Simple Prediction of the Survival of Follicles in Cryopreserved Human Ovarian Tissue

Abstract

Abstract. This study examines the possible predictive value of the LIVE/DEAD fluorescence viability assay for evaluation of survival of cryopreserved human ovarian tissue. Ovarian tissue from ten patients was examined by LIVE/DEAD viability staining before and after cryopreservation and after freezing in a -20 C freezer (negative control). After cryopreservation with a slow freezing protocol and cryoprotectant the LIVE/DEAD assay showed 86% viable follicles (an intact oocyte and at least more than 50% of the granulosa cells alive), whereas after freezing at -20 C the survival rate was 67%. The healthy follicular loss after cryopreservation was 4%, whereas with freezing at -20 C, it was 25%. Although this assay overestimates the survival rate of cryopreserved primordial follicles, if the LIVE/ DEAD assay yields greater than approximately 85% viable follicles, it can be assumed that the follicles in the cryopreserved tissue have maintained their developmental potential and that the tissue is suitable for retransplantation.

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].…”

Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ethidium homodimer-1)

“…Therefore we evaluated the reproducibility of the two staining methods used most often (TB and CaAM/EthD-1) [16][17][18][19], by an inter-observer reliability study. Our data showed excellent agreement between the two observers' results for each staining method.…”

“…To evaluate the potential of viable follicles before grafting, a reliable and rapid method is essential. Previous studies reported mainly two methods to measure the viability of these follicles: trypan blue (TB) and calcein AM/ethidium homodimer-1 (CaAM/EthD-1) [16][17][18][19]. TB is a vital dye which is only able to enter cells with compromised membranes and color them blue [16,17].…”

“…Cell-permeant CaAM is enzymatically converted into calcein in the cytoplasm of living cells, turning these fluorescent green. EthD-1, on the other hand, enters cells with damaged membranes and takes on a bright red fluorescence upon binding to nucleic acids [18,19]. Therefore this work aimed to determine the most appropriate and reliable method for measuring follicles viability (TB versus CaAM/EthD-1) to assess the quality of ovarian cryopreservation.…”