Apoptosis and ultrastructural assessment after cryopreservation of whole human ovaries with their vascular pedicle  Belen Martinez-Madrid, V.M.D., Ph.D., Alessandra Camboni, M.D., Marie-Madeleine Dolmans, M.D., Ph.D., Stefania Nottola, M.D., Ph.D., Anne Van Langendonckt, Ph.D., Jacques Donnez, M.D., Ph.D.  Fertility and Sterility  Volume 87, Issue 5, Pages 1153-1165 (May 2007) DOI: 10.1016/j.fertnstert.2006.11.019 Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Assessment of apoptosis. Analysis of DNA strand breaks by TUNEL in frozen-thawed whole ovary. (A) Primary follicle and stromal cells: (A1) DAPI counterstaining, and (A2) TUNEL staining. (B) Antral follicle: (B1) DAPI counterstaining, and (B2) TUNEL staining. (C and D) Blood vessels of different sizes: (C1 and D1) DAPI counterstaining, and (C2 and D2) TUNEL staining. Bars = 100 μm (A, C, and D), and 50 μm (B). Martinez-Madrid. Freezing whole human ovaries. Fertil Steril 2007. Fertility and Sterility 2007 87, 1153-1165DOI: (10.1016/j.fertnstert.2006.11.019) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Assessment of apoptosis. Immunohistochemical detection of active caspase-3 in frozen-thawed whole ovary. (A) Arteriole negative for active caspase-3 staining. (B) Vein negative for active caspase-3 staining. (C) Corpus luteum with luteal cells positive for active caspase-3 staining. (D) Antral follicle with GCs positive for active caspase-3 staining. Bars = 100 μm (A and B), and 50 μm (C and D). Martinez-Madrid. Freezing whole human ovaries. Fertil Steril 2007. Fertility and Sterility 2007 87, 1153-1165DOI: (10.1016/j.fertnstert.2006.11.019) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Primary follicle with well-preserved features in frozen-thawed whole ovary. (A) The oocyte (O) is surrounded by a single layer of cuboidal follicular cells (Fc) on a continuous basal membrane (bm). Note the mitochondria (m) clustered in clouds in the oocyte cytoplasm, and the nucleolus (nu) visible in the oocyte nucleus (N). *Two stromal cells are visible close to the basal membrane. (B) Detail of a rounded mitochondrion with a pale matrix and peripheral cristae in the oocyte cytoplasm. (C) Detail of close interdigitations observed between follicular cell (Fc) projections and oocyte (O) microvilli. Note a desmosome connecting two follicular cells (arrow). (D) Detail of follicular cells, showing indented nuclei with peripheral patches of heterochromatin and numerous rod-shaped mitochondria (m) in the cytoplasm. bm = continuous basal membrane. Bars = 1 μm (A), 0.25 μm (B), 0.43 μm (C), and 0.62 μm (D). Martinez-Madrid. Freezing whole human ovaries. Fertil Steril 2007. Fertility and Sterility 2007 87, 1153-1165DOI: (10.1016/j.fertnstert.2006.11.019) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Primordial follicle with altered features in frozen-thawed whole ovary (in total, 3.3% of primordial and primary follicles showed ultrastructural alterations). In the oocyte (O), note rupture of the nuclear membrane and condensation of chromatin (arrow), and among the follicular cells (Fc), one altered follicular cell with condensation of chromatin (arrowhead). N = oocyte nucleus. Bar = 2.27 μm. Martinez-Madrid. Freezing whole human ovaries. Fertil Steril 2007. Fertility and Sterility 2007 87, 1153-1165DOI: (10.1016/j.fertnstert.2006.11.019) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Blood vessels showing normal ultrastructure in frozen-thawed whole ovary. (A) Venule with flattened endothelial cells. (B) Arteriole containing thickened endothelial cells protruding into the lumen, with abundant cytoplasmic processes toward either the lumen or the basal membrane. The cytoplasm is filled with organelles and intact connections between endothelial cells. (C) Detail of mitochondria (m) with intact membranes from an endothelial cell. N = endothelial cell nucleus. L = vascular lumen. Bars = 1.56 μm (A), 1 μm (B), and 0.3 μm (C). Martinez-Madrid. Freezing whole human ovaries. Fertil Steril 2007. Fertility and Sterility 2007 87, 1153-1165DOI: (10.1016/j.fertnstert.2006.11.019) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 6 Arterioles with a few altered endothelial cells in frozen-thawed whole ovary (in total, 3.7% of endothelial cells showed ultrastructural alterations). (A) Separation of intercellular connections. *An endothelial cell with nuclear and cytoplasmic condensation (arrow) in an arteriole. (B) Swollen endothelial cell with electron-translucent cytoplasm in an arteriole. L = vascular lumen. Bars = 2.2 μm (A) and 0.75 μm (B). Martinez-Madrid. Freezing whole human ovaries. Fertil Steril 2007. Fertility and Sterility 2007 87, 1153-1165DOI: (10.1016/j.fertnstert.2006.11.019) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions