Keros Victoria, Xella Susanna, Hultenby Kjell, Pettersson Karin, Sheikhi Maryam, Volpe Annibale, Hreinsson Julius, Hovatta Outi
Department of Clinical Science, Technology and Intervention, Division of Obstetrics and Gynaecology, Karolinska Institutet, Karolinska University Hospital Huddinge, K 57, Stockholm SE 141 86, Sweden.
Hum Reprod. 2009 Jul;24(7):1670-83. doi: 10.1093/humrep/dep079. Epub 2009 Apr 9.
Controlled-rate freezing of ovarian cortical tissue for preservation of fertility among young women facing chemo- or radio-therapy is a widely accepted procedure. To improve the method for cryopreservation of ovarian tissue, particularly the stroma, we carried out a systematic comparison of vitrification versus slow programmed freezing.
Ovarian tissue from 20 women, donated during Caesarean section, was used for parallel comparison of survival and detailed light and electron microscopic (EM) morphology of oocytes, granulosa cells and ovarian stroma after freezing (slow freezing and vitrification), thawing and 24-h culture. Using tissue obtained from the same patient, we compared four cryopreservation protocols and fresh tissue. The cryoprotectants used in slow freezing were 1,2-propanediol (PrOH)-sucrose and ethylene glycol (EG)-sucrose. For vitrification, tissues were incubated for 5 or 10 min in three solutions containing a combination of dimethyl sulphoxide (DMSO), PrOH, EG and polyvinylpyrrolidone (PVP).
Cryopreservation using controlled-rate freezing and vitrification preserved the morphological characteristics of ovarian tissue generally well. As revealed by morphological analysis, particularly EM, the ovarian stroma was significantly better preserved after vitrification than after slow freezing (P < 0.001). The follicles were similarly preserved after all freezing methods.
Vitrification using a combination of PrOH, EG, DMSO and PVP was comparable to slow freezing in terms of preserving follicles in human ovarian tissue. Ovarian stroma had significantly better morphological integrity after vitrification than after controlled-rate freezing.
对于面临化疗或放疗的年轻女性，采用控速冷冻卵巢皮质组织以保存生育能力是一种广泛接受的方法。为了改进卵巢组织的冷冻保存方法，特别是基质的保存方法，我们对玻璃化冷冻与慢速程序冷冻进行了系统比较。
使用20名女性在剖宫产时捐献的卵巢组织，对冷冻（慢速冷冻和玻璃化冷冻）、解冻及24小时培养后卵母细胞、颗粒细胞和卵巢基质的存活情况以及详细的光镜和电镜形态进行平行比较。利用同一患者的组织，我们比较了四种冷冻保存方案与新鲜组织。慢速冷冻中使用的冷冻保护剂是1,2 - 丙二醇（PrOH）-蔗糖和乙二醇（EG）-蔗糖。对于玻璃化冷冻，组织在含有二甲亚砜（DMSO）、PrOH、EG和聚乙烯吡咯烷酮（PVP）组合的三种溶液中孵育5或10分钟。
采用控速冷冻和玻璃化冷冻保存卵巢组织的形态特征总体良好。形态学分析尤其是电镜分析显示，玻璃化冷冻后卵巢基质的保存明显优于慢速冷冻（P < 0.001）。所有冷冻方法对卵泡的保存效果相似。
在保存人类卵巢组织中的卵泡方面，使用PrOH、EG、DMSO和PVP组合的玻璃化冷冻与慢速冷冻相当。玻璃化冷冻后卵巢基质的形态完整性明显优于控速冷冻。
