Sperm Chromatin Structure Assay

From WikiProjectMed
Jump to navigation Jump to search

Sperm Chromatin Structure Assay (SCSA) is a diagnostic approach that detects sperm abnormality with a large extent of DNA fragmentation.[1] First described by Evenson in 1980, the assay is a flow cytometric test that detects the vulnerability of sperm DNA to acid-induced denaturation DNA in situ.[2] SCSA measures sperm DNA fragmentation attributed to intrinsic and extrinsic factors and reports the degree of fragmentation in terms of DNA Fragmentation Index (DFI). The use of SCSA expands from evaluation of male infertility and subfertility, toxicology studies and evaluation of quality of laboratory semen samples. Notably, SCSA outcompetes other convention sperm DNA fragmentation (sDF) assays such as TUNEL and COMET in terms of efficiency, objectivity, and repeatability.

Schematic representation of the SCSA. Use of flow cytometry in differentiating intact sperms from defected sperms. Laser beam strikes each sperm cell. Photomultiplier tube identifies and quantifies the results.

History

Before the development of SCSA, diagnosis or prognosis of male infertility/subfertility was principally referenced the World Health Organisation (WHO) manual-based semen parameters,[3] including semen concentration, motility, and morphology. Yet, several reports of pregnancy failure had the parameters within normal range, suggesting that none of these measurements has drawn a reliable conclusion to reflect chance of fertility of a couple.[4] Furthermore, such parameters are often associated with high labour intensity and lack of statistical power.

In the late 1970s, Donald P. Evenson at Memorial Sloan Kettering Cancer Centre in the United States received an NIH Research Project Grant (RO1) for mammalian sperm chromatin structure study.[5][6] Various techniques have since been adopted to gain access to sperm DNA integrity. In particular, transmission electron microscopy reflected a significant amount of sperm chromatin heterogeneity.[4][7]

The heterogeneity was then confirmed through flow cytometry by contrasting AO staining results between human and mouse sperm nuclei. Homogeneous results were observed in the mouse sample while heterogeneous fluorescence intensity varied among the human sample. A hypothesis was proposed “single-stranded/double-stranded DNA breaks-induced sperm DNA fragmentation is correlated to male infertility.”[4][5] In 1980, Evenson et al. published papers that synthesise this knowledge into clinical tests and found SCSA.

Initially, utilization of thermal energy in buffer (100 °C, 5 min) was proposed and used for denaturation of DNA at sites DNA damage.[8] However, the heated sperm protocol was time-consuming and induced random loss of sperm sample. Therefore, acid-induced denaturation has replaced heat-induced denaturation due to greater convenience of low pH technique and similarity in results.[5]

Principle

SCSA is a widespread diagnostic tool in detection of sperm samples with a high degree of DNA fragmentation and absence of histone-to-protamine proteins exchange in sperm nuclei.[9] SCSA defines sperm abnormality as an increased vulnerability of sperm DNA to in-situ heat/acid-induced denaturation.[4] Theoretically, a completely mature and healthy sperm nuclei, which is rich in disulfide bond (S-S), shall have its DNA preserved in double-stranded form.[5] A low pH treatment opens up defective sperm DNA at the sites of damage. Through acridine orange (AO) staining, AO molecules are intercalated into double-stranded DNA in intact sperms while aggregation of AO molecules occurs at single-stranded DNA in defective sperms.[4][5] Undergoing flow cytometry (blue light), green (native DNA) and red (damaged DNA) fluorescence will be emitted from intact and defective sperms respectively.[2][4][10] Signals will be analysed with software programming in examination of both sperm DNA fragmentation (sDF) and atypical chromatin structure.

Causes for sperm DNA damage

The integrity of sperm DNA is in close correlation with the transfer of paternal DNA into the oocyte during fertilisation. The etiology of sperm DNA damage can be subdivided into intrinsic and extrinsic factors. The former is attributed to a series of pathophysiological phenomena during spermatogenesis; the latter is caused by postnatal exposure to endogenous sources of DNA breaks.

