Improving ovarian tissue cryopreservation for oncologic patients: slow freezing versus vitrification, effect of different procedures and devices  Sonia Herraiz, Ph.D., Edurne Novella-Maestre, Ph.D., Beatriz Rodríguez, B.Sc., César Díaz, M.D., María Sánchez-Serrano, Ph.D., M.D., Vicente Mirabet, Ph.D., Antonio Pellicer, Ph.D., M.D.  Fertility and Sterility  Volume 101, Issue 3, Pages 775-784.e1 (March 2014) DOI: 10.1016/j.fertnstert.2013.11.016 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A, B, C, D, E, F) Histologic assessment. The structure of primordial and primary follicles are shown in fresh tissue, slow freezing (SF), vitrification protocol 1 (VT1), VT2, VT3, and VT4, respectively. Groups VT3 and VT4 show abnormal features; VT3 was characterized by an abnormal detachment of the ooplasm from the surrounding granulosa cells in the H&E-stained sections. (G) Cell death index. The apoptotic area percentage statistically significantly increased in groups VT2, VT3, and VT4 when compared with fresh ones. Note also the increase in group SF, which was not statistically significant. *P<.05 when compared with the fresh group. (H) The GAPDH gene expression assay. Tissue variability, measured as the expression levels of the GAPDH gene by quantitative PCR of VT3 samples (*P<.05), statistically significantly decreased after the cryopreservation procedure. SF showed a lowered (but no statistically significant) gene expression, same phenomenon was observed in VT4. Fertility and Sterility 2014 101, 775-784.e1DOI: (10.1016/j.fertnstert.2013.11.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Primordial follicular density. A reduced primordial population was observed after cryopreservation in both groups when compared with fresh fixed tissue (*P<.05). When cryopreserved and xenotransplanted samples were compared between groups, the primordial density was statistically significantly reduced in the slow freezing (SF) group when compared with the vitrification protocol 1 (VT1) group (¥P<.05). (B) In an SF sample, primary follicle proliferation shows Ki-67 staining in granulosa cells and oocyte nuclei. (C) In a vitrified sample, a primary follicle shows Ki-67-positive cells in granulosa cells and oocytes. (D) CD31 staining in the SF group. (E) In group VT1, the small vessels show a complete endothelial layer with positive CD31 immunostained cells. (F) Although no statistically significant differences were detected, the fibrotic area increased in the SF grafts when compared with the VT1 group. (G–I) In the SF graft containing a primordial-transitional follicle, cell nuclei were stained with DAPI (blue) to perform TUNEL. The TUNEL + signal was found in stroma cells (red), but was absent in oocytes and granulosa cells. (H) In the VT1 group, the primary follicle surrounded by cuboidal granulosa cells and stoma nuclei was stained with DAPI (blue). (J) TUNEL-positive cells were stained red in the stroma. The follicle was negative for DNA damage. Fertility and Sterility 2014 101, 775-784.e1DOI: (10.1016/j.fertnstert.2013.11.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Diagram showing the distribution of solutions and cryopreservation devices in all experimental groups of the in vitro study performed with bovine ovarian tissue. For the slow freezing (SF) group, 10% dimethyl sulfoxide (DMSO) was employed as the SF solution and an ethyl vinyl acetate (EVA) bag as the cryopreservation device. Vitrification conditions were established by combination of two vitrification solutions (VS) and two cryopreservation devices to obtain four experimental vitrification protocol (VT) groups. In the VT1 group, VS1 composed of 20% DMSO + 20% ethylene glycol (EG) was used in combination with a Cryotissue metallic grid (MG). In the VT2 group, VS2 composed of 10% DMSO, 10% EG, 10% 1,2-propanediol (PrOH), and 10% polyvinylpyrrolidone (PVP) was used in combination with an MG as the device. The VT3 group used a combination of VS1 and an EVA bag as the device. The VT4 group used a combination of VS2 and an EVA bag as the device. Fertility and Sterility 2014 101, 775-784.e1DOI: (10.1016/j.fertnstert.2013.11.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions