Human Reproduction Archives
https://humanreproductionarchives.com/article/doi/10.4322/hra.001118
Human Reproduction Archives
Original Article Assisted Reproduction

Vitrification of blastocysts at various degrees of blastocoele expansion using different exposure times to the equilibration solution

Tamara Lamin, Rodrigo Sant’Ana, Alfred Paul Senn, Rafael Alonso Salvador, David Til, Larissa Benvenutti, Vera Lucia Langaro Amaral

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Abstract

Objectives: To evaluate the efficiency of a vitrification protocol of murine blastocysts, varying the exposure time to the equilibrium solution, at different degrees of blastocoel expansion. Methods: Sixty female mice were superovulated with 10 IU of equine chorionic gonadotropin (eCG, Novormon, Syntex, Argentina) and after 48 hours with 10 IU of human chorionic gonadotropin (hCG, Vetecor, Calier, Barcelona, Spain). Females were then coupled with males overnight, and the presence of vaginal plug indicated that mating had occurred. Three days later, females were euthanized and the embryos at the morula stage (n = 925) were recovered by flushing the uterine tubes with GV-Hepes medium (Ingámed, Maringá, Brazil). Embryos were then cultivated until the blastocyst stage and classified, at the time the vitrification process was initiated, into 3 groups (G1, G2, G4) according to the degree of blastocoele expansion. A control group remained in culture until hatching. All embryos, after warming, were put back into culture for measurement of the rehydration (RR) and hatching (HR) rates. Results: For G1 group, the mean RR and HR obtained over a range of exposure times to the equilibration solution of 94.0 and 84.4%, respectively. For G2, these rates were 95.2 and 87.1%, respectively. For G1 and G2, RR and HR obtained after an equilibration time of 9 min were statistically higher than those obtained after 10, 11, 12, 13 and 14 min. For G4, RR and HR were 76.4 and 69.9%, respectively, but in this case, an equilibration time of 15 min presented statistically higher survival rates compared to shorter exposition times. Conclusions: The results of this study indicate that the variation in the exposure time of the embryos to the equilibration solution influences significantly the rehydration and hatching of the blastocysts, and the degree of blastocoel expansion is inversely correlated with the survival and development potential after warming.

Keywords

vitrification; blastocysts; blastocoel; cryoprotectant.

References

1. Almodin CG, Costa RR. Criopreservação em reprodução. Maringá: DentalPress; 2014. 292 p.

2. Wong KM, Mastenbroek S, Repping S. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates. Fertil Steril. 2014;102(1):19-26. http://dx.doi.org/10.1016/j.fertnstert.2014.05.027. PMid:24890275.

3. Papadopoulos S, Rizos D, Duffy P, Wade M, Quinn K, Boland MP, et al. Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified, in vivo or in vitro produced ovine blastocysts. Anim Reprod Sci. 2002;74(1-2):35-44. http://dx.doi.org/10.1016/S0378-4320(02)00162-8. PMid:12379373.

4. Kader AA, Choi A, Orief Y, Agarwal A. Factors affecting the outcome of human blastocyst vitrification. Reprod Biol Endocrinol. 2009;7(1):99. http://dx.doi.org/10.1186/1477-7827-7-99. PMid:19758458.

5. Carvalho AA, Faustino LR, Figueiredo JR, Rodrigues APR, Costa APR. Vitrificação: uma alternativa para a preservação de embriões e material genético de fêmeas mamíferas em criobancos. Acta Vet Bras. 2012;5(3):236-48.

6. Pereira RM, Marques CC. Animal oocyte and embryo cryopreservation. Cell Tissue Bank. 2008;9(4):267-77. http://dx.doi.org/9.1/s10561-008-9075-2. PMid:18496769.

7. Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online. 2004;9(2):164-70. http://dx.doi.org/10.1016/S1472-6483(10)62125-6. PMid:15333245.

8. Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ. Potential importance of vitrification in reproductive medicine. Biol Reprod. 2002;67(6):1671-80. http://dx.doi.org/10.1095/biolreprod.102.006833. PMid:12444040.

9. Costa EP, Guimarães JD, Torres CAA, Fagundes LM, Gioso MM. Criopreservação de ovócitos de bovinos imaturos desnudados ou não, utilizando o etilenoglicol pelo método da vitrificação. Rev Bras Zootec. 2002;31(3):1122-9. http://dx.doi.org/10.1590/S1516-35982002000500008.

10. Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online. 2006;12(6):779-96. http://dx.doi.org/10.1016/S1472-6483(10)61091-7. PMid:16792858.

11. Sadler TW, Langman J. Langman’s medical embryology. 12th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2012. 384 p.

12. Levi-Setti PE, Menduni F, Smeraldi A, Patrizio P, Morenghi E, Albani E. Artificial shrinkage of blastocysts prior to vitrification improves pregnancy outcome: analysis of 1028 consecutive warming cycles. J Assist Reprod Genet. 2016;33(4):461-6. http://dx.doi.org/10.1007/s10815-016-0655-y. PMid:26781264.

13. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, van Roosendaal E, Vandervorst M, et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod. 2002;17(3):744-51. http://dx.doi.org/10.1093/humrep/17.3.744. PMid:11870130.

14. Neto JAL, Almodin CG, da Rosa VB, Amaral VLL, Frajblat M, Câmara VCM. Embryo viability after revitrification with Vitri-ingá method. JBRA Assist Reprod. 2011;15:15-8.

15. Gardner DK, Schoolcraft WB. In vitro culture of human blastocysts. Reprod Certain Fertil Genet. 1999;1999:378-88.

16. Damy SB, Camargo RS, Chammas R, Figueiredo LFP. Fundamental aspects on animal research as applied to experimental surgery. Rev Assoc Med Bras. 2010;56(1):103-11. http://dx.doi.org/10.1590/S0104-42302010000100024. PMid:20339795.

17. Nagy A, editor. Manipulating the mouse embryo: a laboratory manual. 3rd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2003. 764 p.

18. Nelder JA, Wedderburn RWM. Generalized linear models. J R Stat Soc Ser Gen. 1972;135(3):370. http://dx.doi.org/10.2307/2344614.

19. McCullagh P, Nelder JA. Generalized linear models. 2nd ed. London: Chapman and Hall; 1989. 513 p. (Monographs on Statistics and Applied Probability). http://dx.doi.org/10.1007/978-1-4899-3242-6.

20. Shu Y, Watt J, Gebhardt J, Dasig J, Appling J, Behr B. The value of fast blastocoele re-expansion in the selection of a viable thawed blastocyst for transfer. Fertil Steril. 2009;91(2):401-6. http://dx.doi.org/10.1016/j.fertnstert.2007.11.083. PMid:18304536.

21. Hashimoto S, Amo A, Hama S, Ito K, Nakaoka Y, Morimoto Y. Growth retardation in human blastocysts increases the incidence of abnormal spindles and decreases implantation potential after vitrification. Hum Reprod. 2013;28(6):1528-35. http://dx.doi.org/10.1093/humrep/det059. PMid:23482339.

22. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod. 2006;21(12):3246-52. http://dx.doi.org/10.1093/humrep/del285. PMid:16936299.

23. Dalcin L, Silva RC, Paulini F, Silva BDM, Neves JP, Lucci CM. Cytoskeleton structure, pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos. Cryobiology. 2013;67(2):137-45. http://dx.doi.org/10.1016/j.cryobiol.2013.05.012. PMid:23770514.

24. Yamanaka M, Hashimoto S, Amo A, Ito-Sasaki T, Abe H, Morimoto Y. Developmental assessment of human vitrified-warmed blastocysts based on oxygen consumption. Hum Reprod. 2011;26(12):3366-71. http://dx.doi.org/10.1093/humrep/der324. PMid:21972254.

25. Dobrinsky JR. Cellular approach to cryopreservation of embryos. Theriogenology. 1996;45(1):17-26. http://dx.doi.org/10.1016/0093-691X(95)00351-8.

26. Hendriks WK, Roelen BA, Colenbrander B, Stout TA. Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow-freezing or vitrification. Equine Vet J. 2015;47(6):701-7. http://dx.doi.org/10.1111/evj.12341. PMid:25187202.

27. Coello A, Meseguer M, Galán A, Alegre L, Remohí J, Cobo A. Analysis of the morphological dynamics of blastocysts after vitrification/ warming: defining new predictive variables of implantation. Fertil Steril. 2017;108(4):659-66.e4. http://dx.doi.org/10.1016/j.fertnstert.2017.07.1157. PMid:28863936.

