/s40659-022-00409-y
Casanovas, A., Ribas-Maynou, J., Lara-Cerrillo, S., Jimenez-Macedo, A. R., Hortal, O., Benet, J., Carrera, J., & García-Peiró, A. (2019a). Double-stranded sperm DNA damage is a cause of delay in embryo development and can impair implantation rates. Fertility and Sterility, 111(4), 699- 707.e1. https://doi.org/10.1016/j.fertnstert.2018.11.035
How Is Sperm DNA Fragmentation Tested and Interpreted in Clinical Practice?
Male fertility is usually assessed using a semen analysis, which looks at both visible (macroscopic) and microscopic features of semen. Macroscopic features include things like volume, pH, colour, and how the sample liquefies. Microscopic features include sperm count, movement (motility), shape (morphology), and other factors such as signs of infection or the presence of antibodies.
While these tests are important, they do not always give a complete picture of sperm DNA quality (Wen et al., 2025). Sperm DNA fragmentation (SDF) refers to damage in sperm DNA and can involve either single-strand breaks or double-strand breaks, both of which are linked to reduced fertility and poorer reproductive outcomes (Agarwal et al., 2020). Testing for SDF can therefore provide additional useful information, especially in cases of unexplained infertility (Ribas-Maynou & Benet, 2019).
There are several laboratory tests used to measure sperm DNA damage, and each detects different types of damage. Tests such as the SCSA, SCD test, TUNEL assay, and alkaline Comet assay mainly detect single-strand DNA damage. In contrast, double-strand DNA damage can only be reliably measured using the neutral Comet assay, which is currently the only widely accepted method for detecting this type of damage (Ribas-Maynou, García-Peiró, Fernandez-Encinas, et al., 2012b).
The TUNEL assay is a commonly used test that detects DNA breaks by attaching fluorescent markers to damaged areas of DNA. These markers can then be measured using specialised equipment, allowing doctors to estimate how much DNA damage is present (Pardiñas et al., 2022).
Another widely used test is the Sperm Chromatin Structure Assay (SCSA), which uses a special dye to assess how stable the DNA is. Healthy DNA glows green, while damaged DNA glows red under analysis. The result is reported as a DNA Fragmentation Index (DFI), which shows the proportion of damaged DNA (Evenson, 2016).
The Sperm Chromatin Dispersion (SCD) test works by observing how sperm DNA spreads out after treatment. Sperm with healthy DNA form a visible “halo,” while those with damaged DNA do not. This test is quick, simple, and relatively inexpensive, although interpreting the results can sometimes be subjective (Fernández et al., 2003; Agarwal et al., 2020).
The Comet assay looks at DNA damage in individual sperm cells. During the test, damaged DNA moves away from the main part of the cell, forming a shape that looks like a comet tail. The longer the tail, the more damage is present. The alkaline version of this test detects single-strand damage, while the neutral version specifically detects double-strand damage (Ribas-Maynou, García-Peiró, Abad, et al., 2012a).
Overall, SDF testing provides important information about sperm DNA quality and fertility potential. However, while these tests can identify problems, they do not directly treat or reduce DNA damage themselves.
Wen, Z.-N., Duan, L., Chen, Y., Qiu, Q.-H., Liu, G., Luo, N., Li, P.-H., Tian, E.-P., & Ge, R.-S. (2025). Comparative Efficacy of Swim-Up, Density-Gradient Centrifugation, and Microfluidic Sorting in Sperm Preparation, and the Impact on Motility, Morphology, and DNA Integrity. International Journal of General Medicine, 18, 2355–2366. https://doi.org/10.2147/IJGM.S517575
Ribas-Maynou, J., García-Peiró, A., Abad, C., Amengual, M. J., Navarro, J., & Benet, J. (2012a). Alkaline and neutral Comet assay profiles of sperm DNA damage in clinical groups. Human Reproduction (Oxford, England), 27(3), 652–658. https://doi.org/10.1093/humrep/der461
Agarwal, A., Majzoub, A., Baskaran, S., Panner Selvam, M. K., Cho, C. L., Henkel, R., Finelli, R., Leisegang, K., Sengupta, P., Barbarosie, C., Parekh, N., Alves, M. G., Ko, E., Arafa, M., Tadros, N., Ramasamy, R., Kavoussi, P., Ambar, R., Kuchakulla, M., ... Shah, R. (2020). Sperm DNA Fragmentation: A New Guideline for Clinicians. The World Journal of Men’s Health, 38(4), 412–471. https://doi.org/10.5534/wjmh.200128
Pardiñas, M. L., Martin, A., Ortega-Jaén, D., De los Santos, J. M., Viloria, T., Gamiz, P., & De los Santos, M. J. (2022). Sperm DNA fragmentation and microfluidics: A new era in human sperm selection. Medicina Reproductiva y Embriología Clínica, 9(3), 100121. https://doi.org/10.1016/j.medre.2022.100121
Evenson, D. P. (2016). The Sperm Chromatin Structure Assay (SCSA®) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Animal Reproduction Science, 169, 56–75. https://doi.org/10.1016/j.anireprosci.2016.01.017
Fernández, J. L., Muriel, L., Rivero, M. T., Goyanes, V., Vazquez, R., & Alvarez, J. G. (2003). The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation.Journal of Andrology, 24(1), 59–66. https://doi.org/10.1002/j.1939- 4640.2003.tb02641.x
What Are the Most Effective Treatment Strategies for Reducing DNA Fragmentation?
