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

PURPOSE: To assess the reliability of trypan blue (TB) and calcein AM/ethidium homodimer-1 (CaAM/EthD-1) staining to evaluate the viability of fresh and thawed human ovarian follicles. METHODS: Isolated follicles from fresh and thawed cortex were stained using TB versus CaAM/EthD-1 methods (n = 10 patients). Measurements were performed by two independent observers. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements were tested by the Wilcoxon test. RESULTS: Inter-observer reliability was excellent for each method. Nevertheless, it was even better with the TB method (ICC = 0.83) compared with CaAM/EthD-1 (ICC = 0.75). Moreover, the ICCs for viability measurements using the two methods were good for each observer (observer 1: ICC = 0.49; observer 2: ICC = 0.40). CONCLUSION: Compared with CaAM/EthD-1, TB appears to be more reliable as a staining method for follicle viability evaluation. TB staining is a quick and useful method, complementary to histological analysis for quality control in ovarian tissue cryopreservation.