Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

The Effects of In Vitro Incubation of Asthenoteratozoospermic Semen after Density Gradient Centrifugation at Room Temperature and 37oC on Sperm Parameters, Chromatin Quality and DNA Fragmentation in a Short Time Period

Authors
Motahareh Karimi Zarchi 1
Behnam Maleki 2
Mahmood Dehghani Ashkezari 1
Leila Motamed Zadeh 2
Azam Agha-Rahimi 2
1 Department of Biology, Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Yazd, Iran
2 Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
10.18502/jri.v21i4.4332
Abstract
Background: Sperm quality is an important factor in assisted reproductive technology (ART) that affects the success rate of infertile couples treatment. In vitro incubation of sperm can influence its parameters and DNA integrity. The present study focused on the effect of different incubation temperatures sperm parameters on asthenoteratozoospermia semen prepared with density gradient centrifugation at different times. Methods: Twenty-seven samples were collected and prepared. Then, the suspension was divided into two parts. One part was incubated at room temperature (RT), and another was incubated at 37 o C. Immediately and after 2 hr (2H) and 4 hr (4H), spermatozoa were evaluated regarding motility, viability, morphology, sperm protamine deficiency, chromatin and DNA fragmentation. Statistical analysis was performed using paired t-test and repeated measures. The p<0.05 was considered statistically significant. Results: Our results showed that following 2 and 4 hr of incubation at RT, sperm progressive motility and viability decreased significantly. Sperm DNA fragmentation increased significantly following 2 and 4 hr of incubation at RT and 37 o C. The Trend analysis confirmed that there were no significant differences between sperm parameters and DNA fragmentation after different times at RT and 37 o C. Conclusion: Incubation of sperm at RT in comparison to 37 o C didn’t preserve sperm parameters and DNA efficiently. Therefore, IVF, ICSI and IUI procedure should be performed in the soonest possible time after sperm preparation.
Keywords
In vitro incubation
Asthenoteratozoospermia
DNA Fragmentation
Room temperature
  1. Elder K, Dale B. In-vitro fertilization. 3rd ed. Cambridge: Cambridge University Press; 2010. 252 p.
  2. World health organization, department of reproductive health and research. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: World health organization; 2010. 287 p.
  3. Holmes E, Björndahl L, Kvist U. Post‐ejaculatory increase in human semen osmolality in vitro. Andrologia. 2019;51(7):e13311.
  4. Ahmed I, Abdelateef S, Laqqan M, Amor H, Abdel‐Lah MA, Hammadeh ME, et al. Influence of extended incubation time on Human sperm chromatin condensation, sperm DNA strand breaks and their effect on fertilisation rate. Andrologia. 2018;50(4)e12960.
  5. Çok T, Aytaç PÇ, Şimşek E, Haydardedeoğlu B, Kalaycı H, Özdemir H, et al. The effect of preserving prepared sperm samples at room temperature or at 37oC before intrauterine insemination (IUI) on clinical pregnancy rate. Turk J Obstet Gynecol. 2015;12(1):6-10.
  6. Thijssen A, Klerkx E, Huyser C, Bosmans E, Campo R, Ombelet W, et al. Influence of temperature and sperm preparation on the quality of spermatozoa. Reprod Biomed Online. 2014;28(4):436-42.
  7. Nabi A, Khalili MA, Halvaei I, Roodbari F. Prolonged incubation of processed human spermatozoa will increase DNA fragmentation. Andrologia. 2014;46(4):374-9.
  8. Rougier N, Uriondo H, Papier S, Checa MA, Sueldo C, Sedó CA, et al. Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time. Fertil Stril. 2013;100(1):69-74.
  9. Nagy ZP, Varghese AC, Agarwal A. In Vitro Fertilization: a textbook of current and emerging methods and devices. 2nd ed. switzerland: Springer; 2019. 947 p.
  10. Bourne H, Archer J, Edgar DH, Gordon Baker HW. Sperm preparation techniques. In: Textbook of assisted reproductive techniques. US: CRC Press; 2012. p. 61-74.
  11. Schuffner A, Morshedi M, Vaamonde D, Duran EH, Oehninger S. Effect of different incubation conditions on phosphatidylserine externalization and motion parameters of purified fractions of highly motile human spermatozoa. J Androl. 2002;23(2):194-201.
  12. Chen MJ, Bongso A. Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception. Hum Reprod. 1999;14(3):759-64.
  13. 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.
  14. Mangoli E, Talebi AR, Anvari M, Pourentezari M. Effects of experimentally-induced diabetes on sperm parameters and chromatin quality in mice. Iran J Reprod Med. 2013;11(1):53-60.
  15. Gosálvez J, Rodríguez‐Predreira M, Mosquera A, López‐Fernández C, Esteves SC, Agarwal A, et al. Characterisation of a subpopulation of sperm with massive nuclear damage, as recognised with the sperm chromatin dispersion test. Andrologia. 2014;46(6):602-9.
  16. Gallup Jr GG, Finn MM, Sammis B. On the origin of descended scrotal testicles: the activation hypothesis. Evolutionary psychology. 2009;7(4):147470490900700402.
  17. Matsuura R, Takeuchi T, Yoshida A. Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa. Asian J Androl. 2010;12(5):753-9.
  18. Guthrie HD, Welch GR. Effects of reactive oxygen species on sperm function. Theriogenology. 2012;78(8):1700-8.
  19. Peer S, Eltes F, Berkovitz A, Yehuda R, Itsykson P, Bartoov B. Is fine morphology of the human sperm nuclei affected by in vitro incubation at 37° C? Fertil Steril. 2007;88(6):1589-94.
  20. Agarwal A, Sharma R, Ahmad G. Sperm chromatin assessment. In: Textbook of assisted reproductive techniques. 5th ed. US: CRC Press; 2017. p. 65-87.
  21. Irez T, Dayioglu N, Alagöz M, Karatas S, Güralp O. The use of aniline blue chromatin condensation test on prediction of pregnancy in mild male factor and unexplained male infertility. Andrologia. 2018;50(10):e13111.
  22. Dadoune JP, Mayaux MJ, Guihard‐Moscato ML. Correlation between defects in chromatin condensation of human spermatozoa stained by aniline blue and semen characteristics: Beziehungen zwischen Defekten der chromatinkondensation menschlicher spermatozoen, die mittels anilinblau gefärbt wurden und spermacharakteristika. Andrologia. 1988;20(3):211-7.
  23. Hassanen E, Elqusi K, Zaki H, Henkel R, Agarwal A. TUNEL assay: Establishing a sperm DNA fragmentation cut‐off value for Egyptian infertile men. Andrologia. 2019;51(10):e13375.
  24. Liffner S, Pehrson I, García‐Calvo L, Nedstrand E, Zalavary S, Hammar M, et al. Diagnostics of DNA fragmentation in human spermatozoa: Are sperm chromatin structure analysis and sperm chromatin dispersion tests (SCD‐HaloSpermG2®) comparable? Andrologia. 2019;51(8):e13316.
  25. Kumar D, Kalthur G, Mascarenhas C, Kumar P, Adiga SK. Ejaculate fractions of asthenozoospermic and teratozoospermic patients have differences in the sperm DNA integrity. Andrologia. 2011;43(6):416-21.
  26. Delbès G, Herrero MB, Troeung ET, Chan PT. The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin‐V magnetic activated cell sorting. Andrology. 2013;1(5):698-706.
  27. Pujol A, García D, Obradors A, Rodríguez A, Vassena R. Is there a relation between the time to ICSI and the reproductive outcomes? Hum Reprod. 2018;33(5):797-806.
Journal of Reproduction & Infertility
Volume 21, Issue 4
Autumn 2020
Pages 275-282