Shandana Jadoon, Muhammad Adeel


Background: Various approaches have been utilized in attempting to cryopreserve oocytes, beginning with slow cooling and more recently the advent of technique of vitrification. Now it seems that oocyte cryopreservation is no longer an experimental technique and it is being increasingly utilized in clinics around the world. As successful outcome in oocyte cryopreservation can be assessed by survival through the freeze-thaw process, potential for fertilization, embryo development and dynamics of meiotic spindles. This study aimed to analyse these features in context of vitrification and slow freezing. Methods: In this laboratory based study, mature MII mouse oocytes from F1(C57BL6/J X CBA) mice (n=43) were divided randomly into two groups of equal numbers and were cryopreserved by slow freezing and by vitrification. Upon re-warming these oocytes were assessed for survival and for fertilization potential. Oocytes were fixed and stained to compare the effect of both protocols on spindle reassembly and chromosome configuration 10min, 1h and 3h after warming. Unfrozen oocytes were used as controls. Results: A greater number of vitrified oocytes survived cryopreservation than slow frozen oocytes (70.3% vs. 12.5%, p=0.024). After insemination, fertilization rates were higher for vitrified oocytes as compared to slow frozen oocytes (15.86% vs. 4.6%, p=0.046). Morphology of the meiotic spindle was found to be in a disorganized configuration in slow frozen oocytes at all-time points 10 mins, 1 h and 3h), whereas in vitrified oocytes the spindles were found to be aligned at all-time points. Chromosomes were seen to be displaced from equatorial region in both groups. Conclusion: Cryopreservation of mouse oocytes was conducted with greater success using vitrification, compared to slow freezing, with survival, fertilization, and spindle assembly more favourable to a successful outcome in this model.

Keywords: Oocyte cryopreservation, Cryoprotectant, Vitrification, polarization microscopy (POLSCOPE), immuno-staining, fluorescent conjugated antibodies, slow freezing

Full Text:



Sanders JE, Buckner CD, Amos D, Levy W, Appelbaum FR, Doney K, et al. Ovarian function following marrow transplantation for aplastic anemia or leukemia. J Clin Oncol 1988;6(5):813–8.

Wallance W, Shalet SM, Hendry JH, Morris-Jones PH, Gattamaneni H. Ovarian failure following abdominal irradiation in childhood: the radiosensitivity of the human oocyte. Br J Radiol 1989;62(743):995–8.

Chian RC, Wang Y, Li YR. Oocyte vitrification: advances, progress and future goals. J Assist Reprod Genet 2014;31(4):411–20.

Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949;164(4172):666.

Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update 2012;18(5):536–54.

Lane M, Maybach JM, Gardner DK. Addition of ascorbate during cryopreservation stimulates subsequent embryo development. Hum Reprod 2002;17(10):2686–93.

Ruffing N, Steponkus PL, Pitt RE, Parks JE. Osmometric behavior, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stages. Cryobiology 1993;30(6):562–80.

Schalkoff ME, Oskowitz SP, Powers RD. Ultrastructural observations of human and mouse oocytes treated with cryopreservatives. Biol Reprod 1989;40(2):379–93.

Carroll J, Depypere H, Matthews CD. Freeze–thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen–thawed mouse oocytes. J Reprod Fertil 1990;90(2):547–53.

Mavrides A, Morroll D. Bypassing the effect of zona pellucida changes on embryo formation following cryopreservation of bovine oocytes. Eur J Obstet Gynecol Reprod Biol 2005;118(1):66–70.

Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993;8(7):1101–9.

Gook DA, Edgar DH. Human oocyte cryopreservation. Hum Reprod Update 2007;13(6):591–605.

Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002;78(3):449–54.

Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe M, Larman MG. Analysis of oocyte physiology to improve cryopreservation procedures. Theriogenology 2007;67(1):64–72.

Arav, A. Vitrification of oocytes and embryos. In: Lauria, A., Gandolfi, F. (Eds.), New Trends in Embryo Transfer. Portland Press, Cambridge, 1992;pp.255–64

Valojerdi MR, Salehnia M. Developmental potential and ultrastructural injuries of metaphase II (MII) mouse oocytes after slow freezing or vitrification. J Assist Reprod Genet 2005;22(3):119–27.

Todorow SJ, Siebzehnriibl ER, Spitzer M, Koch R, Wildt L, Lang N. Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. II. Human. Hum Reprod 1989;4(7):812–6.

Pickering SJ, Johnson MH. The influence of cooling on the oranization of meiotic spindle of the mouse oocyte. Hum Reprod 1987;2(3):207–16.

Chen SU, Yang YS. Slow freezing or vitrification of oocytes: their effects on survival and meiotic spindles, and the time schedule for clinical practice. Taiwan J Obstet Gynecol 2009;48(1):15–22.

Ciotti PM, Porcu E, Notarangelo L, Magrini O, Bazzocchi A, Venturoli S. Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing. Fertil Steril 2009;91(6):2399–407.

Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril 2011;96(2):277–85.

Larman MG, Katz-Jaffe MG, Sheehan CB, Gardner DK. 1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology. Hum Reprod 2007;22(1):250–9.

Renard JP, Babinet C. High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1–2 propanediol as cryoprotectant. J Exp Zool 1984;230(3):443–8.

Cha SK, Kim BY, Kim MK, Kim YS, Lee WS, Yoon TK, et al. Effects of various combinations of cryoprotectants and cooling speed on the survival and further development of mouse oocytes after vitrification. Clin Exp Reprod Med 2011;38(1):24–30.

Stachecki JJ, Cohen J, Willadsen S. Detrimental effects of sodium during mouse oocyte cryopreservation. Biol Reprod 1998;59(2):395–400.

Gook DA, CSchiewe MC, Osborn SM, Asch RH, Jansen RP, Johnston WI. Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum Reprod 1995;10(10):2637–41.

Joly C, Bchini O, Boulekbache H, Testart J, Bernard M. Effects of 1,2-propanediol on the cytoskeletal organization of the mouse oocyte. Hum Reprod 1992;7(3):374–8.

Eroglu A, Toth TL, Toner M. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil Steril 1998;69(5):944–57.

Aigner S, Van der Elst J, Siebzehnriibl E, Wildt L, Lang N, Van Steirteghem A. The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 1992;7(6):857–64.


  • There are currently no refbacks.

Contact Number: +92-992-382571

email: [jamc] [@] []