Hostname: page-component-586b7cd67f-t7fkt Total loading time: 0 Render date: 2024-11-23T02:10:07.953Z Has data issue: false hasContentIssue false
  • English
  • Français

A modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes

Published online by Cambridge University Press:  01 August 2009

Zengyan Wang ,
Zhengyi Sun ,
Ying Chen  and
Fangfang He
Zengyan Wang
Affiliation:
Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
Zhengyi Sun
Affiliation:
Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
Ying Chen
Affiliation:
Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
Fangfang He*
Affiliation:
Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, 1 Shuaifuyuan Alley, Dongcheng District, Beijing, China. Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
*
All correspondence to Fangfang He, Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, 1 Shuaifuyuan Alley, Dongcheng District, Beijing, China. Tel: +610 65296223. Fax: +8610 65296223. e-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

In this study, we examined a modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes. The mature mouse oocytes were first vitrified and then warmed up in a modified cryoloop vitrification medium [15% ethylene glycol (EG) + 15% dimethyl sulphoxide (ME2SO) + 5.8 mg/ml Ficoll 400 (F) + 0.58 mol/l sucrose (S)]. These oocytes were later studied along with fresh oocytes, which served as the control group.

Based on the post-warm-up incubation time, the oocytes in the study group were divided into three subgroups: 0 h, 1 h and 2 h. We then examined the configurations of spindles and chromosomes, the fragmentation of DNA, and the oocyte's ability to be fertilized and developed into blastocysts. By evaluating the vitrified oocytes' morphology, we confirmed that 601 out of 612 (98.2%) oocytes survived this protocol. The percentage of oocytes with normal spindle and chromosome configurations in the study groups 0 h, 1 h and 2 h were all quite similar to each other and not statistically different from that of the control group. Similar results were also observed in the percentage of oocytes containing fragmented DNA. The fertilization rate and blastocyst formation rate of the thawed oocytes were not statistically different from that of the control group either. However, if not handled properly (too much remnant medium on oocytes in the process of freezing or too long a time of oocytes in the vitrification medium before freezing), the cryopreserved oocytes could show dramatic difference from the control group in terms of the morphologically survival rate, the configuration of the spindles and chromosomes, and the DNA fragmentation. In conclusion, when followed correctly, this modified cryoloop vitrification protocol had little effect on the survival rate and development potential of mature mouse oocytes.

Keywords

ChromosomeDNA fragmentationOocytesSpindleVitrification
Type
Research Article
Information
Zygote , Volume 17 , Issue 3 , August 2009 , pp. 217 - 224
Copyright
Copyright © Cambridge University Press 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Almeida, P.A. & Bolton, V.N. (1995). The effect of temperature fluctuations on the cytoskeletal organisation and chromosomal constitution of the human oocyte. Zygote 3, 357–65.CrossRefGoogle ScholarPubMed
Bianchi, V., Coticchio, G., Fava, L., Flamigni, C. & Borini, A. (2005). Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. Hum. Reprod. 20, 1078–83.CrossRefGoogle ScholarPubMed
Cai, X.Y., Chen, G.A., Lian, Y., Zheng, X.Y. & Peng, H.M. (2005). Cryoloop vitrification of rabbit oocytes. Hum. Reprod. 20, 1969–74.CrossRefGoogle ScholarPubMed
Carroll, J., Wood, M.J. & Whittingham, D.G. (1993). Normal fertilization and development of frozen–thawed mouse oocytes: protective action of certain macromolecules. Biol. Reprod. 48, 606–12.CrossRefGoogle ScholarPubMed
Chen, C.K., Wang, C.W., Tsai, W.J., Hsieh, L.L., Wang, H.S. & Soong, Y.K. (2004). Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy. Fertil. Steril. 82, 666–72.CrossRefGoogle ScholarPubMed
Coticchio, G. De., Santis, L., Rossi, G., Borini, A., Albertini, D., Scaravelli, G., Alecci, C., Bianchi, V., Nottola, S. & Cecconi, S. (2006). Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation. Hum. Reprod. 21, 1771–6.CrossRefGoogle ScholarPubMed
Fabbri, R., Porcu, E., Marsella, T., Rocchetta, G., Venturoli, S. & Flamigni, C. (2001). Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum. Reprod. 16, 411–6.CrossRefGoogle ScholarPubMed
Frydman, N., Selva, J., Bergere, M., Auroux, M. & Maro, B. (1997). Cryopreserved immature mouse oocytes: a chromosomal and spindle study. J. Assist. Reprod. Genet. 14, 617–23.CrossRefGoogle ScholarPubMed
Ghetler, Y., Skutelsky, E., Ben Nun, I., Ben Dor, L., Amihai, D. & Shalgi, R. (2006). Human oocyte cryopreservation and the fate of cortical granules. Fertil. Steril. 86, 210–16.CrossRefGoogle ScholarPubMed
Gook, D.A. & Edgar, D.H. (2007). Human oocyte cryopreservation. Hum. Reprod. Update 13, 591–605.CrossRefGoogle ScholarPubMed
Jain, J.K. & Paulson, R.J. (2006). Oocyte cryopreservation. Fertil. Steril. 86, 1037–46.CrossRefGoogle ScholarPubMed
Kazem, R., Thompson, L.A., Srikantharajah, A., Laing, M.A., Hamilton, M.P., & Templeton, A. (1995). Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection. Hum. Reprod. 10, 2650–4.CrossRefGoogle ScholarPubMed
Lane, M. & Gardner, D.K. (2001). Vitrification of mouse oocytes using a nylon loop. Mol. Reprod. Dev. 58, 342–7.3.0.CO;2-X>CrossRefGoogle ScholarPubMed
Larman, M.G., Sheehan, C.B. & Gardner, D.K. (2006). Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes. Reproduction 131, 53–61.CrossRefGoogle ScholarPubMed
Larman, M.G., Minasi, M.G., Rienzi, L. & Gardner, D.K. (2007). Maintenance of the meiotic spindle during vitrification in human and mouse oocytes. Reprod. Biomed. Online 15, 692–700.CrossRefGoogle ScholarPubMed
Men, H., Monson, R.L., Parrish, J.J., Rutledge, J.J. (2003). Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation. Mol. Reprod. Dev. 64, 245–50.CrossRefGoogle ScholarPubMed
Oktay, K., Cil, A.P. & Bang, H. (2006). Efficiency of oocyte cryopreservation: a meta-analysis. Fertil. Steril. 86, 70–80.CrossRefGoogle ScholarPubMed
Palermo, G., Joris, H., Devroey, P. & Van Steirteghem, A.C. (1992). Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 8810, 17–18.CrossRefGoogle Scholar
Porcu, E., Fabbri, R., Seracchioli, R., Ciotti, P.M., Magrini, O. & Flamigni, C. (1997). Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil. Steril. 68, 724–6.CrossRefGoogle ScholarPubMed
Porcu, E., Fabbri, R., Damiano, G., Giunchi, S., Fratto, R., Ciotti, P.M., Venturoli, S. & Flamigni, C. (2000). Clinical experience and applications of oocyte cryopreservation. Mol. Cell. Endocrinol. 169, 33–7.CrossRefGoogle Scholar
Quintans, C.J., Donaldson, M.J., Bertolino, M.V. & Pasqualini, R.S. (2002). Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum. Reprod. 17, 3149–52.CrossRefGoogle Scholar
Rienzi, L., Martinez, F., Ubaldi, F., minasi, M.G., Iacobelli, M., Tesarik, J. & Greco, E. (2004). PolScope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum. Reprod. 19, 655–9.CrossRefGoogle ScholarPubMed
Stachecki, J.J., Cohen, J. & Willadsen, S.M. (1998). Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 37, 346–54.CrossRefGoogle ScholarPubMed
Stachecki, J.J., Munné, S. & Cohen, J. (2004). Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reprod. Biomed. Online 8, 664–72.CrossRefGoogle ScholarPubMed
Suikkari, A.M. & Söderström-Anttila, V. (2007). In-vitro maturation of eggs: is it really useful? Best. Pract. Res. Clin. Obstet. Gynaecol. 21, 145–55.CrossRefGoogle ScholarPubMed
Sun, Z.Y., He, F.F., Yu, Q., Deng, C.Y. & Liu, M.Z. (2005). The outcomes of human blastocyst cryopreservation, vitrification using cryoloop versus slow-freezing method. J. Reprod. Med. 14, 29–32.Google Scholar