Lifestyle factors play a major role in sperm DNA fragmentation, and improving these can help reduce DNA damage. Factors linked to higher SDF include obesity, smoking, alcohol use, exposure to toxins, increasing age, long periods without ejaculation, illness, varicocele, and infections (Ribas-Maynou & Benet, 2019; Agarwal et al., 2014; Gosálvez et al., 2024; Pfeiffer et al., 2000). Making changes such as maintaining a healthy weight, stopping smoking, reducing alcohol intake, and avoiding environmental toxins can improve sperm quality and may lower DNA damage levels.
Antioxidant supplements are commonly used to reduce DNA damage. Studies show that antioxidants, either taken alone or in combination, can improve sperm quality and reduce DNA fragmentation. Common supplements include vitamins C and E, zinc, selenium, coenzyme Q10, and B vitamins. Other helpful compounds include omega-3 fatty acids, N-acetyl-cysteine (NAC), and L-carnitine, which support sperm health and energy production. Overall, research suggests that antioxidant therapy can be an effective and practical option for men with high SDF, either on its own or alongside other treatments (Noegroho et al., 2022).
Treating infections in the male reproductive system is also important. Infections can increase DNA damage by causing inflammation. Identifying and treating these infections, often with antibiotics, can reduce inflammation and improve sperm DNA quality (Gallegos et al., 2008).
Surgical treatment may be recommended for men with varicocele, a condition where enlarged veins in the testicles increase heat and damage sperm production. Repairing a varicocele has been shown to improve sperm quality, reduce DNA fragmentation, and increase the chances of natural pregnancy in some cases (Gill et al., 2021).
In summary, the most effective ways to reduce sperm DNA fragmentation include improving lifestyle habits, using antioxidant supplements, treating infections, and correcting varicocele when present. Combining these approaches can improve sperm health and increase the chances of successful conception.
Ribas-Maynou, J., & Benet, J. (2019). Single and Double Strand Sperm DNA Damage: Different Reproductive Effects on Male Fertility. Genes, 10(2), 105. https://doi.org/10.3390/genes10020105
Agarwal, A., Virk, G., Ong, C., & du Plessis, S. S. (2014). Effect of Oxidative Stress on Male Reproduction. The World Journal of Men’s Health, 32(1), 1–17. https://doi.org/10.5534/wjmh.2014.32.1.1
Gosálvez, J., Johnston, S. D., Prado, A., López-Fernández, C., Contreras, P., Bartolomé-Nebreda, J., González-Martínez, M., Fernández, J. L., De La Vega, C. G., & Góngora, A. (2024). Strong Correlation Between Double-Strand DNA Breaks and Total Sperm DNA Fragmentation in the Human Ejaculate. Archives of Medical Research, 55(8), 103122. https://doi.org/10.1016/j.arcmed.2024.103122
Pfeiffer, P., Goedecke, W., & Obe, G. (2000). Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis, 15(4), 289–302. https://doi.org/10.1093/mutage/15.4.289
Noegroho BS, Siregar S, Tampubolon KAG. Antioxidant Supplementation on Sperm DNA Fragmentation and Sperm Parameters: A Systematic Review and Meta-Analysis. Turk J Urol. 2022 Sep;48(5):375-384. doi: 10.5152/tud.2022.22058. PMID: 36197144; PMCID: PMC9623341.
Sperm DNA fragmentation in infertile men with genitourinary infection by Chlamydia trachomatis and Mycoplasma. Gallegos, Guadalupe, Benito Ramos, M.D.a ∙ Rebeca Santiso, Ph.D.b,c ∙ Vicente Goyanes, M.D., Ph.D.b ∙ Jaime Gosálvez, Ph.D.d ∙ José Luis Fernández, M.D., Ph.D Fertility and Sterility, Volume 90, Issue 2, 328 – 334
Gill, K., Kups, M., Harasny, P., Machalowski, T., Grabowska, M., Lukaszuk, M., Matuszewski, M., Duchnik, E., Fraczek, M., Kurpisz, M., & Piasecka, M. (2021). The Negative Impact of Varicocele on Basic Semen Parameters, Sperm Nuclear DNA Dispersion and Oxidation- Reduction Potential in Semen. International Journal of Environmental Research and Public Health, 18(11), 5977. https://doi.org/10.3390/ijerph18115977
