Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum

Abstract

Chemotherapy and/or radiotherapy protocols have improved the long-term survival of cancer patients. Frequent consequences of antiblastic treatments, used to eradicate malignancies, are the partial loss of ovarian function, which in children and young women can result in permanent sterility. Ovarian tissue cryopreservation implemented before the beginning of treatment may potentially restore fertility. However, the physical effects of cryopreservation can damage oocyte survival and decrease follicular cell integrity and stromal preservation. The aim of this study was to examine the effects of different concentrations of 1,2-propanediol (PROH) and sucrose as cryoprotectants and human serum as protein support. Particular concentrations tested were 1.26, 1.5 and 1.08 mol/l PROH, 0.175, 0.2, 0.224 and 0.3 mol/l of sucrose and 20%, 30% and 40% human serum in the freezing solutions and normal or raised sucrose concentrations in the dilution solutions. Ovarian cortical slices from 13 patients, aged 5-38 years, were cryopreserved using slow freezing-rapid thawing. Tests were conducted using light and transmission electron microscopy. Cryo-damage occurred predominantly in the stromal and follicular cells. The best preservation of morphological characteristics was obtained using the freeze-thaw protocol in which concentrations of cryoprotectants were among the lowest (1.26 mol/l PROH+0.175 mol/l sucrose) with 30% human serum.

Efficacy of ovarian tissue cryopreservation in a major European center

“…Although various large centers for cryopreservation of ovarian tissue exist, [12,13,15,17,18,21,24] to the best of our knowledge, cohort studies in which the impact of currently used cryopreservation/ thawing protocols on both follicle and stromal cell survival in young cancer patients is measured have not been published before. Earlier studies considering the impact of slow freezing techniques other than evaluated here, generally focused on follicle viability only [27][28][29][30][31][32][33][34][35][36][37][38] and often described patient populations without an indication for fertility preservation (e.g. patients applying for a sterilization, cystectomy, or caesarean section) [8, 27-29, 32, 34, 35, 38].…”

“…Although there are studies that quantify the impact of slow-freezing on human ovarian tissue viability, these studies do not per definition focus on the protocols that are currently being used by the major centers. [27][28][29][30][31][32][33][34][35][36][37][38] Moreover, most of these studies did not take the viability of the stromal cell compartment into account. [27][28][29][30][31][32][33][34][35][36][37][38] This compartment, however, is essential for post-autotransplantation neovascularization, follicle survival, and life span of the ovarian graft [39] and is considered to be more sensitive to ischemic and cryoinjury than primordial follicles [8,40].…”

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

“…The use of PrOH in clinical freezing protocols gives rise to the birth of healthy babies following cryopreservation of both embryos and mature oocytes [3,26,44]. Human ovarian tissue cryopreserved with PrOH as cryoprotectant exhibits a satisfactory morphological preservation of follicles in comparison with fresh tissue [11,14]. Moreover, Abir et al [1] reported better survival and development of follicles in grafts when fetal ovarian tissue was cryopreserved using PrOH compared to dimethylsulfoxide (DMSO).…”

“…Although we observed a higher percentage of stroma cells with DNA fragmentation after thawing, this effect remains slight compared with a previous report [49] since it concerns only 16.6 % of stroma cells. The explanation for this observation is most likely linked to the higher sensitivity of these cells to cryoinjuries [11,28]. A better understanding of the cellular pathways activated by the freezing/ thawing process would enable better optimization of preservation of stroma cells, whose functions are important for follicular development [47].…”

“…Despite encouraging results [9], adverse effects on structure and functionality of human ovarian tissue have been reported after cryopreservation [41]. Previous studies reported the beneficial effects of 1,2-propanediol (PrOH) in ovarian tissue cryopreservation [11,14] and raffinose for cold storage of various human tissues before graft [29,51]. Moreover, antioxidant supplements such as taurine and L-glutamine in freezing media have been found to reduce cryoinjuries [4,40].…”