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


CONCLUSIONS
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.
Laboratoire de biologie du développement et de la reproduction, Université Clermont 1, UFR Médecine, E.A. 975, 28 Place Henri Dunant, 63001, Clermont-Ferrand, Cedex 1, France. sanfilipposandra@gmail.com
Sandra
S
Sanfilippo
Michel
M
Canis
Lemlih
L
Ouchchane
Revaz
R
Botchorishvili
Christine
C
Artonne
Laurent
L
Janny
Florence
F
Brugnon
D000328:Adult; D004396:Coloring Agents; D015925:Cryopreservation / Q000379:methods*; D004996:Ethidium / Q000031:analogs & derivatives*; D005260:Female; D005452:Fluoresceins; D006801:Humans; D006080:Ovarian Follicle; D015203:Reproducibility of Results; D014022:Tissue Survival; D014343:Trypan Blue
D004396:Coloring Agents; D005452:Fluoresceins; C085925:calcein AM; C018533:ethidium homodimer; D004996:Ethidium; D014343:Trypan Blue