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

  • Fertility Preservation
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

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.

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Acknowledgements

We would like to thank all the surgical team in the department of gynecology, CHU Estaing, Clermont-Ferrand (France) for providing ovarian tissue for research, Dr Benoît Sion for his critical recommendations on this study and Mrs Elizabeth Petit for language revision of the manuscript. Thanks are also due to Mr Eric Pernet (CICE) for advice and technical assistance.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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Authors and Affiliations

  1. 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

    Sandra Sanfilippo, Michel Canis, Christine Artonne, Laurent Janny & Florence Brugnon

  2. Centre International de Chirurgie Endoscopique (CICE), 28 Place Henri Dunant, 63001, Clermont-Ferrand, Cedex 1, France

    Sandra Sanfilippo, Michel Canis & Revaz Botchorishvili

  3. CHU Clermont-Ferrand, Gynécologie obstétrique, Estaing, 1 Place Lucie Aubrac, 63058, Clermont-Ferrand, Cedex 1, France

    Michel Canis & Revaz Botchorishvili

  4. Laboratoire de biostatistiques, télématique et traitement d’image, Université Clermont 1, UFR Médecine, 28 Place Henri Dunant, 63001, Clermont-Ferrand, Cedex 1, France

    Lemlih Ouchchane

  5. CHU Clermont-Ferrand, Biologie et médecine de la reproduction, AMP-CECOS, Estaing, 1 Place Lucie Aubrac, 63058, Clermont-Ferrand, Cedex 1, France

    Laurent Janny & Florence Brugnon

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  1. Sandra Sanfilippo
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  2. Michel Canis
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  5. Christine Artonne
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Corresponding author

Correspondence to Sandra Sanfilippo.

Additional information

Capsule Trypan blue appears to be a more reliable staining method for follicle viability evaluation compared with calcein AM/ethidium homodimer-1

Supported by: an industrial doctoral grant (Convention Industrielle de Formation par la Recherche, CIFRE) with the Centre International de Chirurgie Endoscopique (CICE), France (Grant No: 176/2009).

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Sanfilippo, S., Canis, M., Ouchchane, L. et al. Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ethidium homodimer-1). J Assist Reprod Genet 28, 1151–1156 (2011). https://doi.org/10.1007/s10815-011-9649-y

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  • DOI: https://doi.org/10.1007/s10815-011-9649-y

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