Cubillos GS, Caldiño SF, Pedraza CJ, Cuneo PS

Language: Spanish
References: 21
Page: 63-67
PDF size: 190.19 Kb.

ABSTRACT

Background: Nowadays, little is known about the effect of vitrification method on spindle and its recovery time in terms of functionality.
Objective: To evaluate the process of meiotic spindle reassembly and chromosomal alignment of metaphase II in vitrified human oocyte. We monitored the time course and chromosome-MT (microtube) characteristics of human meiotic spindles from zero to three hours after thawing using confocal microscopy.
Material and method: Oocytes were vitrified with the Cryotop method, 36 oocytes were vitrified with 7.5% ethylene glycol, 7.5% propanediol, and 0.5 mol/L sucrose. After thawing, six oocytes were fixed to zero hours, 12 at one hour, 12 at two hours and six at three hours. As control four extra unfrozen oocytes were compared with frozen oocyte fixed al 0, 1, 2 and 3 hours after thawing. For immunostaining of microtubules, oocytes were first incubated with mouse monoclonal anti tubulin followed by Alexa 488-labeled goat antimouse IgG. Oocytes were analyzed by confocal imaging system. Mann-Whitney U-tests were carried out. Significance was defined as p ‹ 0.05.
Results: We analyzed 36 oocytes. Unfrozen control oocytes (100%) displayed bipolar spindles. After thawing there was a significant reduction of oocyte with bipolar spindle: (T0) 66.7%, after 1 h of culture (T1): 91.6%, after 1.5 h (T2): 75%, and after 2 h (T3): 67%. One hour following thawing, most oocytes recovered spindle bipolarity and equatorial chromosomal alignment. After warming of vitrified metaphase II oocytes, tubulin was depolymerized and chromatin remained condensed on the metaphase plate. Within a 1-hour period, tubulin repolymerized, forming morphologically normal metaphase spindles with properly aligned chromatin.
Conclusions: We found that the mean time of recovery of the spindle of thawed human oocytes after vitrification was one hour. For that reason we suggest to perform ICSI procedures after one hour of thawing. More studies should be done to confirm these results in this specific protocol.

REFERENCES

1. Coticchio G, Sciajno R, Hutt K, Bromfield J, et al. Comparative analysis of the metaphase II spindle of human oocytes through polarized light and high-performance confocal microscopy. Fertil Steril 2009;93(6):2056-2064.

2. Cao YX, Xing Q, Li L, Cong L, et al. Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification. Fertil Steril 2009;92(4):1306-1311.

3. Boiso I, Martí M, Santaló J, Ponsá M, et al. A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod 2002;17(7):1885-1891.

4. Bromfield JJ, Coticchio G, Hutt K, Sciajno R, et al. Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation. Hum Reprod 2009;24(9):2114-2123.

5. Chang C, Sung LY, Lin CJ, Kort HI, et al. The oocyte spindle is preserved by 1,2-propanediol during slow freezing. Fertil Steril 2010;93(5):1430-1439.

6. Chang CC, Tian CX, Yang X, Straub RJ, et al. The effect of cryoprotectants on preserving oocyte spindle following sub-physiological temperature treatment. 2007;88(Suppl. 1):S94.

7. Chen C. Pregnancy after human oocytes cryopreservation. Lancet 1986;11:884-886.

8. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta-analysis. Fertil Steril 2006;86:78-80.

9. Al-Hasani S, Diedrich K, van Der Ven H, Reinecke A, et al. Cryopreservation of human oocyte. Hum Reprod 1987;2:695-700.

10. Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human eggs cryopreservation. Hum Reprod 1998;13:3156-3159.

11. Fabbri R, Porcu E, Marsella T, Rocchetta G, et al. Human oocyte cryopreservation: new perspective regarding oocyte survival. Hum Reprod 2001;16:411-416.

12. Porcu E, Fabbri R, Damiano G, Giunchi S, et al. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 2000;169:33-37.

13. Chen SU, Lien YR, Chen HF, Chang LJ, et al. Observational clinical follow-up of oocyte cryopreservation using a slowfreezing method with 1,2 propanediol plus sucrose followed by ICSI. Hum Reprod 2005;20(7):1975-1980.

14. Bianchi V, Coticchio G, Fava L, Flamigni C, Borini A. Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. Hum Reprod 2005;20(4):1078-1083.

15. Pickering SJ, Johnson MH. The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 2007;2:207-216.

16. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990;54:102-108.

17. Zenses MT, Bielecki R, Casper RF, Leibo SP. Effect of chilling to 0 degrees C on the morphology of meiotic spindle in human metaphase II oocytes. Fertil Steril 2001;75:769-777.

18. Boiso I, Marti M, Santalo J, Ponsa M, et al. A confocal microscopy analysis of the spindle and chromosome configuration of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod 2002;17:1885-1891.

19. Cobo A, Perez S, De los Santos MJ, Zulategui J, et al. Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes. Reprod BioMed Online 2008;1(3):350-359.

20. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: The CryoTop method. Theriogenology 2007;67:73-80.

21. Duque C, Alfonso J, Cervera R, Monzó A, y col. Efecto del proceso de vitrificación sobre la estructura del huso meiótico y organización cromosómica en ovocitos humanos en metafase. Rev Asoc Est Biol Rep 2010;15(1):15.