Research question

Can the low-temperature transport time of removed human ovarian tissue be prolonged until cryopreservation?

Design

Fresh ovarian cortex from nine premenopausal patients was either slow-frozen immediately or stored at 4°C for 24 or 48 h before slow-freezing. The fresh and frozen–thawed biopsies were evaluated by follicle counting via calcein staining, histologic analyses via haematoxylin and eosin staining, and apoptosis via terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL). The fresh cortex was assessed by reactive oxygen species (ROS) and total antioxidant capacity (TAC) assay to detect oxidative stress. The frozen–thawed cortex biopsies were also evaluated by quantitative PCR for messenger RNA (mRNA) expression of BCL-2, BAX, TNFa, HIF-1a, BMP15 and GDF9, and Western blot for detection of BCL-2, BMP15, GDF9 and CASPASE-3. The frozen–thawed cortex was cultured in vitro for 4 days, anti-Müllerian hormone and glucose were assessed in the supernatant, and ROS and TAC assay detected any oxidative stress in the cortex.

Results

In the fresh cortex, there were no significant differences between the three groups. In the frozen–thawed cortex, there were no significant differences between the three groups regarding follicle viability, TUNEL, mRNA expression of TNFa, HIF-1a or BMP15. GDF9 mRNA and BAX/BCL-2 were lower and higher at 48 h than at 0 h, respectively. However, the protein expression of BCL-2, CASPASE-3, GDF9 and BMP15 were no different. In the cultured cortex, ROS, TAC and glucose uptake were no different across the three groups.

Conclusion

Ovarian tissue transportation was validated for 24 h in the procedure used in clinical practice. This study showed that 4–8°C transportation for 24 or 48 h does not seem to damage the ovarian tissue. However, ovarian tissue transportation beyond 48 h needs to be further studied for conclusions to be made.

中文翻译：

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冷冻保存前人体卵巢组织的长期低温运输

研究问题

取出的人卵巢组织的低温运输时间可以延长到冷冻保存吗？

设计

来自 9 名绝经前患者的新鲜卵巢皮质要么立即缓慢冷冻，要么在缓慢冷冻前在 4°C 下储存 24 或 48 小时。通过钙黄绿素染色对卵泡计数、通过苏木精和伊红染色进行组织学分析以及通过末端脱氧核苷酸转移酶介导的 dUDP 缺口末端标记 (TUNEL) 进行细胞凋亡来评估新鲜和冷冻解冻的活检。通过活性氧（ROS）和总抗氧化能力（TAC）测定评估新鲜皮层以检测氧化应激。还通过定量 PCR 对BCL-2、BAX、TNFa、HIF-1a、BMP15和GDF9的信使 RNA (mRNA) 表达以及用于检测 BCL-2、BMP15、GDF9 和CASPASE-3。培养冻融的皮层体外4 天，在上清液中评估抗苗勒管激素和葡萄糖，ROS 和 TAC 测定检测皮质中的任何氧化应激。

结果

在新鲜皮层中，三组之间没有显着差异。在冻融皮质中，三组之间在卵泡活力、TUNEL、TNFa、HIF-1a或BMP15 的 mRNA 表达方面没有显着差异。GDF9 mRNA 和BAX / BCL-2在 48 h 时分别低于和高于 0 h。然而，BCL-2、CASPASE-3、GDF9 和 BMP15 的蛋白表达没有差异。在培养的皮层中，ROS、TAC 和葡萄糖摄取在三组之间没有差异。

结论

在临床实践中使用的程序中验证了卵巢组织运输 24 小时。这项研究表明，4-8°C 运输 24 或 48 小时似乎不会损伤卵巢组织。然而，超过 48 小时的卵巢组织运输需要进一步研究才能得出结论。