Evidence of metabolic activity during low-temperature ovarian tissue preservation in different media.,Journal of Assisted Reproduction and Genetics

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Evidence of metabolic activity during low-temperature ovarian tissue preservation in different media.
Journal of Assisted Reproduction and Genetics ( IF 3.1 ) Pub Date : 2020-09-04 , DOI: 10.1007/s10815-020-01935-y
Janice de M V Vilela ₁ , Marie-Madeleine Dolmans _{1,

2} , Emi Maruhashi ₁ , Marine C N M Blackman ₃ , Pierre Sonveaux ₃ , Ana Luisa Miranda-Vilela ₄ , Christiani A Amorim ₁

Affiliation

Purpose

Although ovarian tissue transportation has been validated for up to 24 h, there is no standard protocol to date. We aimed to elucidate how existing media currently used for ovarian tissue transportation affect ovarian tissue metabolism and cell viability.

Methods

Cow ovarian fragments were immersed in 0.9% NaCl solution, IVF medium, Leibovitz 15 medium (L-15), or PBS for 1, 4, or 24 h at 4 °C. Media were analyzed for pH, lactate dehydrogenase (LDH) activity, and glucose, pyruvate, and lactate concentrations, while apoptosis was assessed by TUNEL assays in fixed fragments. Viability rates were assessed by flow cytometry (FACS).

Results

There were lower pH levels in NaCl at all time points compared with other media. LDH activity increased with time and was lowest in NaCl at 1 and 4 h. There was no significant difference in glucose levels, but a significant pyruvate decrease in L-15 and a significant lactate increase in all media. TUNEL showed apoptosis rates ranging from 0 to 5%. FACS showed a mean of 4% necrotic cells and 15–19% apoptotic cells after 1 h of incubation, but less than 1% necrotic cells and 2–6% apoptotic cells after 24 h in all media.

Conclusion

Our results indicate marked metabolic activity in ovarian tissue at 4 °C and suggest that cells use internal sources of energy, which may influence transplantation outcomes. This highlights the importance of better understanding whole tissue dynamics to develop a standard protocol for ovarian tissue transportation.

Graphical abstract

中文翻译：

不同介质中低温卵巢组织保存过程中代谢活动的证据。

目的

尽管卵巢组织运输已被验证长达 24 小时，但迄今为止还没有标准方案。我们的目的是阐明目前用于卵巢组织运输的现有介质如何影响卵巢组织代谢和细胞活力。

方法

将牛卵巢碎片在 4°C 下浸入 0.9% NaCl 溶液、IVF 培养基、Leibovitz 15 培养基 (L-15) 或 PBS 中 1、4 或 24 小时。分析培养基的 pH、乳酸脱氢酶 (LDH) 活性以及葡萄糖、丙酮酸和乳酸浓度，同时通过固定片段中的 TUNEL 测定评估细胞凋亡。通过流式细胞术（FACS）评估存活率。

结果

与其他介质相比，氯化钠在所有时间点的 pH 水平都较低。LDH 活性随着时间的推移而增加，并且在 1 小时和 4 小时时在 NaCl 中最低。葡萄糖水平没有显着差异，但所有培养基中 L-15 的丙酮酸显着降低，乳酸显着增加。TUNEL 显示细胞凋亡率范围为 0% 至 5%。FACS 显示，孵育 1 小时后，所有培养基中平均有 4% 的坏死细胞和 15-19% 的凋亡细胞，但 24 小时后，坏死细胞和 2-6% 的凋亡细胞均低于 1%。