Purpose

The aim of this study was to elucidate whether ovarian tissue is able to withstand a double freezing-thawing procedure.

Methods

Human ovarian cortical biopsies from 4 thawed whole ovaries were divided into 4 experimental subgroups: (a) frozen-thawed non-grafted group, (b) frozen-thawed xenografted group, (c) refrozen-rethawed non-grafted group, and (d) refrozen-rethawed xenografted group. Xenografting was performed using 8 severe combined immunodeficient mice for a total duration of 21 days. The following analyses were conducted: classic hematoxylin and eosin staining, Ki67 immunolabeling, transmission electron microscopy, Masson’s green trichrome, and double CD34 immunostaining.

Results

Morphologically normal preantral follicles were detected in all groups. We observed a dramatic decline of more than 65% in early preantral follicle survival rates after grafting of both frozen-thawed (p < 0.0001) and refrozen-rethawed (p < 0.0001) ovarian tissue. However, mean follicle densities remained comparable between the frozen-thawed and refrozen-rethawed non-grafted groups, as well as both grafted groups. Equivalent proportions of proliferating early preantral follicles were identified in frozen-thawed and refrozen-rethawed samples, whether the tissue was grafted or not. Furthermore, we did not observe any significant difference in atretic follicle rates between any of the four groups, and the ultrastructural quality of follicles appeared unaffected by the refreezing procedure. Similar proportions of fibrosis were noted in the frozen-thawed and refrozen-rethawed groups, irrespective of grafting. Finally, no significant differences were witnessed in terms of vascularization.

Conclusion

We were able to demonstrate, for the first time, that refrozen-rethawed ovarian tissue has the same functional characteristics as frozen-thawed ovarian tissue.

中文翻译：

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冻融的人类卵巢能否承受皮质条形式的再次冻融？

 目的

本研究的目的是阐明卵巢组织是否能够承受双重冻融过程。

 方法

将来自 4 个解冻的完整卵巢的人卵巢皮质活检分为 4 个实验亚组：（a）冻融非移植组，（b）冻融异种移植组，（c）重新冻融非移植组，（d） ）再冻融异种移植组。使用8只严重联合免疫缺陷小鼠进行异种移植，总持续时间为21天。进行以下分析：经典苏木精和伊红染色、Ki67 免疫标记、透射电子显微镜、马森绿三色和双 CD34 免疫染色。

 结果

所有组中均检测到形态正常的腔前卵泡。我们观察到，移植冷冻解冻 ( p < 0.0001) 和重新冷冻再解冻 ( p < 0.0001) 卵巢组织后，早期窦前卵泡存活率急剧下降超过 65%。然而，冻融和重新冻融的非移植组以及两个移植组之间的平均卵泡密度仍然相当。无论组织是否移植，在冷冻解冻和重新冷冻再解冻的样本中都鉴定出相同比例的增殖早期窦前卵泡。此外，我们没有观察到四组之间闭锁卵泡率有任何显着差异，并且卵泡的超微结构质量似乎不受再冷冻程序的影响。无论移植情况如何，冻融组和重新冻融组的纤维化比例相似。最后，在血管化方面没有观察到显着差异。

 结论

我们首次证明，重新冷冻和解冻的卵巢组织具有与冷冻解冻的卵巢组织相同的功能特征。