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Hum. Reprod. Advance Access published online on March 20, 2006
Human Reproduction, doi:10.1093/humrep/del073
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© The Author 2006. 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
Received December 7, 2005
Revised February 11, 2006
Accepted February 15, 2006

Article

Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation

G. Coticchio 1 *, L.De Santis 2, G. Rossi 3, A. Borini 1, D. Albertini 4, G. Scaravelli 5, C. Alecci 6, V. Bianchi 1, S. Nottola 7, and S. Cecconi 3

1 Tecnobios Procreazione, Bologna
2 Vita-Salute University, H.S.Raffaele, Milan
3 University of L’Aquila, L’Aquila
4 University of Kansas Medical Center, Kansas City, KS
5 National Health Institute, Rome
6 UMR - Associazione HERA, Catania
7 ‘La Sapienza’ University, Rome, Italy

* To whom correspondence should be addressed.
G. Coticchio, E-mail: coticchio{at}tecnobiosprocreazione.it


  Abstract

BACKGROUND: Recently described slow-cooling cryopreservation protocols involving elevated sucrose concentration have improved survival frequencies of human oocytes, potentially overcoming a major hurdle that has limited the adoption of oocyte storage. Because implantation rates of embryos from frozen oocytes remain generally low, it is still debated whether, irrespective of survival rates, this form of cryopreservation leads inevitably to the disruption or complete loss of the metaphase II (MII) spindle. METHODS: Human oocytes with an extruded polar body I (PBI) were cryopreserved using a slow-cooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution. Fresh control and frozen-thawed survived oocytes were analysed by confocal microscopy to evaluate MII spindle and chromosome organizations. RESULTS: Of the 104 oocytes included in the unfrozen group, 76 (73.1%) displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol (152 oocytes). CONCLUSIONS: Partial disruption of the MII spindle and associated chromosomes accompanies inadequate cryopreservation during slow cooling. However, protocols adopting higher sucrose concentration in the freezing solution promote the retention of an intact chromosome segregation apparatus comparable in incidence to freshly collected oocytes.

Keywords: oocyte cryopreservation/slow cooling/spindle apparatus/chromosomes/sucrose.
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