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Hum. Reprod. Advance Access originally published online on August 3, 2007
Human Reproduction 2007 22(10):2776-2783; doi:10.1093/humrep/dem240
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation

L. De Santis1, G. Coticchio2,6, S. Paynter3, D. Albertini4, K. Hutt4, I. Cino1, M. Iaccarino5, A. Gambardella2, C. Flamigni2 and A. Borini2

1 IVF Unit, Vita-Salute University, H. S. Raffaele, Milan, Italy 2 Tecnobios Procreazione, Via Dante 15, 40125 Bologna, Italy 3 Cardiff University, Cardiff, UK 4 University of Kansas Medical Center, Kansas City, KS, USA 5 Clinica Mediterranea, Napoli, Italy

6 Correspondence address. Tel: +39-051-2867511; Fax: +39-051-2867512; E-mail: coticchio{at}tecnobiosprocreazione.it

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and PEG (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant.

METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle.

RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 ± 0.29 µm min–1 atm–1 (mean ± SD) and PEG 0.10 ± 0.01 µm s–1 were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05).

CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.

Key words: cryopreservation/oocytes/ethylene glycol/slow cooling/meiotic spindle

Submitted on September 1, 2006; resubmitted on May 29, 2007; accepted on June 7, 2007.


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J.J. Bromfield, G. Coticchio, K. Hutt, R. Sciajno, A. Borini, and D.F. Albertini
Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation
Hum. Reprod., September 1, 2009; 24(9): 2114 - 2123.
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