Intrinsic factors

Extrinsic factors

  • Age: Although males produce sperm throughout their adulthood, older age is associated with increased number of DNA double-stranded breaks and decreased frequency of sperm apoptosis.[13] Such observation is implicative of deterioration of sperm selection, quality, and integrity.[20]
  • Heat stress: High temperatures cause adverse effects to sperm DNA and male fertility. Excessive heat is related to impaired sperm chromatin integrity,[21] and testis overheating is associated with reduced fertility potential.[22]
  • Smoking: Toxins in common tobaccos may increase the prevalence of fragmented DNA.[4] Smoking is associated with significantly escalated levels of seminal ROS and oxidative stress.[23] Increased ROS activity leads to apoptosis and increased fragmentation of DNA.[16]

Procedure

Currently, only the SCSA protocol developed by Evenson et al. has received trademark protection in achievement of clinical relevance between different laboratories.[4] The individual steps of SCSA are as follows:

  1. Freezing/Thawing: After ejaculation, human sperm samples are subjected to a 30-minute semen liquefaction at 37 °C, followed by cryopreservation in an ultra-low temperature freezer (–70 to –110 °C) or placed directly into liquefied nitrogen cryovial.[24] Frozen or fresh sperm samples are thawed in a 37 °C water bath and diluted with a TNE buffer to obtain 200 μl suspension (sperm concentration: 1-2 x 10^6 mL).[24]
  2. Acid-induced denaturation: 400 μl acidic solution (pH 1.2) containing 0.15M sodium chloride solution, 0.08M Tris hydrochloric acid, and 0.1% Triton-X 100 are added into 200 μl sperm suspension,[4][24] and the solution is mixed strictly for 30 seconds. Such a process enables denaturation of sperm nuclei with DNA damage.
  3. Acridine orange (AO) staining: Next, 1.20 ml of AO staining solution with 6 µg AO/ml staining buffer is added into the mixture.[4][24] The small AO molecules penetrate through the sperm chromatin in access to double-stranded DNA and single-stranded DNA in intact and defective sperm nuclei respectively.
  4. Flow cytometry (FCM): Using a flow cytometer, 500-1000 sperms can be examined within minutes on a 1024 x 1024 gradation scale through a dual parameter.[9] Visualized under blue light at the wavelengths of 450-490 nm, double-stranded DNA from intact sperms emit green fluorescence (488 nm) while aggregation of AO molecules single-stranded DNA from defective sperms leads to metachromatic shift to red fluorescence (>630 nm).[9][4]
  5. Data analysis: A scattergram (cytogram) will be generated from the flow cytometer reflecting DNA stainability from red (X-axis) and green (Y-axis) dots to single out the heterogeneity;[9] With SCSAsoft® software, data from the scattergram will be converted into frequency histograms in calculation of DNA fragmentation index (DFI) / Cells Outside the Main Peak of αt (COMPαt), alpha t (αt), and High DNA Stainable fraction (HDS).[4][5]

Parameters

SCSA consists of a fixed flow cytometry protocol and a specific computing program, SCSAsoft ®. Measurements include DNA fragmentation index (DFI) and High DNA Stainable (HDS) fraction, which represent the percentage of sperm with DNA breaks/protamine defects and immature spermatozoa without full protamination respectively.[10]

DNA fragmentation index (DFI)

Also known as Cells Outside the Main Peak of αt (COMPαt), DFI can be further sub-classified into mean DFI (X DFI) and standard deviation DFI (SD DFI).[5] The index has been determined as the most sensitive criteria for fertility assessment in reflection of sperm DNA integrity. Normal DFI implies no measurable value; moderate DFI sample infers normal sperm morphology; and high DFI fractions exhibited elongated nuclei and signs of apoptosis. In general, the greater the DFI, the higher the chance of infertility or subfecundity.

