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Sperm DNA Fragmentation: Comparison
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Infertility is defined as the inability to conceive after 12 months of regular, unprotected intercourse. Approximately 15% of all couples are affected by infertility, with a male factor being solely responsible in about 20% of couples and contributory in another 30–40%. While a semen analysis is an important initial part of a male fertility evaluation, this test alone cannot differentiate those who are fertile versus infertile (except in cases of azoospermia). Specifically, while a standard semen analysis (SA) provides information regarding the patency of the reproductive tract, sperm production, sperm motility, and sperm viability, it does not provide insight into the functional potential of sperm, including its ability to fertilize an oocyte and contribute to normal embryonic development. Consequently, several tests have been developed to better assess the fertility potential of sperm, including those designed to measure sperm deoxyribonucleic acid (DNA) fragmentation (SDF). While sperm DNA damage is broadly defined as any defect in sperm chromatin structure, SDF relates specifically to the single- or double-stranded breaks (SSBs or DSBs) within DNA strands. 

  • sperm
  • infertility
  • cell

1. Introduction

Sperm DNA fragmentation may be associated with a variety of intrinsic (intratesticular) and extrinsic (post-testicular) factors [1]. Intrinsically, such factors may include defective germ cell maturation, abortive apoptosis, and oxidative stress, while extrinsically, lifestyle factors (i.e., smoking, heat exposure, etc.), medications, environmental pollutants, and more may contribute to DNA damage primarily via oxidative stress [1][2]. Regardless of the factor associated with the induction of SDF, at the cellular level, such damage may act through at least one of three proposed mechanisms: abortive apoptosis, defective chromatin maturation, and/or oxidative attack [2].
Intrinsically, sperm are typically protected from DNA damage by the tight compaction of DNA allowed by replacement of somatic histone proteins by protamines during spermatogenesis. This process is facilitated by topoisomerase enzymes, which create DNA breaks to reduce torsional stress and allow for histone to protamine substitution [1][2]. If these breaks are not repaired, impaired chromatin packaging may result in defective sperm maturation and sperm with persistent DNA breakage [1][2].
Such chromatin immaturity may also induce SDF through the activation of apoptosis [3]. In a 2015 study by Muratori et al., the authors demonstrated that a large fraction of sperm concomitantly showed sperm DNA breaks, apoptotic traits (as indicated by the presence of caspase activity and cleaved PARP), and incomplete protamination (as indicated by aniline blue staining) [3]. Since SDF was rarely associated with markers of oxidative damage (as indicated by the presence of 8-hydroxyguanosine and malondialdehyde), the authors concluded that apoptosis plays a major role in the sperm DNA fragmentation that occurs within the testis [3].
While the onset of SDF in the testis is primarily due to defective chromatin maturation or apoptosis, such processes often lead to unviable sperm [2]. In contrast, among the viable sperm with DNA fragmentation, SDF is primarily due to the oxidative stress that occurs during transit through the reproductive tract [2]. In this case, reactive oxygen species (ROS) may induce DNA breaks by directly attacking the DNA backbone or triggering apoptosis, though either the direct activation of caspases and endonucleases or the MAPK pathway [1][2]. Clinically, delays in sperm transport that may subsequently increase the risk of SDF can occur with neurologic abnormalities [4], after microsurgical reconstruction [5], or due to abnormalities in emission and ejaculation associated with medications such as selective serotonin reuptake inhibitors (SSRIs) [6].
Regardless of the source of the DNA damage, several tests have been developed to effectively measure it. The four assays most commonly used to clinically evaluate SDF include: (1) terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay (TUNEL), (2) sperm chromatin structure assay (SCSA), (3) sperm chromatin dispersion (SCD) test, and (4) single-cell gel electrophoresis assay (SCGE/Comet) [1][7]. While all are recognized as common clinical tests for measuring SDF, they have different mechanisms for assessing DNA damage, leading to several unique pros and cons. 

2. Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End Labeling Assay

The terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay (TUNEL) is the most commonly used test for evaluating SDF [8]. TUNEL uses the terminal deoxynucleotidyl transferase (TdT) enzyme to directly label the free 3′ ends of SSBs and DSBs [7]. Specifically, TdT is a polymerase that catalyzes the addition of fluoresceinated-dUTP at the 3′-OH end of the damaged DNA fragments [8]. These breaks are then identified via optical fluorescent microscopy or flow cytometry. Results are reported as the percentage of sperm exhibiting DNA fragmentation out of the total number of sperm analyzed (%SDF) [7]. If manual fluorescent microscopy is used, this test has the advantage of being performed on relatively few (one to several hundred) fresh or frozen sperm, making it suitable for patients with severe oligospermia [7][9]. The main disadvantages of TUNEL are: variable assay protocols among laboratories, a lack of standardization of normal versus abnormal thresholds, and the need for special equipment (i.e., fluorescent microscopy or flow cytometry) [10]. In meta-analyses of clinical data, TUNEL was most predictive of miscarriage rate [11] and birth rate with ART [12] compared to other SDF tests. Similarly, Cissen et al. found that the TUNEL test demonstrated a predictive value in clinical pregnancy rates after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), unlike SCSA and SCD tests, which only demonstrated a weak predictive value [8][13]. One study estimated the cost of this test to be approximately USD 330 [14].

