Restoration of fertility to oophorectomized sheep by ovarian autografts stored at-196°C

Abstract

Cortical slices were prepared from the right ovaries of six lambs and either grafted directly to the ovarian pedicles of origin or cooled slowly to liquid nitrogen temperatures in medium containing dimethylsulphoxide. Three weeks later, the contra-lateral ovary was removed and replaced with frozen-thawed slices from the same animal. Two of the animals mated during their second oestrous cycle 3-4 months later and the remainder had at least one ovulatory cycle. The pregnancies reached full-term development, one lamb being derived from an ovulation in a fresh graft and the other from a frozen-thawed graft. None of the sheep had peripheral plasma concentrations of follicle stimulating hormone or luteinizing hormone consistently in the castrate range, and only one graft was devoid of follicles when the animals were slaughtered 9 months after the operations. Grafts with primordial follicles always contained developing follicles, which occasionally attained pre-ovulatory sizes of 7 mm in diameter. A corpus albicans was present in five grafts. Since all developing follicles had degenerated 1 week after grafting in an additional ewe, the large follicles in long-term grafts had presumably commenced growing after the operation. There were no obvious differences between fresh and frozen-thawed grafts in either appearance or weight, and all had apparently grown since implantation. Despite substantial depletion of primordial follicle numbers, the results indicated that frozen storage and replacement of a patient's own ovarian tissue might be practicable when fertility potential is threatened by chemotherapy/radiotherapy.

Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure

“…Most investigators use a standard freezing protocol described more than 15 years ago for cryopreserving ovarian tissue of sheep [15]. In this protocol manual seeding is performed to induce extracellular ice formation and a very slow post-seeding freezing rate of 0.3°C/min is applied.…”

“…Moreover, antioxidant supplements such as taurine and L-glutamine in freezing media have been found to reduce cryoinjuries [4,40]. In an earlier study, follicular growth was observed after subcutaneous xenografting of human ovarian tissue cryopreserved according an original protocol using a serumfree freezing medium supplemented with PrOH and raffinose as well as antioxidants, and a faster freezing program than the usual one [15,39]. Nevertheless, further studies of the quality and functionality of thawed ovarian tissue are needed to support the feasibility of this freezing protocol in clinical settings.…”

Efficacy of ovarian tissue cryopreservation in a major European center

“…[41,42]. Nunc CryoTubes (Sigma-Aldrich, St. Louis, MO, USA) were filled with 1.7 ml Leibovitz's L-15 GlutaMAX cryomedium (Gibco, Carisbad, CA, USA), 10 % CryoSure-DMSO (Wak-Chemie Medical GmbH, Steinbach, Germany), and 10 % serum substitute supplement (SSS; Irvine Scientific, Santa Ana, CA, USA) and precooled to 2°C.…”

Is transplantation of cryopreserved ovarian tissue from patients with advanced-stage breast cancer safe? A pilot study

“…Freezing and thawing of ovarian tissue was undertaken according to the protocol described by Gosden et al [21], as detailed in previous papers [14,44]. Briefly, biopsy samples were cut into small strips (+/− 4×1-2 mm).…”