Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
Laboratory for Veterinary Physiology and Biochemistry, Gamete Research Centre, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Belgium.
Cryobiology. 2021 Jun;100:164-172. doi: 10.1016/j.cryobiol.2021.01.013. Epub 2021 Jan 21.
We aimed to assess the feasibility of passive slow freezing (PSF using Mr. Frosty container, Nalgene) as an alternative to controlled slow rate freezing (CSF using (Freezal™, Air liquide)) for human ovarian tissue (OT) cryopreservation. Validation studies needed were determined after assessing the risk associated (EuroGTP-II ART tool) and were conducted in 66 OT samples from 10 transgender men aged 23.4 ± 5.1 y. Folliculogenesis was assessed in vitro (after 2 h and 2 days of culture) and in vivo (2, 4 and 6 weeks xenotransplantation in Balbc/nude mice) by haematoxilin-eosin staining. Fibrosis was assessed by Masson's trichrome staining. Immunohistochemistry was used to study cell proliferation (PCNA and Ki-67) and apoptosis (caspase-3 and TUNEL). Differences in percentages were estimated using a generalized estimated equations method. After 2 days of in vitro culture, higher odds of primordial follicles (PF) (OR 1.626; 95%CI (1.162-2.266); P = 0.004) and lower odds of growing follicles (GF) (OR 0.616; 95%CI (0.441-0.861); P = 0.004) were associated with the established CSF technique. No statistical differences were found in the mean estimated proportion of proliferating (Ki-67+ or PCNA+) or apoptotic (caspase-3+ or Tunel+) follicles. Two and 6 weeks after xenotransplantation, respectively lower odds of GF (OR 0.419; 95%CI (0.217-0.809); P = 0.010) and secondary follicles (OR 0.135; 95%CI (0.071-0.255); P < 0.001) were associated with CSF. Proportion of fibrosis was similar. This validation study shows a higher follicle activation after 2 days in vitro and after 2 weeks following xenotransplantation in mice using PSF. PSF may be an easy, cost-effective low-risk alternative to CSF for cryopreservation of human OT.
我们旨在评估使用 Mr. Frosty 容器（Nalgene）的被动慢速冷冻（PSF）作为替代控制慢速速率冷冻（CSF 使用 Freezal™，Air Liquide）的可行性，用于人类卵巢组织（OT）的冷冻保存。在评估相关风险（EuroGTP-II ART 工具）后，确定了需要进行的验证研究，并在 10 名年龄 23.4±5.1 岁的跨性别男性的 66 份 OT 样本中进行。通过苏木精-伊红染色在体外（培养 2 小时和 2 天后）和体内（在 Balbc/nude 小鼠中进行 2、4 和 6 周异种移植）评估卵泡发生。通过 Masson 三色染色评估纤维化。免疫组织化学用于研究细胞增殖（PCNA 和 Ki-67）和细胞凋亡（caspase-3 和 TUNEL）。使用广义估计方程方法估计百分比差异。在体外培养 2 天后，原始卵泡（PF）的出现几率更高（OR 1.626；95%CI（1.162-2.266）；P=0.004），生长卵泡（GF）的出现几率更低（OR 0.616；95%CI（0.441-0.861）；P=0.004）与既定的 CSF 技术相关。在增殖（Ki-67+或 PCNA+）或凋亡（caspase-3+或 Tunel+）卵泡的平均估计比例方面未发现统计学差异。异种移植后 2 周和 6 周，GF（OR 0.419；95%CI（0.217-0.809）；P=0.010）和次级卵泡（OR 0.135；95%CI（0.071-0.255）；P<0.001）的出现几率较低与 CSF 相关。纤维化比例相似。这项验证研究表明，在体外培养 2 天和在小鼠中进行异种移植后 2 周，PSF 后体外和体内的卵泡激活更高。PSF 可能是 CSF 用于人类 OT 冷冻保存的一种简单、具有成本效益且风险较低的替代方法。