3. Sperm Chromatin Structure Assay

Like TUNEL, the sperm chromatin structure assay (SCSA) is also a fluorescent assay [15]. SCSA was developed to assess both sperm DNA fragmentation and chromatin structure [15]. To perform this test, a mild acid treatment is used to denature DNA at the sites of existing SSBs or DSBs; then, the DNA is stained with acridine orange (AO) [7]. When bound to double-stranded (or intact) DNA, AO emits a green fluorescence; in contrast, when bound to single-stranded (or denatured) DNA, AO emits a red fluorescence [7][9][10]. These fluorescence patterns are captured by a flow cytometer, and the ratio of red to total (red + green) fluorescence intensity is used to calculate the DNA fragmentation index (DFI) [7][16]. SCSA also reports the percent of sperm with high DNA stainability (%HDS), which indicates an abnormally high level of DNA staining due to sperm chromatin defects [16]. Specifically, %HDS indicates that excess histones and proteins have prevented full condensation of the sperm chromatin [13]. This test has several benefits, including the ability to be performed on either fresh or frozen samples, a standardized protocol, and well-established clinical thresholds [7][9]. However, it does require expensive equipment, skilled technicians, and large numbers of sperm, making it less suitable for men with severe oligospermia than other SDF tests [9][10]. The cost of SCSA is estimated to be approximately USD 300 [14].

4. Sperm Chromatin Dispersion

The sperm chromatin dispersion test (SCD; also known as the ‘halo test’) is a commercially available kit that assesses the dispersion of DNA fragments after denaturation [9]. This test indirectly estimates the level of DNA fragmentation by quantifying the amount of nuclear dispersion (“halo”) seen after sperm lysis and acid denaturation remove excess nuclear proteins [13]. Specifically, sperm are embedded in agarose gel and treated with a DNA denaturing solution to “melt” the DNA double helix; however, this “melting” will only occur if there is massive DNA breakage [17]. The sperm are then treated with a lysing solution to remove nuclear proteins, stained, and visualized using bright field or fluorescent microscopy [7][8][9][17]. This removal of nuclear proteins results in nucleoids with a central core and a peripheral ‘halo’ of dispersed DNA loops [18]. Sperm with intact DNA demonstrate large halos of dispersed DNA, while sperm with DNA fragmentation will not have a halo since it was dissolved during the denaturation step [15][17]. Ultimately, the size of the halo is proportional to the extent of the DNA damage [9]. While this is a relatively simple and affordable test (average cost USD 175) [14], it can be time consuming and prone to inter-observer variability [9].

5. Comet

Finally, the Comet assay is an assessment of SDF that utilizes electrophoresis to measure sperm DNA strand breaks [9][15]. Specifically, this test quantifies the shape of single cell nuclei after gel electrophoresis, with small, fragmented DNA migrating more quickly towards the anode than larger, non-fragmented DNA. This produces a typical ‘comet’ shape, with fragmented DNA in the ‘tail’ and non-fragmented DNA in the ‘head’ [13]. Mechanistically, sperm are embedded as a single-cell suspension in an agarose matrix and treated with a lysis buffer containing high salt and detergent to remove cell and nuclear membranes [19]. This processing leaves DNA attached to the nuclear matrix as nucleoids, which are then subjected to electrophoresis [19]. Electrophoresis is performed under either alkaline or neutral pH conditions, resulting in the migration of DNA fragments toward the anode. This allows for the migration of DNA fragments to form a comet ‘tail’, while intact DNA remains in the ‘head’. After electrophoresis, the samples are rinsed, stained with fluorescent dye, and quantitatively analyzed based on the distribution of the fluorescence pattern to determine the extent of the DNA damage [19]. The relative fluorescence of the tail versus the head corresponds to the amount of SDF, with increased fluorescence in the tail reflecting high levels of SDF [7]. Both DNA SSBs and DSBs can be detected using this technique if alkaline pH conditions are used, while neutral pH conditions can only detect DSBs [7]. Results are reported as average comet score (ACS), which represents the average SDF across 100 individual comets (individual sperm) analyzed [7]. The proportion of sperm with high DNA damage (high Comet score, HCS) and proportion of sperm with low DNA damage (low Comet Score, LCS) are also reported [7]. While this assay can be performed with only a small number of sperm, they must be from a fresh sample, and—like the SCD test—interpretation of the results may be time-consuming and prone to inter-observer variability if a manual analysis is performed [9][10]. To reduce such variability, a computerized image analysis system can be used to obtain images, compute the fluorescent intensity profile for each nucleoid, and estimate the relevant comet components [19]. Regarding cost, this test has been estimated to be approximately USD 400 [14].

6. Summary

Ultimately, while the mechanisms and results of the four clinical tests for sperm DNA fragmentation are not identical, there is generally a good correlation between them [7]. Despite these corollary results, there is no consensus regarding the normal reference range(s) for these tests. There is also little evidence for whether these tests are cost-effective in the management of infertile couples [20]. Given this controversy, wresearchers recently reviewed the current evidence regarding the impact of SDF on reproductive outcomes, as well as how SDF test results are best used in modern clinical practice.

References

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  2. Muratori, M.; Marchiani, S.; Tamburrino, L.; Baldi, E. Sperm DNA fragmentation: Mechanisms of origin. Adv. Exp. Med. Biol. 2019, 1166, 75–85.
  3. Muratori, M.; Tamburrino, L.; Marchiani, S.; Cambi, M.; Olivito, B.; Azzari, C.; Forti, G.; Baldi, E. Investigation on the origin of sperm DNA fragmentation: Role of apoptosis, immaturity and oxidative stress. Mol. Med. 2015, 21, 109–122.
  4. Vargas-Baquero, E.; Johnston, S.; Sánchez-Ramos, A.; Arévalo-Martín, A.; Wilson, R.; Gosálvez, J. The incidence and etiology of sperm DNA fragmentation in the ejaculates of males with spinal cord injuries. Spinal Cord 2020, 58, 803–810.
  5. Smit, M.; Wissenburg, O.G.; Romijn, J.C.; Dohle, G.R. Increased sperm DNA fragmentation in patients with vasectomy reversal has no prognostic value for pregnancy rate. J. Urol. 2010, 183, 662–665.
  6. Tanrikut, C.; Feldman, A.S.; Altemus, M.; Paduch, D.A.; Schlegel, P.N. Adverse effect of paroxetine on sperm. Fertil. Steril. 2010, 94, 1021–1026.
  7. Esteves, S.C.; Zini, A.; Coward, R.M.; Evenson, D.P.; Gosálvez, J.; Lewis, S.E.M.; Sharma, R.; Humaidan, P. Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations. Andrologia 2021, 53, e13874.
  8. Sharma, R.; Iovine, C.; Agarwal, A.; Henkel, R. TUNEL assay-Standardized method for testing sperm DNA fragmentation. Andrologia 2021, 53, e13738.
  9. Agarwal, A.; Panner Selvam, M.K.; Baskaran, S.; Cho, C.L. Sperm DNA damage and its impact on male reproductive health: A critical review for clinicians, reproductive professionals and researchers. Expert Rev. Mol. Diagn 2019, 19, 443–457.
  10. Sharma, R.; Masaki, J.; Agarwal, A. Sperm DNA fragmentation analysis using the TUNEL assay. Methods Mol. Biol. 2013, 927, 121–136.
  11. Robinson, L.; Gallos, I.D.; Conner, S.J.; Rajkhowa, M.; Miller, D.; Lewis, S.; Kirkman-Brown, J.; Coomarasamy, A. The effect of sperm DNA fragmentation on miscarriage rates: A systematic review and meta-analysis. Hum. Reprod. 2012, 27, 2908–2917.
  12. Osman, A.; Alsomait, H.; Seshadri, S.; El-Toukhy, T.; Khalaf, Y. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: A systematic review and meta-analysis. Reprod. Biomed. Online 2015, 30, 120–127.
  13. Cissen, M.; Wely, M.V.; Scholten, I.; Mansell, S.; Bruin, J.P.; Mol, B.W.; Braat, D.; Repping, S.; Hamer, G. Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: A systematic review and meta-analysis. PLoS ONE 2016, 11, e0165125.
  14. Loloi, J.; Petrella, F.; Kresch, E.; Ibrahim, E.; Zini, A.; Ramasamy, R. The effect of sperm DNA fragmentation on male fertility and strategies for improvement: A narrative review. Urology 2022, 168, 3–9.
  15. Karavolos, S. Sperm DNA fragmentation. Semin. Reprod. Med. 2021, 39, 194–199.
  16. Evenson, D.P. The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Anim. Reprod. Sci. 2016, 169, 56–75.
  17. Fernández, J.L.; Johnston, S.; Gosálvez, J. Sperm chromatin dispersion (SCD) assay. In A Clinician’s Guide to Sperm DNA and Chromatin Damage; Zini, A., Agarwal, A., Eds.; Springer International Publishing: Cham, Switzerland, 2018; pp. 137–152.
  18. Fernández, J.L.; Muriel, L.; Goyanes, V.; Segrelles, E.; Gosálvez, J.; Enciso, M.; LaFromboise, M.; De Jonge, C. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil. Steril. 2005, 84, 833–842.
  19. Gajski, G.; Ravlić, S.; Godschalk, R.; Collins, A.; Dusinska, M.; Brunborg, G. Application of the comet assay for the evaluation of DNA damage in mature sperm. Mutat. Res. Rev. Mutat. Res. 2021, 788, 108398.
  20. Minhas, S.; Bettocchi, C.; Boeri, L.; Capogrosso, P.; Carvalho, J.; Cilesiz, N.C.; Cocci, A.; Corona, G.; Dimitropoulos, K.; Gül, M.; et al. European association of urology guidelines on male sexual and reproductive health: 2021 update on male infertility. Eur. Urol. 2021, 80, 603–620.
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