Title: The importance of ischemia time and tissue size in the preparation of ovarian tissue for cryopreservation.
Abstract: Objective: Recent reports on ovarian tissue banking have raised many hopes and expectations for women facing premature ovarian failure. However, little is known about optimizing procedure sequencing and the additional effects mechanical or ischemic injuries may have on ovarian tissue processed for cryopreservation. The objectives of this study were to evaluate the impact of tissue size and the processing time interval before cryopreservation (warm ischemia time) on follicular survival. Design: Animal study using a porcine model. Materials/Methods: Bilateral oophorectomy was performed in eight sows. The harvested ovaries were divided into five segments and subjected to assigned time intervals in warm ischemia (1, 5, 10, 20 and 30 min), after which one part was immediately fixed for histological evaluation (F) and the other cryopreserved according to standard protocol and after thawing evaluated in the same fashion (C). For all samples a 1mm thick cortex layer was obtained. Within the cryopreservation group one sample measuring 1-2 mm2 (C-s) and a 5 mm2 section (C-l) were taken and compared. Results: Although some histological features of the cryopreserved samples were not as well preserved, with the follicles displaying some degree of dyshesion and disorganization, overall the changes did not amount to significant autolysis. The number of primordial follicles per high magnification field were significantly reduced in the cryopreserved group (C: 4.9 ± 5.3 vs. F: 7.2 ± 5.4, p = 0.03∗), however, stratifying the cryopreserved group according to size showed a significant higher follicle count for larger tissue sections (C-l: 9.3 ± 6.5 vs. C-s: 2.1 ± 2.4, p = 0.002∗). The number of primordial follicles was not significantly changed when comparing cryopreserved tissue segments measuring 5 mm2 to sections which were fixed without cryopreservation (C-l: 9.3 ± 6.5 vs. F: 7.2 ± 5.4, NS). Changes in the percentage of primary and secondary follicles were not significant. The time factor had no additional effect on any of the described changes in the cryopreserved group, nor did increasing exposure to warm ischemia (up to 30 min) result in significant damage to the freshly fixed samples (Figure 1). Conclusions: Handling larger ovarian cortex pieces facilitates cryopreservation as well as autotransplantation procedures. Our results show that cryopreserved pieces measuring 5 mm2 maintain more of their histological features compared to smaller pieces subject to more mechanical damage. Although the cryopreserved ovarian tissue displayed subtle histological changes, these did not amount to significant cell injury. Processing time up to 30 minutes will not have significant damaging effects on the ovarian tissue. Supported By: Minimally Invasive Surgery Center, Cleveland Clinic Foundation.