Direct comparative analysis of conventional and directional freezing for the cryopreservation of whole ovaries  Sara Maffei, M.Sc., Maike Hanenberg, M.Sc.,

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Direct comparative analysis of conventional and directional freezing for the cryopreservation of whole ovaries  Sara Maffei, M.Sc., Maike Hanenberg, M.Sc., Georgia Pennarossa, Ph.D., José Roberto V. Silva, D.V.M., Ph.D., Tiziana A.L. Brevini, D.Pharm., Ph.D., Amir Arav, D.V.M., Ph.D., Fulvio Gandolfi, D.V.M.  Fertility and Sterility  Volume 100, Issue 4, Pages 1122-1131 (October 2013) DOI: 10.1016/j.fertnstert.2013.06.003 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Morphologic analysis of ovarian cortical and medullar regions in fresh control and frozen–thawed ovaries. (A) Overall distribution of morphologically normal follicles in CTR and in DF or CF groups after thawing. (B, C) Representative pictures of the medullar region of the different experimental groups (B) and the quantitative results of image analysis (C). Values with different indices (a–b) are significantly different (P<.05). Fertility and Sterility 2013 100, 1122-1131DOI: (10.1016/j.fertnstert.2013.06.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A, B) Representative pictures of blood vessels in the different experimental groups classified in three diameter categories (A) and their distribution in the medulla and cortex regions (B). Values with different indices (a–b) are significantly different (P<.05). Scale bars = 100 μm. Fertility and Sterility 2013 100, 1122-1131DOI: (10.1016/j.fertnstert.2013.06.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Functional assessment of cortical fragments cultured in vitro for 7 days. (A) Distribution of primordial, intermediate, and primary follicles at the beginning (day 0) and at the end (day 7) of the culture period. (B) Overall distribution of morphologically normal follicles during culture in the three experimental groups. (C) Quantitative evaluation of cell proliferation assessed both as Ki67 messenger RNA levels (left) and as percentage of immunopositive cells for the encoded protein (right). Values with different indices (a–c) are significantly different (P<.05). Fertility and Sterility 2013 100, 1122-1131DOI: (10.1016/j.fertnstert.2013.06.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Representative pictures of cells positive for γH2AX (marker of double-strand DNA breaks; green) and RAD51 (marker of repair ability; red). (A) Immunostaining was carried out at day 0 and day 7 of in vitro culture. (B) Quantitative assessment by image analysis. Values with different indices (a–c) are significantly different (P<.05). Scale bars = 100 μm. Fertility and Sterility 2013 100, 1122-1131DOI: (10.1016/j.fertnstert.2013.06.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions