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玻璃化冷冻保存对人卵巢组织卵泡形态和应力松弛行为的影响:蔗糖与海藻糖作为非渗透性保护剂的比较。

Effects of vitrification cryopreservation on follicular morphology and stress relaxation behaviors of human ovarian tissues: sucrose versus trehalose as the non-permeable protective agent.

机构信息

Center for Reproductive Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, China.

Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, China Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, China.

出版信息

Hum Reprod. 2015 Apr;30(4):877-83. doi: 10.1093/humrep/dev012. Epub 2015 Feb 5.

Abstract

STUDY QUESTION

Is sucrose more effective than trehalose in human ovarian tissue cryopreservation?

SUMMARY ANSWER

The effect of sucrose as a cryoprotective agent (CPA) was not significantly different from that of trehalose in human ovarian tissue cryopreservation.

WHAT IS KNOWN ALREADY

Sugars have the ability to keep the cell membrane intact and can decrease the toxicity of CPAs. Sucrose is the most commonly used non-permeable CPA, while trehalose is rarely used in human ovarian tissue cryopreservation. Although various methods are utilized to evaluate the efficiency of human ovarian tissue cryopreservation, few studies have evaluated the effect of cryopreservation from the viewpoint of biomechanics.

STUDY DESIGN, SIZE, DURATION: A total of 15 ovarian tissue samples were collected from 15 patients (20-41 years old) with benign ovarian tumors or malignancies, and each was dissected into six slices. Two slices were taken as the fresh control group. The remaining four slices were vitrified using different vitrification protocols. After warming, samples in each group were either fixed for histological evaluation or destined for stress relaxation test.

PARTICIPANTS/MATERIALS, SETTING, METHODS: The CPA solutions for the control and vitrified groups were composed of EDS and EDT (E, ethylene glycol; D, dimethylsulphoxide; S, sucrose; T, trehalose). The stress relaxation experiments were carried out at room temperature using a dynamic mechanical analyzer. Ovarian tissue samples were assessed for both their morphology and viscoelasticity. Stress relaxation data (SRD) were calculated as a percentage, representing the ability to maintain the initial stress after stretching. The percentage of morphologically normal follicles was compared between groups, which was represented by morphologic preservation ratio.

MAIN RESULTS AND THE ROLE OF CHANCE

The morphologic preservation ratio of the primordial follicles in the fresh control group (87.58%) was higher than that in group S (72.33%) (P = 0.000) and group T (79.56%) (P = 0.002). Although not statistically significant, compared with the S group, vitrification with T suggested a trend toward a higher morphologic preservation ratio of the primordial follicles. The SRD in the fresh control group (0.6433 ± 0.7233) was significantly different from that in group S (0.5200 ± 0.8331, P = 0.000) or in group T (0.5667 ± 0.6415, P = 0.000). However, no significant difference was found between groups S and T.

LIMITATIONS, REASONS FOR CAUTION: Experimental samples were directly exposed to the air, which will result in a discrepancy in the viscoelastic properties between experimental tissues and in vivo tissues.

WIDER IMPLICATIONS OF THE FINDINGS

Our study suggested a trend toward a higher morphologic preservation ratio of the primordial follicles after vitrification in trehalose compared with sucrose, which may provide a basis for further optimizing human ovarian tissue vitrification. In addition, it was possible to evaluate the effect of ovarian tissue cryopreservation from a biomechanics perspective.

STUDY FUNDING/COMPETING INTERESTS: This study was supported by the grants from the Medical Scientific Research Subject, Health Ministry of Anhui Province (2010B014) and National Basic Research Program of China (973 Program) (2012CB944704), and the National Natural Science Foundation of China (Nos. 51276179 and 51476160). The authors declare that there is no conflict of interests regarding the publication of this original paper.

摘要

研究问题

蔗糖在人类卵巢组织冷冻保存中是否比海藻糖更有效?

总结答案

蔗糖作为一种冷冻保护剂(CPA)的效果与海藻糖在人类卵巢组织冷冻保存中没有显著差异。

已知情况

糖具有保持细胞膜完整的能力,并能降低 CPAs 的毒性。蔗糖是最常用的非渗透型 CPA,而海藻糖很少用于人类卵巢组织冷冻保存。虽然有多种方法用于评估人类卵巢组织冷冻保存的效率,但很少有研究从生物力学的角度评估冷冻保存的效果。

研究设计、大小、持续时间:共从 15 名患有良性卵巢肿瘤或恶性肿瘤的患者(年龄 20-41 岁)中采集了 15 个卵巢组织样本,每个样本均被解剖成 6 个切片。其中 2 个切片作为新鲜对照组。剩余的 4 个切片用不同的玻璃化方案进行了玻璃化处理。在解冻后,每组的样本要么固定用于组织学评估,要么用于应力松弛测试。

参与者/材料、设置、方法:对照组和玻璃化组的 CPA 溶液由 EDS 和 EDT(E,乙二醇;D,二甲基亚砜;S,蔗糖;T,海藻糖)组成。在室温下使用动态力学分析仪进行了应力松弛实验。评估了卵巢组织样本的形态和粘弹性。计算了代表拉伸后保持初始应力的能力的应力松弛数据(SRD),以百分比表示。各组之间的原始卵泡形态正常比例(形态保存比)进行了比较。

主要结果及其机会

新鲜对照组(87.58%)原始卵泡的形态保存比高于 S 组(72.33%)(P=0.000)和 T 组(79.56%)(P=0.002)。虽然没有统计学意义,但与 S 组相比,用 T 进行玻璃化处理后,原始卵泡的形态保存比有升高的趋势。新鲜对照组的 SRD(0.6433±0.7233)与 S 组(0.5200±0.8331,P=0.000)或 T 组(0.5667±0.6415,P=0.000)有显著差异。然而,S 组和 T 组之间没有显著差异。

局限性、谨慎的原因:实验样本直接暴露在空气中,这将导致实验组织和体内组织之间的粘弹性存在差异。

研究结果的更广泛意义

我们的研究表明,在用海藻糖进行玻璃化处理后,原始卵泡的形态保存比用蔗糖有升高的趋势,这可能为进一步优化人类卵巢组织玻璃化提供依据。此外,有可能从生物力学的角度评估卵巢组织冷冻保存的效果。

研究资金/利益冲突:本研究得到了安徽省医学科学研究重点项目(2010B014)和国家重点基础研究发展计划(973 计划)(2012CB944704)以及国家自然科学基金(No.51276179 和 51476160)的支持。作者声明,在发表这篇原始论文方面没有利益冲突。

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