Vitrification and xenografting of human ovarian tissue

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Vitrification and xenografting of human ovarian tissue Christiani Andrade Amorim, V.M.D., Ph.D., Marie-Madeleine Dolmans, M.D., Ph.D., Anu David, Ph.D., Jonathan Jaeger, M.Sc., Julie Vanacker, B.Sc., Alessandra Camboni, M.D., Ph.D., Jacques Donnez, M.D., Ph.D., Anne Van Langendonckt, Ph.D. Fertility and Sterility Volume 98, Issue 5, Pages 1291-1298.e2 (November 2012) DOI: 10.1016/j.fertnstert.2012.07.1109 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A–C) Preantral follicles stained with hematoxylin-eosin from frozen-thawed and vitrified-warmed fragments of ovarian tissue after 1 week of xenografting to nude mice: (A) primordial follicle from frozen-thawed tissue; (B) primary and secondary follicles from vitrified-warmed tissue (vitrification protocol 1); (C) primary and secondary follicles from vitrified-warmed tissue (vitrification protocol 2). (D–F) Ki-67 immunostaining: (D) positive staining in growing follicles from frozen-thawed grafted tissue; (E) positive staining in vitrified-warmed grafted tissue (vitrification protocol 1); (F) positive staining in and vitrified-warmed grafted tissue (vitrification protocol 2). Fertility and Sterility 2012 98, 1291-1298.e2DOI: (10.1016/j.fertnstert.2012.07.1109) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Hematoxylin-eosin (A) and Masson trichrome (B) staining on ovarian tissue after freezing, thawing, and xenografting. Fibrotic areas were evidenced by Masson trichrome strongly stained blue due to collagen fibers present in higher numbers among stromal cells. Fertility and Sterility 2012 98, 1291-1298.e2DOI: (10.1016/j.fertnstert.2012.07.1109) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Analysis of DNA strand breaks by TUNEL in grafted ovarian tissue. Pictures of DAPI counterstaining (cells in blue) and TUNEL staining (cells in red) are merged. Stromal cells in: (A) frozen-thawed tissue; (B) vitrified-warmed tissue (vitrification protocol 1); (C) vitrified-warmed tissue (vitrification protocol 2). Original magnification ×20. Fertility and Sterility 2012 98, 1291-1298.e2DOI: (10.1016/j.fertnstert.2012.07.1109) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Proportions of primordial and primary and secondary follicles in the three treatment groups (slow freezing, vitrification protocol 1, and vitrification protocol 2) after 1 week of xenografting to nude mice. a,bDifferent letters are significantly different (P<.05). Fertility and Sterility 2012 98, 1291-1298.e2DOI: (10.1016/j.fertnstert.2012.07.1109) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Fibrotic and total areas (in μm²) in the three treatment groups (slow freezing, vitrification protocol 1, and vitrification protocol 2) after 1 week of xenografting to nude mice. Fertility and Sterility 2012 98, 1291-1298.e2DOI: (10.1016/j.fertnstert.2012.07.1109) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions