Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

Vitrification Increased Vacuolization of Human Spermatozoa: Application of MSOME Technology

Authors
Sara Taherzadeh 1
Mohammad Ali Khalili 1
Azam Agha-Rahimi 1
Fatemeh Anbari 1
Shahin Ghazali 1
Guido Macchiarelli 2
1 Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2 Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy
Abstract
Background: Sperm vitrification is a technique of ice and cryoprotectant free cryopreservation by direct plunging of sperm suspension into liquid nitrogen (LN2). The aim of this study was to investigate the influence of cryoprotectant free-vitrification on human sperm fine structure by MSOME technology and the fertility potential by zona binding assay (ZBA). Methods: 20 normo-ejaculates were prepared by swim up technique, and supernatants were divided into two parts of fresh and vitrified groups. For vitrification, sperm was dropped into LN2. Sperm motility, morphology, viability and MSOME were evaluated for each sample. In MSOM morphologically normal sperm (class 1), ≤2 small vacuoles (class 2), and one large vacuole or >2 small vacuoles (class 3) were evaluated. Also, fertility potential was evaluated by zona binding assay. Data was analyzed using paired t-test or Willcoxon’s test and p-value <0.05 was considered significant. Results: Vitrification significantly reduced both progressive motility, viability and morphology. Also, normal morphology of spermatozoa decreased significantly after vitrification. In MSOME evaluation, normal motile spermatozoa (Class 1) decreased from 23.00±12.44 to 16.00.56±10.79 after vitrification (p=0.008). Although spermatozoa classes 2 and 3 were increased, the difference was not significant. Moreover, fertility potential of motile spermatozoa was reduced after vitrification (9.0±13.87 vs. 13.40±22.73; p=0.07). Conclusion: Vitrification increased the rate of vacuolization in motile sperm head. Therefore, MSOME technology is recommended for assessment of sperm fine morphology in ICSI program used cryopreserved spermatozoa.
Keywords
MSOME
Sperm
Vitrification
Zona binding assay
  1. Mossad H, Morshedi M, Toner JP, Oehninger S. Impact of cryopreservation on spermatozoa from infertile men: impli-cations for artificial insemination. Arch Androl. 1994;33(1):51-7.
  2. O'Connell M, McClure N, Lewis SE. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Hum Reprod. 2002;17(3):704-9.
  3. Punyatanasakchai P, Sophonsritsuk A, Weerakiet S, Wansumrit S, Chompurat D. Comparison of cryopreserved hu-man sperm in vapor and liquid phases of liquid nitrogen: effect on motility parameters, morphology, and sperm function. Fertil Steril. 2008;90(5):1978-82.
  4. Lee CY, Lee CT, Wu CH, Hsu CS, Hsu MI. Kruger strict morphology and post-thaw progressive motility in cryo-preserved human spermatozoa. Andrologia. 2012;44 Suppl 1:81-6.
  5. Isachenko E, Isachenko V, Katkov II, Dessole S, Nawroth F. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success. Reprod Biomed Online. 2003;6(2):191-200.
  6. Agha-Rahimi A, Khalili MA, Nabi A, Ashourzadeh S. Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm par-ameters, DNA fragmentation and hyaluronan binding. Reprod Biomed Online. 2014;28(3):352-8.
  7. Oliveira JB, Petersen CG, Massaro FC, Baruffi RL, Mauri AL, Silva LF, et al. Motile sperm organelle morphology ex-amination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles. Reprod Biol Endocrinol. 2010;8:56.
  8. Luna D, Hilario R, Dueñas-Chacón J, Romero R, Zavala P, Villegas L, et al. The IMSI procedure improves laboratory and clinical outcomes without compromising the aneuploidy rate when compared to the classical ICSI procedure. Clin Med Insights Reprod Health. 2015;9:29-37.
  9. Shalom-Paz E, Anabusi S, Michaeli M, Karchovsky-Shoshan E, Rothfarb N, Shavit T, et al. Can intra cytoplasmatic morphologically selected sperm injection (IMSI) technique improve outcome in patients with repeated IVF-ICSI fail-ure? a comparative study. Gynecol Endocrinol. 2015;31(3):247-51.
  10. Orief YI, Elabd MM, Said THM, Ahmed NAAJ. Comparative study between intracytoplasmic morphologically selected sperm injection versus intra-cytoplasmic sperm injection in patients with severe male factor infertility and repeated intra cytoplasmic sperm injection failure. Int J Reprod Contra-cept Obstet Gynecol. 2016;5(4):1102-7.
  11. Perdrix A, Rives N. Motile sperm organelle morphology examination (MSOME) and sperm head vacuoles: state of the art in 2013. Hum Reprod Update. 2013;19(5):527-41.
  12. Garolla A, Fortini D, Menegazzo M, De Toni L, Nicoletti V, Moretti A, et al. High-power microscopy for selecting spermatozoa for ICSI by physiological status. Reprod Biomed Online. 2008;17(5):610-6.
  13. Berkovitz A, Eltes F, Lederman H, Peer S, Ellen-bogen A, Feldberg B, et al. How to improve IVF-ICSI outcome by sperm selection. Reprod Biomed Online. 2006;12(5):634-8.
  14. Antinori M, Licata E, Dani G, Cerusico F, Versaci C, d'Angelo D, et al. Intracytoplasmic morphologically selected sperm injection: a prospective randomized trial. Reprod Biomed Online. 2008;16(6):835-41.
  15. Ghazali S, Talebi AR, Khalili MA, Aflatoonian A, Esfandiari N. Large nuclear vacuoles in spermatozoa negatively af-fect pregnancy rate in IVF cycles. Iran J Reprod Med. 2015;13(7):425-32.
  16. Boitrelle F, Albert M, Theillac C, Ferfouri F, Ber-gere M, Vialard F, et al. Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl. 2012;33(6):1371-8.
  17. World Health Organization, Department of Reproductive Health and Research. WHO laboratory manual for the examination and processing of human semen. Geneva: WHO Press; 2010. 287 p.
  18. Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73.
  19. Sermondade N, Dupont C, Faure C, Boubaya M, Cédrin-Durnerin I, Chavatte-Palmer P, et al. Body mass index is not associated with sperm-zona pel-lucida binding ability in subfertile males. Asian J Androl. 2013;15(5):626-9.
  20. Cassuto NG, Bouret D, Plouchart JM, Jellad S,Vanderzwalmen P, Balet R, et al. A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality. Fertil Steril. 2009;92(5):1616-25.
  21. Khalili MA, Adib M, Halvaei I, Nabi A. Vitrification of neat semen alters sperm parameters and DNA integrity. Urol J. 2014;11(2):1465-70.
  22. Cassuto NG, Hazout A, Hammoud I, Balet R, Bou-ret D, Barak Y, et al. Correlation between DNA defect and sperm-head morphology. Reprod Biomed Online. 2012;24(2):211-8.
  23. Fortunato A, Boni R, Leo R, Nacchia G, Liguori F, Casale S, et al. Vacuoles in sperm head are not associated with head morphology, DNA damage and reproductive success. Reprod Biomed Online. 2016;32(2):154-61.
  24. Gatimel N, Leandri R, Parinaud J. Sperm vacuoles are not modified by freezing--thawing procedures. Reprod Biomed Online. 2013;26(3):240-6.
  25. Agha-Rahimi A, Khalili MA, Nottola SA, Migliet-ta S, Moradi A. Cryoprotectant-free vitrification of human spermato-zoa in new artificial seminal fluid. Andrology. Andrology. 2016;4(6):1037-44.
  26. Green CE, Watson PF. Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. Reproduction. 2001;122(6):889-98.
  27. Vadnais ML, Roberts KP. Seminal plasma proteins inhibit in vitro- and cooling-induced capacitation in boar spermato-zoa. Reprod Fertil Dev. 2010;22(6):893-900.
  28. Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahu-seyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod Genet. 2008;25(8):403-11.
Journal of Reproduction & Infertility
Volume 18, Issue 2
Spring 2017
Pages 225-230