Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation

Abstract

Cryopreservation as a process can be divided into two methods: conventional freezing and vitrification. The high effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos is shown, whereas data on human ovarian tissue are limited. The aim of this study was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Ovarian tissue fragments from 15 patients were transported to the laboratory within 22-25 h in a special, isolated transport box that can maintain a stable temperature of between 5 and 8 8C for 36 h. Small pieces of ovarian tissue (0.3-1!1-1.5!0.7-1 mm) were randomly distributed into three groups: group 1, fresh pieces immediately after receiving transport box (control); group 2, pieces after vitrification; and group 3, pieces after conventional freezing. After thawing, all the pieces were cultured in vitro. The viability and proliferative capacity of the tissue by in vitro production of hormones, development of follicles, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression after culture were evaluated. A difference between freezing and vitrification was not found in respect to hormonal activity and follicle quality. The supernatants showed 17-b estradiol concentrations of 365, 285, and 300 pg/ml respectively, and progesterone concentrations of 3.82, 1.99, and 1.95 ng/ml respectively. It was detected that 95, 80, and 83% follicles respectively were morphologically normal. The molecular biological analysis, however, demonstrated that the GAPDH gene expression in ovarian tissue after vitrification was dramatically decreased in contrast to conventional freezing. For cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification, because of higher developmental potential.

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

“…Indeed, aromatase activity, which is required for the synthesis of E2, is present only from secondary follicular stages onwards [17]. Our data are in agreement with previous findings showing a correlation between E2 secretions and follicular growth in cultures of frozen/thawed tissues [2,21,24]. PCNA is a nuclear protein that plays an essential role in cell cycle regulation [50].…”

Efficacy of ovarian tissue cryopreservation in a major European center

“…The observed percentages of tissue viability impairment as a result of cryopreservation and thawing are roughly consistent with the 70-80 % follicle survival reported earlier for various slowfreezing protocols. [8,27,29,30,35,38,39] In addition, we observed a 24-27 % reduction in overall tissue viability using the glucose uptake assay. This decreased glucose uptake indicates a reduced metabolism of the tissue during culture as a result of cryodamage.…”

“…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 steroid hormone production has earlier been associated with ovarian tissue damage. [30,37,[54][55][56], The clinical relevance of measuring steroid hormone production is severely restricted. Namely, as developing follicles produce larger amounts of steroid hormones than primordial and primary follicles, [57] whereas one specifically aims to preserve the more numerous small follicles that have the ability to mature after autotransplantation.…”

“…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].…”