28. Lin R, Feng G, Shu J, Zhang B, Zhou H, Gan X, et al. Blastocoele re‐expansion time in vitrified–warmed cycles is a strong predictor of clinical pregnancy outcome. J Obstet Gynaecol Res. 2017;43(4):689-95. http://dx.doi.org/10.1111/jog.13257. PMid:28127833.

29. Yin H, Jiang H, He R, Wang C, Zhu J, Li Y. The effects of blastocyst morphological score and blastocoele re-expansion speed after warming on pregnancy outcomes. Clin Exp Reprod Med. 2016;43(1):31-7. http://dx.doi.org/10.5653/cerm.2016.43.1.31. PMid:27104155.

30. Cobo A, de los Santos MJ, Castellò D, Gámiz P, Campos P, Remohí J. Outcomes of vitrified early cleavage-stage and blastocyststage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertil Steril. 2012;98(5):1138-46.e1. http://dx.doi.org/10.1016/j.fertnstert.2012.07.1107. PMid:22862909.

31. Ahlström A, Westin C, Wikland M, Hardarson T. Prediction of live birth in frozen–thawed single blastocyst transfer cycles by prefreeze and post-thaw morphology. Hum Reprod. 2013;28(5):1199-209. http://dx.doi.org/10.1093/humrep/det054. PMid:23477908.

32. Du Q-Y, Wang E-Y, Huang Y, Guo X-Y, Xiong Y-J, Yu Y-P, et al. Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitrified/warmed single blastocyst transfer cycles. Fertil Steril. 2016;105(4):910-9.e1. http://dx.doi.org/10.1016/j.fertnstert.2015.12.014. PMid:26776910.

33. Goto S, Kadowaki T, Tanaka S, Hashimoto H, Kokeguchi S, Shiotani M. Prediction of pregnancy rate by blastocyst morphological score and age, based on 1,488 single frozen-thawed blastocyst transfer cycles. Fertil Steril. 2011;95(3):948-52. http://dx.doi.org/10.1016/j.fertnstert.2010.06.067. PMid:20674914.

34. Hur YS, Park JH, Ryu EK, Yoon HJ, Yoon SH, Hur CY, et al. Effect of artificial shrinkage on clinical outcome in fresh blastocyst transfer cycles. Clin Exp Reprod Med. 2011;38(2):87-92. http://dx.doi.org/10.5653/cerm.2011.38.2.87. PMid:22384424.

35. Song W, Wang X, Jin H, Yao G, Zhang X, Shi S, et al. Comparison of vitrified outcomes between human early blastocysts and expanded blastocysts. Vitro Cell Dev Biol - Anim. 2016;52(5):522-9. http://dx.doi.org/10.1007/s11626-016-0009-1.

36. Bertolini M, Costa Lange M, Rodrigues JL. In vitro and in vivo survival of mouse morulas and blastocysts following vitrification in 45% glycerol. Acta Sci Vet. 2005;33(3):245-51. https://doi.org/10.22456/1679-9216.14944.

37. Bogliolo L, Ledda S, Innocenzi P, Ariu F, Bebbere D, Rosati I, et al. Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida. Cryobiology. 2012;64(3):267-72. http://dx.doi.org/10.1016/j.cryobiol.2012.02.010. PMid:22387147.

38. Elnahas T, Tawab N, Azmy O, Elnoury A, El-Faissal Y, Fouad T, et al. Prospective randomized trial on the use of laser assisted hatching for transfer of frozen/thawed embryos in human Intracytoplasmic Sperm injection. Middle East Fertil Soc J. 22(4), 309-12.

39. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril. 2006;86(1):20-6. http://dx.doi.org/10.1016/j.fertnstert.2006.01.029. PMid:16762345.

40. Raju GAR, Prakash GJ, Krishna KM, Madan K. Vitrification of human early cavitating and deflated expanded blastocysts: clinical outcome of 474 cycles. J Assist Reprod Genet. 2009;26(9-10):523-9. http://dx.doi.org/10.1007/s10815-009-9356-0. PMid:19876729.

41. Kader A, Choi A, Sharma RK, Falcone T, Agarwal A. Effect of varying equilibration time in a two-step vitrification method on the postwarming DNA integrity of mouse blastocysts. Fertil Steril. 2010;93(8):2640-5. http://dx.doi.org/10.1016/j.fertnstert.2009.07.991. PMid:19732878.

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