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

被引:34
作者
De Santis, L.
Coticchio, G. [1 ]
Paynter, S.
Albertini, D.
Hutt, K.
Cino, I.
Iaccarino, M.
Gambardella, A.
Flamigni, C.
Borini, A.
机构
[1] Univ Vita Salute San Raffaele, H S Raffaele, IVF Unit, Milan, Italy
[2] Tecnobios Procreazione, I-40125 Bologna, Italy
[3] Cardiff Univ, Cardiff, Wales
[4] Univ Kansas, Med Ctr, Lawrence, KS 66045 USA
[5] Clin Mediterranea, Naples, Italy
关键词
cryopreservation; oocytes; ethylene glycol; slow cooling; meiotic spindle;
D O I
10.1093/humrep/dem240
中图分类号
R71 [妇产科学];
学科分类号
100211 ;
摘要
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 5 0.82 +/- 0.29 mm min(-1) atm(-1) (mean +/- SD) and PEG 0.10 +/- 0.01 mm 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.
引用
收藏
页码:2776 / 2783
页数:8
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