64 EX VIVO CULTURE OF FRESH AND FROZEN-THAWED SHEEP WHOLE OVARIES

Abstract

Cryopreservation and retransplantation of ovarian tissue is a real option to preserve fertility in young cancer patients. However, a high risk of retransmission of malignancy exists in several tumours. In these patients, cryopreserved whole ovaries could provide an appealing source of oocytes to be grown and matured in vitro. The aim of this study was to develop a perfusion system for ex vivo culture of fresh and cryopreserved whole ovaries. Upon arrival to the laboratory, all ovaries were perfused via the ovarian artery with Ringer's solution and 10 UI L^–1 of heparin for 10 min. Ovaries to be frozen were subsequently perfused with cryoprotectant solution [L-15 medium, 10% FBS, and 1.5 M dimethyl sulfoxide (DMSO)] and then frozen using Multi Thermal Gradient freezing technology (Core Dynamics Ltd., Ness Ziona, Israel), pushing the samples along the thermal gradient (4 to –70°C) at 0.01 mm s^–1, resulting in a cooling rate of 0.3°C min^–1. Samples were thawed at 37°C and immediately perfused with L-15 medium supplemented with decreasing sucrose concentrations (0.25, 0.125, and 0 M). In a closed-circuit perfusion system, 100 mL of recirculating medium (M199, 25 mM HEPES, 1% BSA, 2 mM glutamine, and antibiotic/antimycotic) was pumped into the ovarian artery using a peristaltic pump. The flow rate through the ovary was maintained between 1 and 1.5 mL min^–1. Whole sheep ovaries were cultured at 38.5°C for 1 or 3 days. After culture, ovaries were fixed with 10% formaldehyde. Statistical analysis was performed using Student's t-test (SPSS 20, IBM Corp., Armonk, NY, USA). Morphological analysis showed that the rate of intact follicles was inversely related to the days of culture but was not affected by cryopreservation. In fact, the percentage of morphologically normal follicles in fresh and frozen ovaries cultured for 1 day (87 ± 3.4 and 83 ± 3.2%, respectively; P = 0.058) was higher (P = 0.048) than in ovaries cultured for 3 days (75 ± 2.9 and 71 ± 2.8%, respectively; P = 0.053). Cell proliferation, measured as Ki67-positive stromal cells, decreased during culture (P = 0.028) and was affected by cryopreservation both on Day 1 (13 ± 7 v. 15 ± 4%; P = 0.047) and Day 3 (10 ± 4 v. 12 ± 6%; P = 0.039). Similar results were observed for the apoptotic index that increased during culture both in fresh and cryopreserved ovaries (P = 0.028). The number of apoptotic cells per millimeter squared was lower (P = 0.031) in fresh (23 ± 10%) than in frozen ovaries (27 ± 15%) both on Day 1 and on Day 3 (30 ± 14 v. 33 ± 20%, respectively; P = 0.03). Cell viability and active endocrine function during culture is confirmed by steroid secretion, which is conserved in both fresh and cryopreserved ovaries for up to 3 days. Our results show that it is possible to culture both fresh and cryopreserved whole ovaries for up to 3 days. Although fresh ovaries, on average, did better than cryopreserved ones, we observed large individual variations, with positive and negative results overlapping between fresh and frozen samples. Further studies are in progress to explain the reason of such variations.

Supported by AIRC IG 10376, Carraresi Foundation, and by Legge 7 (R.A.S).