Replacement of sodium with choline in slow-cooling media improves human ovarian tissue cryopreservation  Riccardo Talevi, Vincenza Barbato, Valentina.

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Replacement of sodium with choline in slow-cooling media improves human ovarian tissue cryopreservation  Riccardo Talevi, Vincenza Barbato, Valentina Mollo, Ilaria Fiorentino, Cristoforo De Stefano, Fabio Maria Guarino, Roberto Gualtieri  Reproductive BioMedicine Online  Volume 27, Issue 4, Pages 381-389 (October 2013) DOI: 10.1016/j.rbmo.2013.07.003 Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Human primordial/primary follicle grading of grade-1 (A, D, G, J), grade-2 (B, E, H, K) and grade-3 (C, F, I, L) human ovarian follicles. (A–C) Histology, haematoxylin/eosin staining. (D–L) Immunohistochemical localization of apoptotic markers: p53 (D–F), p21 (G–I) and Apaf-1 (J–L) immunohistochemical labelling. Bar=10μm. Reproductive BioMedicine Online 2013 27, 381-389DOI: (10.1016/j.rbmo.2013.07.003) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Distribution of grade-1–3 follicles in fresh and cryopreserved ovarian tissue. C=fresh control; P-Na=PrOH-Na-based slow cooling; P-Ch=PrOH-choline-based slow cooling; D-Na=DMSO-Na-based slow cooling; D-Ch=DMSO-choline-based slow cooling. ∗P<0.05 versus fresh control; ∗∗P<0.01 versus fresh control. Reproductive BioMedicine Online 2013 27, 381-389DOI: (10.1016/j.rbmo.2013.07.003) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Transmission electron micrographs of fresh (A, B) and cryopreserved (C–J) ovarian tissue slow cooled with PrOH-Na (C, D), PrOH-choline (E, F), DMSO-Na (G, H) and DMSO-choline (I, J). Asterisks indicate swollen mitochondria; arrowheads indicate degenerated mitochondria. Bars=10μm (A, C, E, G), 2μm (B, D, F, H, J), 3μm (I). Reproductive BioMedicine Online 2013 27, 381-389DOI: (10.1016/j.rbmo.2013.07.003) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Proportion of damaged mitochondria in oocytes in fresh and cryopreserved ovarian tissue. C=fresh control; P-Na=PrOH-Na-based slow cooling; P-Ch=PrOH-choline-based slow cooling; D-Na=DMSO-Na-based slow cooling; D-Ch=DMSO-choline-based slow cooling. ∗P<0.05 versus fresh control; ∗∗P<0.01 versus fresh control; ##P<0.01 versus same protocol with sodium-based media. Reproductive BioMedicine Online 2013 27, 381-389DOI: (10.1016/j.rbmo.2013.07.003) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Transmission electron micrographs of fresh (A, D) and cryopreserved (B, C, E, F) ovarian stroma in samples slow cooled with PrOH-Na (b), PrOH-choline (e), DMSO-Na (c) and DMSO-choline (f). Asterisks indicate intermediate stages of stromal cell necrosis. Bars=10μm (A), 2μm (B–F). Reproductive BioMedicine Online 2013 27, 381-389DOI: (10.1016/j.rbmo.2013.07.003) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions