Effects of blastocyst artificial collapse prior to vitrification on hatching and ‎survival rates and the expression of klf4 gene in mouse embryos

Document Type : Short Communication


1 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, ‎Tehran, Iran

2 Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran


Although the rate of blastocysts implantation of embryos is higher than previous stages but their survival rate is lower than them, which could be attributed to the completely filled blastocoel cavity with liquid and increased possibility of the formation of ice crystals. This liquid could prevent the penetration of cryoprotecting materials into the embryos. In this study, we reduced the volume of blastocoel before vitrification and compared survival rate and quality of in vitro embryos through klf4 gene expression with control group. In vitro mouse blastocysts were divided into three groups. In group 1, the blastocoel volume of blastocysts were reduced before vitrification and warming. In group 2, blastocysts were just vitrified and warmed and the blastocysts of group 3 (control group) were not undergone any specific treatment and were not vitrified. The expression ofklf4 gene was assessed using real-time PCR technique. Data were statistically analyzed using one-way ANOVA and Duncan’s post hoc tests. Our results showed that blastocoel volume reduction before vitrification significantly increased the hatching rate of the blastocysts from the zona pellucida and klf4 gene expression compared to vitrified group. Blastocoel volume reduction before vitrification could be used as an efficient method for improving the rate of in vitro fertilization.


Main Subjects


    1. Balaban B, Urman B, Sertac A, et al. Blastocyst quality affects the success of blastocyst-stage embryo transfer. Fertil Steril 2010;74: 282-287.
    2. Guerif F, Bidault R, Gasnier O, et al. Efficacy of blastocyst transfer after implantation failure. Reprod Biomed 2004;9(6):630-636.
    3. Gardner DK1, Vella P, Lane M, Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69(1):84-88.
    4. Kolibianakis EM, Zikopoulos K, Verpoest W, et al. Should we advise patients undergoing IVF to start a cycle leading to a day 3 or a day 5 transfers? Hum Reprod 2004; 19: 2550-2554.
    5. Nijs M, Geerts L, van Roosendaal E, et al. Prevention of multiple pregnancies in an in vitro fertilization program. Fertil Steril 1993; 59:245-260.
    6. Rall WF, Wood MJ. High in vitro and in vivo survival of day 3 mouse embryos vitrified or frozen in a non-toxic solution of glycerol and albumin. J Reprod Fertil 1994; 101:681-688.
    7. Urman B, Sertac A, Alatas C, et al. Progression of excess embryos to the blastocyst stage predicts pregnancy and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1998; 13(9):2564-2567.
    8. Dobrinsky JR, Pursel VG, Long CR, et al. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 2000; 62(3):564-570.
    9. Ema M, Mori D,  Niwa H, et al. Krüppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs. Cell Stem Cell 2008; 3 (5): 555-567.
    10. Tsai SY, Clavel C, Kim S, et al. Oct4 and klf4 repro­gram dermal papilla cells into induced pluripotent stem cells. Stem Cells 2010; 28:221-228.
    11. Quinn P, Barros C, Whittingham DG. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J Reprod and Ferttil 1982; 66(1): 161-168.
    12. Quinn P, Warnes GM, Kerin J F et al. Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann N Y Acad Sci 1985;442:195-204.
    13. Lawitts JA, Biggers JD. Culture of preimplantation embryos. Methods Enzymol 1992; 225: 153-164.
    14. Vanderzwalmen P, Bertin G, Lejeune B, et al. Two essential steps for a successful intracytoplasmic sperm injection: Injection of immobilized spermatozoa after rupture of the oolema. Hum Reprod 1996; 11(3):
    15. Kelly LA, Gardner SP, Sutcliffe MJ. An automated approach for clustering an ensemble of NMR derived protein into conformational related subfamilies. Protein Eng 1996; 9(11): 1063-1065.
    16. Zech NH1, Lejeune B, Zech H, et al. Vitrification of hatching and hatched human blastocysts: Effect of an opening in the zona pellucida before vitrification. Reprod Biomed 2005;11(3):355-361.
    17. Villami PR, Lozano D, Oviedo JM, et al. Developmental rates of in vivo and in vitro produced bovine embryos cryopreserved in ethylene glycol based solution by slow freezing or solid surface vitrification. Anim Reprod 2012;9(2): 86-92.
    18. Giritharan G1, Talbi S, Donjacour A, et al. Effect of in vitro fertilization on gene expression and development of mouse preimplantation embryos. Reproduction 2007; 134(1):63-72.
    19. Wanderzwalmen P, Bertin G, Debauche CH, et al. Birth after vitrification at morula and blastocyst stage: Effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002; 17(3): 744-751.
    20. Quinn P, Warnes GM, Kerin JF, et al. Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann NY Acad Sci 1985; 442:
    21. Lee SY, Kim HJ, Park SJ. Optimization of a dilution method for human expanded blastocysts vitrified using EM grids after artificial shrinkage. J Assist Reprod Genet 2006; 23:87-91.
    22. Chatzimeletiou K, Morrison EE, Panagiotidis Y, et al. Cytoskeletal analysis of human blastocysts by confocal laser scanning microscopy following vitrification. Hum Reprod 2012; 27(1): 106-113.
Volume 9, Issue 1
March 2018
Pages 87-92
  • Receive Date: 03 January 2017
  • Revise Date: 14 May 2017
  • Accept Date: 22 May 2017
  • First Publish Date: 01 March 2018