Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

Evaluation of Sperm Chromatin Integrity Using Aniline Blue and Toluidine Blue Staining in Infertile and Normozoospermic Men

Authors
1 Non-Communicable Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
2 Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
3 Gametogenesis Research Center, Fertility, and Infertility Center, Kashan University of Medical Sciences, Kashan, Iran
4 Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran
Abstract
Background: Male infertility is defined as a man lost his ability to fertilize a fertile female naturally. Diagnosis of male infertility cannot be made just according to basic semen analysis. It is necessity to have specific tests for evaluation of chromatin integrity. In this study, an attempt was made to evaluate the sperm chromatin quality in fertile men and infertile subgroup. Methods: Among 1386 couples, 342 men were categorized into normospermia and 1044 were infertile and they were referred to Yazd Research and Clinical Center for infertility treatment. Standard semen analysis and sperm nuclear maturity tests including aniline blue (AB) and toluidine blue (TB) staining were done. Data were analyzed by SPSS software. The p≤0.05 was considered statistically significant. Results: The mean value of TB staining was significantly higher in infertile group compared to normospermic group (p=0.005). Mean of sperm normal morphology was lower in idiopathic infertile men in comparison with normozoospermic men (p=0.001). The highest negative correlation was obtained between sperm count and AB staining. Progressive motility was negatively correlated with AB and TB staining in both groups but there was no significant difference between AB staining and progressive motility in men normospermia group. Conclusion: Sperm chromatin staining using AB and TB showed a negative association between sperm chromatin condensation with sperm count, normal morphology and progressive motility. It seems that the AB and TB test may be useful for the assessment of male fertility potential.
Keywords

  1. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37.
  2. Lewis SE, Agbaje I, Alvarez J. Sperm DNA tests as useful adjuncts to semen analysis. Syst Biol Reprod Med. 2008;54(3):111-25.
  3. Evgeni E, Charalabopoulos K, Asimakopoulos B. Human sperm DNA fragmentation and its correlation with conventional semen parameters. J Reprod Infertil. 2014;15(1):2-14.
  4. Varghese A, Bragais F, Mukhopadhyay D, Kundu S, Pal M, Bhattacharyya A, et al. Human sperm DNA integrity in normal and abnormal semen samples and its correlation with sperm characteristics. Andrologia. 2009;41(4):207-15.
  5. Lewis S, Aitken R. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res. 2005;322(1):33-41.
  6. Zini A, Boman JM, Belzile E, Ciampi A. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2008;23(12):2663-8.
  7. Pourmasumi S, Ghasemi N, Talebi AR, Mehrabani M, Sabeti P. The effect of vitamin E and selenium on sperm chromatin quality in couples with recurrent miscarriage. Int J Med Lab. 2018;5(1):1-10.
  8. Sivanarayana T, Krishna CR, Prakash GJ, Krishna K, Madan K, Sudhakar G, et al. Sperm DNA fragmentation assay by sperm chromatin dispersion (SCD): correlation between DNA fragmentation and outcome of intracytoplasmic sperm injection. Reprod Med Biol. 2014;13(2):87-94.
  9. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update. 2003;9(4):331-45.
  10. Pourmasumi S, Sabeti P, Rahiminia T, Mangoli E, Tabibnejad N, Talebi AR. The etiologies of DNA abnormalities in male infertility: An assessment and review. Int J Reprod Biomed (Yazd). 2017;15(6):331-44.
  11. Emelyanov AV, Fyodorov DV. Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin. Genes Dev. 2016;30(24):2651-6.
  12. Virro MR, Larson-Cook KL, Evenson DP. Sperm chromatin structure assay (SCSA®) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril. 2004;81(5):1289-95.
  13. Sergerie M, Laforest G, Boulanger K, Bissonnette F, Bleau G. Longitudinal study of sperm DNA fragmentation as measured by terminal uridine nick end-labelling assay. Hum Reprod. 2005;20(7):1921-7.
  14. Mehta A, Sigman M. Identification and preparation of sperm for ART. Urolc Clin North Am. 2014;41 (1):169-80.
  15. Talebi A, Vahidi S, Aflatoonian A, Ghasemi N, Ghasemzadeh J, Firoozabadi R, et al. Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions. Andrologia. 2012;44 Suppl 1:462-70.
  16. Talebi AR, Khalili MA, Vahidi S, Ghasemzadeh J, Tabibnejad N. Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury. J Spinal Cord Med. 2013;36(2):140-6.
  17. Sabeti P, Amidi F, Kalantar SM, Sedighi Gilani MA, Pourmasumi S, Najafi A, et al. Evaluation of intracellular anion superoxide level, heat shock protein A2 and protamine positive spermatozoa percentages in teratoasthenozoospermia. Int J Reprod Biomed (Yazd). 2017;15(5):279-86.
  18. Evenson DP. 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.
  19. Practice committee of the American society for reproductive medicine. The clinical utility of sperm DNA integrity testing: a guideline. Fertil Steril. 2013;99(3):673-7.
  20. Sellami A, Chakroun N, Ben Zarrouk S, Sellami H, Kebaili S, Rebai T, et al. Assessment of chromatin maturity in human spermatozoa: useful aniline blue assay for routine diagnosis of male infertility. Adv Urol. 2013;2013:578631.
  21. Irez T, Sahmay S, Ocal P, Goymen A, Senol H, Erol N, et al. Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests, and assisted reproduction techniques. Andrologia. 2015;47(4):438-47.
  22. Kim HS, Kang MJ, Kim SA, Oh SK, Kim H, Ku SY, et al. The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis. Clin Exp Reprod Med. 2013;40(1):23-8.
  23. Hamidi J, Frainais C, Amar E, Bailly E, Clément P, Ménézo Y. A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index. Zygote. 2015;23(4):556-62.
  24. Varghese AC, Bragais FM, Mukhopadhyay D, Kundu S, Pal M, Bhattacharyya AK, et al. Human sperm DNA integrity in normal and abnormal semen samples and its correlation with sperm characteristics. Andrologia. 2009;41(4):207-15.
  25. Sabeti P, Pourmasumi S, Rahiminia T, Akyash F, Talebi AR. Etiologies of sperm oxidative stress. Int J Reprod Biomed (Yazd). 2016;14(4):231-40.
  26. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Switzerland: WHO press; 2010. 287 p.
  27. Abdulla M, Ahmed M, Barakat A. Comparison of Swim Down and Density Gradient Sperm Preparation Methods in Terms of Motility, Morphology and DNA Fragmentation. Am J Life Sci Res. 2015;3(2):169-77.
  28. Franken DR, Franken CJ, de la Guerre H, de Villiers A. Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining. Andrologia. 1999;31(6):361-6.
  29. Erenpreisa J, Erenpreiss J, Freivalds T, Slaidina M, Krampe R, Butikova J, et al. Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry A. 2003;52(1):19-27.
  30. Al-Fahham AA, Al-Sultani YK, Muhammad-Ali AK. Using Sperm chromatin Staining Techniques as a Predictive Diagnostic Tool for Male Infertility. Kufa J Nursing Sci. 2014;4(2):56-64.
  31. Hammadeh M, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W, Strehler E. Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl. 2001;46(2):99-104.
  32. Kazerooni T, Asadi N, Jadid L, Kazerooni M, Ghanadi A, Ghaffarpasand F, et al. Evaluation of sperm’s chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion. J Assist Reprod Genet. 2009;26(11-12):591-6.
  33. Talebi AR, Vahidi S, Aflatoonian A, Ghasemi N, Ghasemzadeh J, Firoozabadi RD, et al. Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions. Andrologia. 2012;44 Suppl 1:462-70.
  34. Muratori M, Tamburrino L, Marchiani S, Cambi M, Olivito B, Azzari C, et al. Investigation on the origin of sperm DNA fragmentation: role of apoptosis, immaturity and oxidative stress. Mol Med. 2015;21:109-22.
  35. Talebi AR, Sarcheshmeh AA, Khalili MA, Tabibnejad N. Effects of ethanol consumption on chromatin condensation and DNA integrity of epididymal spermatozoa in rat. Alcohol. 2011;45(4):403-9.
  36. Talebi AR, Moein MR, Tabibnejad N, Ghasemzadeh J. Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests. Andrologia. 2008;40(4):245-51.
  37. Talebi AR, Khalili MA, Hossaini A. Assessment of nuclear DNA integrity of epididymal spermatozoa following experimental chronic spinal cord injury in the rat. Int J Androl. 2007;30(3):163-9.
  38. Rahimipour M, Talebi AR, Anvari M, Sarcheshmeh AA, Omidi M. Effects of different doses of ethanol on sperm parameters, chromatin structure and apoptosis in adult mice. Eur J Obstet Gynecol Reprod Biol. 2013;170(2):423-8.
  39. Simon L, Liu L, Murphy K, Ge S, Hotaling J, Aston K, et al. Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment. Hum Reprod. 2014;29(5):904-17.
  40. Dada R, Kumar BS, Kumar M, Oberoi AS, Shamsi MB. Sperm DNA damage assessment: techniques and relevance in male infertility diagnostics. In: Talwar CP, editor. Manual of cytogenetics in reproductive biology. India: Jaypee Brothers meical Publishers (P) Ltd; 2014. p. 135-43.
  41. Pourentezari M, Talebi AR, Mangoli E, Anvari M, Rahimipour M. Additional deleterious effects of alcohol consumption on sperm parameters and DNA integrity in diabetic mice. Andrologia. 2015;48(5):564-9.
  42. Ribas‐Maynou J, García‐Peiró A, Fernández‐Encinas A, Abad C, Amengual M, Prada E, et al. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay. Andrology. 2013;1(5):715-22.
  43. Tsarev I, Bungum M, Giwercman A, Erenpreisa J, Ebessen T, Ernst E, et al. Evaluation of male fertility potential by Toluidine Blue test for sperm chromatin structure assessment. Hum Reprod. 2009;24(7):1569-74.