Cryopreservation of human ovarian tissue and the evaluation methods

doi:10.3969/j.issn.2095-4344.2014.36.025

Abstract

Abstract:

BACKGROUND: How to preserve the fertility and ovarian function in patients with procreation needs has important significance and broad application prospects.
OBJECTIVE: To summarize the recent studies about ovarian tissue cryopreservation, preservation and evaluation methods, and to explore the best solution for ovarian tissue freezing.
METHODS: An online computer-based retrieval of PubMed database and Wanfang database between January 1994 and January 2014 was performed by the first author. The key words were ovarian tissue, ovarian tissue cryopreservation, freezing factors in English and Chinese. Finally 59 literatures on the ovarian tissue cryopreservation technique, influencing factor of cryopreservation, resuscitation, and human ovarian tissue transplantation were included.
RESULTS AND CONCLUSION: Currently, laboratory methods of ovarian tissue cryopreservation are divided into slow-rate freezing, ultra-rapid cooling and vitrification technology. The freezing effect may vary in different patients, tissue sections, cryoprotectants species, penetration time and frozen carrier. After the recovery, the histological morphology of granulosa cells and follicles was observed. The follicles were counted to evaluate ovarian tissue. Freeze-thaw effects were evaluated by observing the changes of subcellular structures. The apoptotic signals were also detected. Immunohistochemical analysis was applied to detect the proliferation after recovery and apoptosis-related information. Variation in the in vitro culture reflected endocrine levels. Genetic level detection was also performed. In recent years, frozen ovarian tissue is clinically applied for the transplantation, to restore reproductive endocrine function of the patients, or get mature germ cells, thereby preserving female fertility. However, there are still some questions about cryopreservation of ovarian tissue, which need further study.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: ovary, freezing, transplantation

CLC Number:

R318

Zhang Ling, Peng Tao, Yu Yan-ling. Cryopreservation of human ovarian tissue and the evaluation methods[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(36): 5885-5890.

Figures/Tables 1

2.1 卵巢组织冷冻技术卵巢组织冷冻技术即将离体的组织或器官保存在低温中并保持其活性。目前主要使用的卵巢组织冷冻方法主要有慢速冷冻、快速冷冻和玻璃化冷冻。 2.1.1 慢速冷冻 1998年，Oktay等[8]首次将取出的人卵巢皮质组织在-197 ℃液氮中保存。至此之后有很多报道这样的冷冻方法。2004年Donnez等[9]、2005年Meirow等[10]、2006年Demeestere等[11]以及2010年Serrano等[12]均报道慢速冷冻卵巢组织，解冻后自体移植，或进行辅助生殖技术获得自然妊娠或活婴分娩。这些研究证实了慢速冷冻法可使卵巢组织保存卵泡质量，并能获得具有生殖潜能的卵巢组织。 2.1.2 快速冷冻快速冷送通常的方法是将卵巢组织置于含低浓度(1.5 mol/L)的冷冻保护剂中。在液氮蒸汽中放置> 12 h后投人液氮中保存[13]。目前对于该方法的报道不多。其有效性需进一步研究。 2.1.3 玻璃化冷冻在20世纪90年代初期，有许多学者将卵巢组织进行玻璃化冷冻，并取得一系列的成功。在1996年出现第1例关于人卵巢组织低温保存的报道[14]。Wang等[15]研究表明，玻璃化冷冻较慢速冷冻法可以更好地保存人卵巢组织的间质细胞、胶原束、细胞间隙及始基卵泡。Keros等[16]报道人卵巢组织玻璃化冷冻后对卵泡的保存效果与慢速冷冻相当，但玻璃化冷冻后卵巢基质保存的结构完整性明显优于慢速冷冻组。玻璃化冷冻法使冷冻过程简化，费用低，效率高，具有巨大的发展潜力[17-19]。 2.2 影响冷冻化效果的因素目前还没有对卵巢组织冷冻的统一方案，可能影响卵巢组织冷冻的因素，总结如下。 2.2.1 患者的选择关于卵巢冻存的适应人群相关文献已进行了一些研究[20-23]，在卵巢冷冻保存过程中，会损失7%的卵泡，移植后新血供建立前还会丧失60%以上的始基卵泡[24]，因此，卵巢组织必须存有相当数量才能有良好的冷冻效果，必须考虑患者的疾病及年龄因素。不同种类的卵巢疾病，会造成卵巢组织不同程度的损伤。Catherine等[25]对卵巢组织冷冻的研究发现，随着患者年龄的增大，其卵巢中始基卵泡和初级卵泡的平均密度在不断下降。而现有文献对于卵巢疾病种类、患者年龄及患者其他因素等对冷冻效果的影响还不是很全面，有待进一步研究论证。 2.2.2 卵巢组织标本的大小卵巢组织块的厚度可能会影响冷冻保护剂的渗透，从而影响冷冻剂的保护作用。目前文献报道大部分卵巢组织块都处理为厚度约1 mm，Meirow等[26]将人卵巢组织切至厚度为1 mm左右，得到较好的保存效果。李宇彬等[27]研究表明，0.5-1.0 mm的厚度，卵巢组织与新鲜组对照始基卵泡正常率没有显著的差异。最近，赵硕[28]对大块人卵巢组织的冻融进行研究表明，15 mm2×(2.0-3.0) mm的人卵巢组织冷冻复苏后卵泡形态及功能良好，冷冻大块卵巢组织具有可行性。 2.2.3 冷冻保护剂 Amorim等[29]的研究显示，二甲基亚砜与乙二醇效果最好。严晓南等[30]将二甲基亚砜与乙二醇联合应用，既促进玻璃化过程，提高冷冻效率，又能减少两者的用量，减轻冷冻保护剂的细胞毒性作用。Sheikhi等[31]联合二甲基亚砜、乙二醇和丙二醇作为冷冻保护剂对人卵巢组织进行玻璃化冷冻，得到形态正常卵泡的比率达为92.31%(图1)。 Isachenko等[32]报道单独用渗透性冷冻保护剂比混合应用蔗糖的冷冻保存效果好。Xiao等[33]研究发现，联合使用低浓度的二甲基亚砜，乙二醇和蔗糖既可以达到很好的冷冻效果，也降低了冷冻保护剂的毒性。因此，冷冻保护剂的选择及其浓度的确定，以及和冷冻方法的结合，需要进一步实验探讨，才能得到最佳冷送效果的保护剂配比。"

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