P-442 A new option to thaw slow-frozen human ovarian tissue in cancer patients: efficacy and safety of the combination of different cryopreservation kits
Authors: Canosa S , Parmegiani L , Evangelista F , Durando S , Salvagno F , Sestero M , Cimadomo D , Vaiarelli A , Rienzi L , Ubaldi F.M , Bongioanni F , Revelli A , Gennarelli G 
Published: June 2022

Abstract

Study question
Is follicular viability of slow-frozen human ovarian tissue preserved if rapid thawing is performed using a solution containing extracellular cryoprotectant only?

Summary answer
Follicular viability is preserved even using thawing solutions containing extracellular cryoprotectant only, combining kits with different composition.

What is known already
Ovarian tissue cryopreservation is an alternative option to oocytes or embryos for fertility preservation in cancer patients facing gonadotoxic treatments. To date, each brand producing kits approved for the slow-freezing of human ovarian tissue recommends the use of its own thawing kit. However, a potential single protocol based on the use of any extracellular cryoprotectant has already been proposed for human oocytes and embryos. The current study aims at finding alternative options to thaw cryopreserved human ovarian tissue when the original kit was withdrawn from the market and only one CE-marked kit was available, even with different composition in cryoprotectants.

Study design, size, duration
Ovarian tissue cryopreservation of ten cancer patients (18.3 ± 7.6 years) undergoing fertility preservation between 2001 and 2012 was performed following a slow-freezing protocol with 1.5M 1,2 PROH and 0.5M Sucrose. Once deceased, for each patient, cortical fragments were prospectively thawed and equally allocated into two groups: i) fragments thawed using 0.5-1M 1,2 PROH and 0.3M Sucrose (PROH+S Group, n = 73); ii) fragments thawed following an adjusted protocol with 0.5M Sucrose only (S Group, n = 73).

Participants/materials, setting, methods
Post thawing follicular density/mm2, integrity (%) and the presence of interstitial oedema were assessed by histological and ultrastructural analysis performed after formalin fixation and haematoxylin/eosin staining. Follicular viability was evaluated by the expression of markers of proliferation (Ki67) and of vascularization (CD31) by immunohistochemistry after a 24h culture in Iscove’s modified Dulbecco’s medium at 37 °C and 6% CO2. A paired comparison was performed referring to the fresh tissue of the same patient as control.

Main results and the role of chance
The histological evaluation performed after thawing revealed that follicles were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. A significant reduction of follicular density per mm2 was observed in both study groups (14.2 ± 12.0 vs. 15.1 ± 14.0 for PROH+S and S Group, respectively; p = 0.4) compared to the fresh tissue (27.2 ± 31.6; p = 0.04) as well as a remarkable decreased of the proportion of intact follicles (39.3 ± 17.1 vs. 25.5 ± 9.8; p = 0.2) compared to the fresh tissue (98.1 ± 1.4; p = 0.002). Thawed samples equally showed interstitial oedema and increased stromal cell vacuolization and chromatin clumping. Ki67 positive staining of active proliferating cells revealed a comparable proportion of viable follicles between thawed samples (46.3 ± 20.8 vs. 28.3 ± 27.9 for PROH+S and S Group, respectively; p = 0.2). Finally, the expression of the endothelial marker CD31 in the thawed samples suggested an equivalent number of blood vessels per mm2 (43.8 ± 34.3 vs. 41.7 ± 44.8; p = 0.6).

Limitations, reasons for caution
Single centre study with a limited sample size. Only 24h of in vitro culture was assessed. The use of the freezing medium corresponding to the Sucrose only solution was not tested. Clinical outcomes after ovarian tissue transplantation should be evaluated before drawing final conclusion.

Wider implications of the findings
First evidence of the feasible application of a “Universal Warming” protocol, irrespective of brand and cryoprotectants, for the rapid thawing of slow-frozen human ovarian tissue. IVF centres would be provided with alternative options to thaw ovarian tissue for restoring reproductive potential in cancer patient undergoing ovarian transplantation.

Trial registration number
Not applicable