De Santis L, Coticchio G, Paynter S, Albertini D, Hutt K, Cino I, Iaccarino M, Gambardella A, Flamigni C, Borini A
IVF Unit, Vita-Salute University, H. S. Raffaele, Milan, Italy.
Hum Reprod. 2007 Oct;22(10):2776-83. doi: 10.1093/humrep/dem240. Epub 2007 Aug 3.
To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (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.
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.
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 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm 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).
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.
为开发新的冷冻保存方法，我们评估了成熟人类卵母细胞在暴露于乙二醇（EG）后的渗透系数Lp（水力传导率）和P(EG)（冷冻保护剂渗透率），并测试了基于这种冷冻保护剂的多步慢速冷却方案的效率。
用1.5mol/L的EG灌注卵母细胞10分钟。计算每个时间点的卵母细胞体积并将其标准化为原始体积。通过将卵母细胞暴露于递增的EG浓度（0.5、1.0和1.5mol/L，n = 155）或1.5mol/L的1,2-丙二醇（PrOH，n = 102）来进行慢速冷却。将冷冻保存后存活的卵母细胞（n = 80）和新鲜卵母细胞（n = 73）用于减数分裂纺锤体的共聚焦显微镜分析。
在暴露于EG期间，卵母细胞体积突然减少50%。未观察到初始体积的完全恢复。从最佳拟合图的值中，得出系数Lp = 0.82±0.29μm min(-1) atm(-1)（平均值±标准差）和P(EG) 0.10±0.01μm s(-1)。用EG冷冻后的存活率低于用PrOH冷冻后的存活率（分别为51.6%和71.5%，P < 0.05）。与新鲜卵母细胞相比，冷冻EG和冷冻PrOH卵母细胞中正常纺锤体构型的频率较低（分别为53.8%、50.9%和66.7%，P < 0.05）。
卵母细胞质膜对EG的渗透性有限，EG暴露会导致相当大的渗透应激。然而，通过设计旨在最小化渗透应激的方案，解冻后的存活率和正常减数分裂纺锤体组织率可能得以保留。
