Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant  Mona Sheikhi, M.Sc., Kjell Hultenby, Ph.D., Boel Niklasson, R.N.M., Monalill Lundqvist, Ph.D., Outi Hovatta, M.D., Ph.D.  Fertility and Sterility  Volume 100, Issue 1, Pages 170-177.e2 (July 2013) DOI: 10.1016/j.fertnstert.2013.03.018 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A–F) Morphology of follicles within human ovarian cortical tissues stained with hematoxylin and eosin, showing normal morphology of primordial, primary, and secondary follicles within vitrified tissues both (A, B, C) in noncultured samples and (D, E, F) after 24 hours in culture. (A, D) Fresh; (B, D) vitrified with dimethyl sulfoxide (DMSO), 1,2-propanediol (PrOH), and ethylene glycol (EG); (C, F) vitrified with only EG. Scale bars: A, B, D, E, F = 50 μm; C = 20 μm. (G–K) Immunohistochemical detection of active caspase-3 in human ovarian tissue (before 24 hours in culture). Active caspase-3 staining showed negative signal in granulosa cells in primary, primordial, and transitional primary follicles. No apoptosis was observed in stromal cells in either (G) fresh tissue or tissue vitrified (H) with DMSO, PrOH, and EG or (I) with only EG. (K) There was no positive signal observed in negative control tissue, and (J) there were three granulosa cells shown in a mouse early antral follicle as a positive control. Scale bars: G, H, I, J = 10 μm; K = 25 μm. Fertility and Sterility 2013 100, 170-177.e2DOI: (10.1016/j.fertnstert.2013.03.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Scores of oocytes (OC), granulosa cells (GC) in follicles, and stroma (SC) within human ovarian tissue evaluated by transmission electron microscopy. Diagrams represent scores of follicles within either noncultured (top) or 24-hour cultured (bottom) vitrified tissue: fresh (control) and vitrified with DMSO, PrOH, and EG or with only EG. The results are presented as fraction of the maximum score of 1. ∗There was a significant difference between the scores of oocytes in the tissue vitrified with DMSO, PrOH, and EG compared with fresh tissue after culture (P=.02). Abbreviations as in Figure 1. Fertility and Sterility 2013 100, 170-177.e2DOI: (10.1016/j.fertnstert.2013.03.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A–F) Transmission electron microscopy of human ovarian follicles within cortical tissues that were (A, D) fresh or vitrified (B, E) with DMSO, PrOH, and EG or (C, F) with only EG. (A, B, C) A primordial follicle contained an oocyte and a single layer of flattened granulosa cells, surrounded by stromal cells. (D, E, F) The nuclei (N) of the oocytes in fresh tissue and tissue vitrified after the use of either cryoprotectant solution showed normal chromatin with a clearly distinguished inner and outer nuclear membrane. The round-shaped mitochondria (M) and Golgi complexes were the most abundant organelles in the OC. (D, E, F) Swollen mitochondrial cristae were occasionally seen in vitrified tissue. The nuclei of the GC in (D) fresh and (E, F) vitrified tissues contained normally distributed chromatin. The contact between OC and GC was well preserved (arrows). In all groups, the stromal tissue contained abundant collagen fibers (CF) and spindle-shaped SC, with no difference between the tissues. Scale bars: A, B, C = 10 μm; D, E, F = 5 μm. (G–L) Transmission electron microscopy of human ovarian follicles within tissues after 24 hours in culture in (G, J) fresh tissue and tissue vitrified (H, K) with DMSO, PrOH, and EG or (I, L) with only EG. Overviews of a (G) transitional primary, (H) primordial, and (I) primary follicles with OC, GC, and SC are shown. (J, K, L) The contacts between OC and GC were well preserved (arrows). Scale bars: G, H, I = 10 μm; J, K, L = 5 μm. Abbreviations as in Figures 1 and 2. Fertility and Sterility 2013 100, 170-177.e2DOI: (10.1016/j.fertnstert.2013.03.018) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions