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Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration.

Bianchi V, Coticchio G, Fava L, Flamigni C, Borini A

Tecnobios Procreazione, Via Dante 15, 40125 Bologna and University of Bologna, 40125 Bologna, Italy.

BACKGROUND: One of the major concerns derived from the cryopreservation of meiotically mature oocytes is possible damage to the cytoskeletal apparatus, and in particular the meiotic spindle. METHODS: One hundred fresh oocytes showing the polar body I and high meiotic spindle birefringence (maximum retardance+/-1.5 mol/l SD = 2.58+/-0.1 nm), assessed through analysis, were included in this study. Oocytes were cryopreserved with a 1.5 mol/l 1,2-propanediol +0.3 mol/l sucrose solution. After thawing, spindles were imaged at 0, 3 and 5 h. Spindle birefringence was quantified by measuring microtubule maximum retardance. Signals of thawed oocytes were classified as absent (non-detectable), weak (1.55+/-0.3 nm) or high (2.50+/-0.2 nm). RESULTS: Immediately after thawing, only 22.9% of oocytes showed a weak birefringence signal, while only 1.2% of oocytes displayed a high signal. Three hours after thawing, the proportion of oocytes exhibiting a weak or high intensity signal was 49.4% and 18.1%, respectively. Finally, after culture for 5 h following thawing, a weak birefringence signal was detected in 51.8% of oocytes, while 24.1% showed a high signal. There was a statistically significant increase in signal restoration after 3 h of culture (P < 0.001). CONCLUSIONS: These results suggest that in mature oocytes stored via slow freezing, the meiotic spindle undergoes transient disappearance immediately after thawing but is reorganized in the majority of oocytes, at least to some extent, after 3-5 h of culture.

Published 24 March 2005 in Hum Reprod, 20(4): 1078-83.
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