Fabbri R, Vicenti R, Macciocca M, Martino N A, Dell'Aquila M E, Pasquinelli G, Morselli-Labate A M, Seracchioli R, Paradisi R
Gynecology and Pathophysiology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital, 40138 Bologna, Italy.
Gynecology and Pathophysiology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital, 40138 Bologna, Italy
Hum Reprod. 2016 Aug;31(8):1838-49. doi: 10.1093/humrep/dew134. Epub 2016 Jun 9.
Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)?
The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol.
Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma.
STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them.
By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section.
LIMITATIONS, REASONS FOR CAUTION: The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed.
The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure.
STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests.
Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.
人类卵巢组织冷冻保存的最佳方法是什么：慢速冷冻/快速解冻（SF/RT）还是玻璃化/复温（V/W）？
本研究中使用的传统SF/RT方案似乎比所采用的开放式载体V/W方案能更好地保存人类冷冻保存卵巢组织的形态功能状态。
人类卵巢组织的冷冻保存通常采用SF/RT方法。然而，经常观察到卵泡池减少和基质损伤。一种新兴的替代方法是V/W，它似乎能维持基质的形态完整性。
研究设计、规模、持续时间：这是一项回顾性队列研究，纳入了6例患有肿瘤疾病的患者，于2014年1月至12月入组。
参与者/材料、环境、方法：通过腹腔镜从左右卵巢采集卵巢组织，使用常规的SF/RT方案或V/W方法进行冷冻保存，V/W方法包括将组织置于两种溶液（含有不同浓度的丙二醇、乙二醇和蔗糖）中孵育并在开放系统中进行玻璃化。对于每位患者，在腹腔镜检查时（新鲜组织）以及储存后（SF/RT或V/W）从每个卵巢收集三块组织，进行光镜检查（LM）和透射电子显微镜检查（TEM），以评估卵泡和基质的形态和超微结构特征，并进行激光扫描共聚焦显微镜检查（LSCM），以确定功能性能量/氧化还原基质状态。将SF/RT和V/W卵巢组织的保存状态与新鲜组织的进行比较，以及两者之间相互比较。
通过LM和TEM，SF/RT和V/W样本显示出轻微的冷冻损伤。在SF/RT样本中观察到间质水肿、基质细胞空泡化增加和染色质凝聚；相比之下，V/W样本显示卵母细胞核染色质略增厚且形状不规则。LSCM的功能成像分析显示，与新鲜样本相比，SF/RT和V/W样本中的线粒体活性和细胞内活性氧水平均降低。该研究还表明，从卵巢皮质的外层到内层连续切片，线粒体活性逐渐功能障碍。与新鲜样本相比，V/W样本在内层切片中的线粒体活性降低明显高于外层切片。
局限性、谨慎原因：结果报告了通过LSCM对新鲜和冷冻保存的人类卵巢组织进行的生物能量和氧化状态评估，LSCM是一种最近应用于组织样本的技术。在SF/RT或V/W后对人类卵巢组织使用LSCM是一种需要验证的新应用。使用功能性探针进行线粒体染色和固定的程序尚未标准化。尚未对冷冻保存的卵巢组织在严重联合免疫缺陷小鼠中的异种移植和体外培养进行研究。
确定一种能够维持卵巢组织形态功能完整性以及与新鲜组织中观察到的卵泡数量相当的冷冻保存方法，可能会在临床实践中优化恢复效果及卵巢功能持续时间，并提高移植后的分娩成功率。SF/RT方案比V/W程序能更好地保持组织的形态功能完整性。
研究资金/利益冲突：资金由意大利博洛尼亚和拉文纳储蓄银行基金会提供。N.A.M.博士获得了项目ONEV MIUR PONa3 00134 - n.254/R&C 18 5 项目编号：2011和项目GR - 20（2011/2012年卫生部青年研究人员资助项目）。不存在利益冲突。
临床试验74/2001/0（批准时间：2002年2月13日）：“人类卵巢组织冷冻保存的初步研究：冷冻保存前后的形态学和免疫组织化学分析”
