Whole-ovary cryopreservation : a direct comparison of conventional and directional freezing

Whole-ovary cryopreservation followed by retransplantation with vascular anastomosis is a promising method to restore cancer-patient fertility. However, protocols need to be improved to minimize the damages related to an inadequate cooling rate caused by the large volume of whole organs. The aim of this study was to compare conventional slow-freezing (SF) versus directional-freezing (DF) techniques. Of 30 sheep ovaries collected at the local abattoir, 6 were used as fresh controls and 24 were perfused for 5min with cryoprotectant solution made of Leibovitz L-15 medium, 10% FCS, and 1.5-M dimethyl sulfoxide. Samples were inserted in glass freezing tubes filled with the same solution. Twelve ovaries were frozen with a Kryo 560M apparatus (Planer, UK) using a cooling rate of 0.5°Cmin(-1) from 4 to -40°C and 5°Cmin(-1) from -40 to -100°C followed by direct plunging in liquid nitrogen. The other 12 ovaries were frozen using an MTG 1315 apparatus (Core Dynamics, Ness Ziona, Israel). Tubes were pushed along the thermal gradient (4 to -70°C) at 0.01mms(-1) resulting in a cooling rate of 0.3°Cmin(-1) and then plunged in liquid nitrogen. After 2 weeks, samples were thawed at 37°C for 2min with gentle shaking. Morphological analysis showed that the rate of intact follicles was 94% in control samples, 87% in DF samples, and 58.3% in SF samples (P<0.05). No significant differences were found in stromal cell density between fresh and DF ovaries (17833 and 17041 nucleimm(-2), respectively), whereas SF ovaries showed a lower density (10875nucleimm(-2); P<0.001). To assess vessel integrity, thawed ovaries were perfused with 25% Indian ink/167UI of heparinmL(-1) saline solution and embedded in paraffin. Fresh and DF ovaries displayed a similar rate of perfused vessels (82.7 and 80.2%, respectively), whereas SF ovaries significantly lower number of perfused vessels (52.3%), indicating the occurrence of vascular injury. Follicle viability after freezing was evaluated by culturing individual cortical fragments (2×2×1mm) for 7 days. A high rate of morphologically normal follicles was found in both fresh (85%) and DF ovaries (78%). The number of intermediate and primary follicles was significantly increased in both groups when compared to Day 0 (P<0.001). By contrast, cortical slices obtained from SF ovaries showed a higher rate of degenerated follicles, oocytes with pyknotic nuclei, and a significant decrease of follicle number. Furthermore, qPCR and immunohistochemical assay for Ki-67 showed a similar cell proliferation in fresh and DF ovary but a significantly lower one in SF samples. We conclude that directional freezing allows a better preservation of both cortical and stromal tissue, as well as the vasculature, thus overcoming 2 of the main restrictions associated with whole-organ freezing.