Within DFI of 0-20%, the occurrence of spontaneous pregnancy remains consistent;[2] when DFI exceeds 20%, the rate of natural fertility gradually declines;[2] when DFI exceeds 30%, the odds ratio for natural or Intrauterine insemination (IUI) fertility is greatly reduced by 8-10 folds, suggesting a close-to-zero chance of pregnancy.[2]

High DNA Stainable (HDS) fraction

The HDS sperm population has a remarkably high degree of DNA staining by AO molecules due to the presence of unprocessed P2 protamines.[9][25] Determination of HDS value reflects structural chromatin abnormalities. A high HDS value is indicative of immature sperm morphology and hence pregnancy failure.[25][26]

Applications

Diagnosis of male infertility or subfertility

Since the SCSA can be performed to assess the sperm abnormality, it is a valid instrument to determine male infertility or subfertility.

Although the causes and events that actuate sperm DNA damage and fragmentation are not yet fathomed, Sperm DNA fragmentation has been shown to be closely correlated with fertility and subfertility in not only humans, but also bulls, boars, and stallions.[5][27][28][29] Such finding asserts the DFI determined by SCSA to be a strong independent predictor of in vivo pregnancy and a clinically useful technique.[13][23][30][31][8]

Currently, 25% DFI is the established clinical threshold in classifying males into statistical probability of: 1) increased time for natural pregnancy, 2) lower chance of Intrauterine insemination (IUI) success, 3) more miscarriage, or 4) infertility. High HDS values are in positive correlation to pregnancy failures.

In such cases, other assisted reproductive technologies (ART) may be performed, including intracytoplasmic sperm injection (ICSI) (for sperm sample with DFI>25%) or testicular sperm extraction (TESE) (for sperm sample with DFI>50%).[9]

Toxicology studies

Sperm DNA damage can be attributed to exposure chemotherapy, radiotherapy, or other environmental toxicants. SCSA is highly dose-responsive to sperm DNA fragmentation induced by chemical toxicants.[13] Therefore, SDαt is the most important variable for toxicology studies.

Evaluation of cool-stored semen

SCSA is also performed to assess the quality of laboratory sperm samples that have been stored for at least 24 hours. Semen samples that have been stored at appropriate conditions will have essentially no change, while greater change in DNA quality indicates an improper handling.[32]

Advantages

SCSA has numerous advantages when compared to other sperm DNA fragmentation (sDF) assays [TUNEL assay, COMET assay, and Sperm Chromatin Dispersion (SCD)], which include:

  • More time and cost efficient: 5000-10000 spermatozoa can be analysed in less than 5 minutes.[5][4] The efficiency is higher than any other existing sperm fragmentation protocols. Moreover, the requirements for equipment and reagents are relatively low. Only 10 cents are required per test for the reagents required.[9]
  • Higher objectivity and accuracy: Conventional sperm analysis includes sperm count, morphology and motility in determination of infertility or subfertility. However, several reports of pregnancy failure had the aforementioned parameters within normal range.[4] For SCSA, machine-guided DFI and HDS values with an unbiased threshold are measured rather than subjective human-eye evaluation, resulting in a higher precision (Coefficient of Variation testing, CV's of 1-3%).
  • Higher repeatability: Since sperm count, morphology and motility of semen samples fluctuate within a short period of time, results of analysis are less repeatable. SCSA has a repeatability of 0.98-0.99 in clinical settings. Unless disruption is made by different lifestyles or medical intervention, experimental results are reproducible.[9][4][13]

Limitations

Despite the objective data and advantages offered, the efficacy of SCSA in fertility assessment remains doubted clinically. Suggested limitations include:

References

  1. ^ "Sperm Chromatin Structure Assay (SCSA) | Center for Women's Health | OHSU". www.ohsu.edu. Retrieved 2021-03-31.
  2. ^ a b c d e Evenson, Donald 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. doi:10.1016/j.anireprosci.2016.01.017. PMID 26919909.
  3. ^ "Aktuelles". Praxis. 92 (20): 978. 2003. doi:10.1024/0369-8394.92.20.978. ISSN 0369-8394.
  4. ^ a b c d e f g h i j k l m n o EVENSON, DONALD P.; LARSON, KJERSTEN L.; JOST, LORNA K. (2002-01-02). "Sperm Chromatin Structure Assay: Its Clinical Use for Detecting Sperm DNA Fragmentation in Male Infertility and Comparisons With Other Techniques". Journal of Andrology. 23 (1): 25–43. doi:10.1002/j.1939-4640.2002.tb02599.x. ISSN 0196-3635. PMID 11780920.
  5. ^ a b c d e f g h i Bungum, Mona; Bungum, Leif; Giwercman, Aleksander (2011). "Sperm chromatin structure assay (SCSA): a tool in diagnosis and treatment of infertility". Asian Journal of Andrology. 13 (1): 69–75. doi:10.1038/aja.2010.73. ISSN 1008-682X. PMC 3739398. PMID 21057512.
  6. ^ Evenson, D. P.; Witkin, S. S.; de Harven, E.; Bendich, A. (1978-05-01). "Ultrastructure of partially decondensed human spermatozoal chromatin". Journal of Ultrastructure Research. 63 (2): 178–187. doi:10.1016/S0022-5320(78)80073-2. ISSN 0022-5320. PMID 671582.
  7. ^ Oleszczuk, K.; Giwercman, A.; Bungum, M. (2016). "Sperm chromatin structure assay in prediction of in vitro fertilization outcome". Andrology. 4 (2): 290–296. doi:10.1111/andr.12153. ISSN 2047-2927. PMID 26757265.
  8. ^ a b Evenson, D.P. (1999-04-01). "Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic". Human Reproduction. 14 (4): 1039–1049. doi:10.1093/humrep/14.4.1039. ISSN 1460-2350. PMID 10221239.
  9. ^ a b c d e f g h Evenson, Donald P. (2013), Carrell, Douglas T.; Aston, Kenneth I. (eds.), "Sperm Chromatin Structure Assay (SCSA®)", Spermatogenesis, Methods in Molecular Biology, vol. 927, Totowa, NJ: Humana Press, pp. 147–164, doi:10.1007/978-1-62703-038-0_14, ISBN 978-1-62703-037-3, PMID 22992911, retrieved 2021-03-31
  10. ^ a b Ajina, T.; Ammar, O.; Haouas, Z.; Sallem, A.; Ezzi, L.; Grissa, I.; Sakly, W.; Jlali, A.; Mehdi, M. (2017). "Assessment of human sperm DNA integrity using two cytochemical tests: Acridine orange test and toluidine blue assay". Andrologia. 49 (10): e12765. doi:10.1111/and.12765. PMID 28224638. S2CID 22415071.
  11. ^ Bannister, Laura A.; Reinholdt, Laura G.; Munroe, Robert J.; Schimenti, John C. (2004). "Positional cloning and characterization of mousemei8, a disrupted allele of the meiotic cohesinRec8". Genesis. 40 (3): 184–194. doi:10.1002/gene.20085. ISSN 1526-954X. PMID 15515002. S2CID 38349506.
  12. ^ Page, Andrea W; Orr-Weaver, Terry L (1997). "Stopping and starting the meiotic cell cycle". Current Opinion in Genetics & Development. 7 (1): 23–31. doi:10.1016/s0959-437x(97)80105-0. ISSN 0959-437X. PMID 9024631.
  13. ^ a b c d e f Oleszczuk, K.; Giwercman, A.; Bungum, M. (2016-01-12). "Sperm chromatin structure assay in prediction of in vitro fertilization outcome". Andrology. 4 (2): 290–296. doi:10.1111/andr.12153. ISSN 2047-2919. PMID 26757265.
  14. ^ Marcon, Ludovic; Boissonneault, Guylain (2004-04-01). "Transient DNA Strand Breaks During Mouse and Human Spermiogenesis:New Insights in Stage Specificity and Link to Chromatin Remodeling1". Biology of Reproduction. 70 (4): 910–918. doi:10.1095/biolreprod.103.022541. ISSN 0006-3363. PMID 14645105.
  15. ^ Laberge, R.-M.; Boissonneault, G. (2005). "Chromatin Remodeling in Spermatids: A Sensitive Step for the Genetic Integrity of the Male Gamete". Archives of Andrology. 51 (2): 125–133. doi:10.1080/014850190518134. ISSN 0148-5016. PMID 15804867.
  16. ^ a b c Sakkas, D (1999-01-01). "Origin of DNA damage in ejaculated human spermatozoa". Reviews of Reproduction. 4 (1): 31–37. doi:10.1530/ror.0.0040031. ISSN 1359-6004. PMID 10051100.
  17. ^ Agarwal, Ashok; Saleh, Ramadan A.; Bedaiwy, Mohamed A. (2003). "Role of reactive oxygen species in the pathophysiology of human reproduction". Fertility and Sterility. 79 (4): 829–843. doi:10.1016/s0015-0282(02)04948-8. ISSN 0015-0282. PMID 12749418.
  18. ^ Aitken, RJ; Krausz, C (2001-10-01). "Oxidative stress, DNA damage and the Y chromosome". Reproduction. 122 (4): 497–506. doi:10.1530/rep.0.1220497. hdl:2158/778616. ISSN 1470-1626. PMID 11570956. S2CID 32081883.
  19. ^ de Lamirande, E (1997-01-01). "Reactive oxygen species and sperm physiology". Reviews of Reproduction. 2 (1): 48–54. doi:10.1530/revreprod/2.1.48. ISSN 1359-6004. PMID 9414465.
  20. ^ Singh, Narendra P; Muller, Charles H; Berger, Richard E (2003). "Effects of age on DNA double-strand breaks and apoptosis in human sperm". Fertility and Sterility. 80 (6): 1420–1430. doi:10.1016/j.fertnstert.2003.04.002. ISSN 0015-0282. PMID 14667878.
  21. ^ Ahmad, Gulfam; Moinard, Nathalie; Esquerré-Lamare, Camille; Mieusset, Roger; Bujan, Louis (2012). "Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men". Fertility and Sterility. 97 (3): 546–553. doi:10.1016/j.fertnstert.2011.12.025. ISSN 0015-0282. PMID 22265039.
  22. ^ Thonneau, P.; Bujan, L.; Multigner, L.; Mieusset, R. (1998-08-01). "Occupational heat exposure and male fertility: a review". Human Reproduction. 13 (8): 2122–2125. doi:10.1093/humrep/13.8.2122. ISSN 0268-1161. PMID 9756281.
  23. ^ a b Saleh, Ramadan A; Agarwal, Ashok; Nelson, David R; Nada, Essam A; El-Tonsy, Mohammed H; Alvarez, Juan G; Thomas, Anthony J; Sharma, Rakesh K (2002). "Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study". Fertility and Sterility. 78 (2): 313–318. doi:10.1016/s0015-0282(02)03219-3. ISSN 0015-0282. PMID 12137868.
  24. ^ a b c d Evenson, Donald; Jost, Lorna (2000). "Sperm chromatin structure assay is useful for fertility assessment". Methods in Cell Science. 22 (2/3): 169–189. doi:10.1023/A:1009844109023. PMID 11264952.
  25. ^ a b Lewis, Sheena E.M. (2013-06-01). "The place of sperm DNA fragmentation testing in current day fertility management". Middle East Fertility Society Journal. 18 (2): 78–82. doi:10.1016/j.mefs.2013.01.010. ISSN 1110-5690. S2CID 53339711.
  26. ^ Evenson, D.; Darzynkiewicz, Z; Melamed, M. (1980-12-05). "Relation of mammalian sperm chromatin heterogeneity to fertility". Science. 210 (4474): 1131–1133. Bibcode:1980Sci...210.1131E. doi:10.1126/science.7444440. ISSN 0036-8075. PMID 7444440.
  27. ^ Knuth, Ulrich A.; Behre, Hermann; Belkien, Lutz; Bents, Hinrich; Nieschlag, Eberhard (1985). "Clinical trial of 19-nortestosterone-hexoxyphenylpropionate (Anadur) for male fertility regulation". Fertility and Sterility. 44 (6): 814–821. doi:10.1016/s0015-0282(16)49043-6. ISSN 0015-0282. PMID 3935486.
  28. ^ Giwercman, Aleksander; Lindstedt, Lars; Larsson, Mattias; Bungum, Mona; Spano, Marcello; Levine, Richard J.; Rylander, Lars (2009-10-15). "Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case-control study". International Journal of Andrology. 33 (1): e221–e227. doi:10.1111/j.1365-2605.2009.00995.x. ISSN 0105-6263. PMID 19840147.
  29. ^ Castilla, J.A.; Zamora, S.; Gonzalvo, M.C.; Luna del Castillo, J.D.; Roldan-Nofuentes, J.A.; Clavero, A.; Björndahl, L.; Martínez, L. (2010). "Sperm chromatin structure assay and classical semen parameters: systematic review". Reproductive BioMedicine Online. 20 (1): 114–124. doi:10.1016/j.rbmo.2009.10.024. ISSN 1472-6483. PMID 20158996.
  30. ^ Spanò, Marcello; Bonde, Jens P; Hjøllund, Henrik I; Kolstad, Henrik A; Cordelli, Eugenia; Leter, Giorgio (2000). "Sperm chromatin damage impairs human fertility‡‡The Danish First Pregnancy Planner Study is a collaborative follow-up study on environmental and biological determinants of fertility. The project is coordinated by the Steno Institute of Public Health, Aarhus University and is undertaken in collaboration with the Department of Growth and Reproduction, National University Hospital, Copenhagen. The team includes Jens Peter E. Bonde, Niels Henrik I. Hjøllund, Tina Kold Jensen, Tine Brink Henriksen, Henrik A. Kolstad, Erik Ernst, Aleksander Giwercman, Niels Erik Skakkebæk, and Jørn Olsen". Fertility and Sterility. 73 (1): 43–50. doi:10.1016/S0015-0282(99)00462-8. PMID 10632410.
  31. ^ Bungum, M. (2004-04-22). "The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI". Human Reproduction. 19 (6): 1401–1408. doi:10.1093/humrep/deh280. ISSN 1460-2350. PMID 15117894.
  32. ^ Love, Charles C. (2005). "The sperm chromatin structure assay: A review of clinical applications". Animal Reproduction Science. 89 (1–4): 39–45. doi:10.1016/j.anireprosci.2005.06.019. ISSN 0378-4320. PMID 16140481.
  33. ^ Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert (2016-11-10). Drevet, Joël R (ed.). "Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis". PLOS ONE. 11 (11): e0165125. Bibcode:2016PLoSO..1165125C. doi:10.1371/journal.pone.0165125. ISSN 1932-6203. PMC 5104467. PMID 27832085.
  34. ^ Simon, Luke; Zini, Armand; Dyachenko, Alina; Ciampi, Antonio; Carrell, DouglasT (2016). "A systematic review and meta-analysis to determine the effect of sperm DNA damage on IVF and ICSI outcome". Asian Journal of Andrology. 19 (1): 80–90. doi:10.4103/1008-682X.182822. ISSN 1008-682X. PMC 5227680. PMID 27345006.
  35. ^ a b Zini, Armand (September 2017). "The benefits and limitations of sperm DNA testing in clinical practice". Translational Andrology and Urology. 6 (S4): S326–S327. doi:10.21037/tau.2017.09.09. PMC 5643662. PMID 29082133.
Retrieved from "https://mdwiki.org/wiki/Sperm_Chromatin_Structure_Assay"