Chick embryo chorioallantoic membrane (CAM) model: a useful tool to study short-term transplantation of cryopreserved human ovarian tissue  Belen Martinez-Madrid, V.M.D., Ph.D., Jacques Donnez, M.D., Ph.D., Anne-Sophie Van Eyck, M.D., Almudena Veiga-Lopez, V.M.D., Ph.D., Marie-Madeleine Dolmans, M.D., Ph.D., Anne Van Langendonckt, Ph.D.  Fertility and Sterility  Volume 91, Issue 1, Pages 285-292 (January 2009) DOI: 10.1016/j.fertnstert.2007.11.026 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Macroscopic aspect of chorioallantoic membrane (CAM) and frozen-thawed human ovarian tissue grafted to it. (A) Rectangular window made in the egg shell of a 3-day-old chick embryo. (B and C) Grafting onto traumatized CAM: (B) diagram of the technique; (C) frozen-thawed human ovarian tissue graft (1 mm3; arrow) placed manually by microsurgical forceps onto the traumatized CAM of a 10-day-old chick embryo. (D and E) Grafting beneath the CAM: (D) diagram of the technique; (E) frozen-thawed human ovarian tissue graft (1 mm3; arrow) placed manually by microsurgical forceps beneath the CAM of a 10-day-old chick embryo between the CAM and the yolk sac (ys). (F–I) Frozen-thawed human ovarian graft (arrow) placed onto traumatized CAM, 5 days after grafting. Note adhesion between the CAM and the ovarian graft and vascularization of the CAM of a 15-day-old chick embryo. (F) Chick embryo (ce) and yolk sac (ys) can be seen under the CAM; (G) note the wheel-spoke pattern of CAM blood vessels around the ovarian graft; (H) detail of the ovarian graft retrieved with the area of adherent CAM; (I) retrieval of the ovarian graft with the area of CAM adherent to it. Fertility and Sterility 2009 91, 285-292DOI: (10.1016/j.fertnstert.2007.11.026) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Viability and histology of cryopreserved human ovarian tissue grafted to chorioallantoic membrane (CAM). (A and B) Viability assessment by live-dead staining (viable cells stained green with calcein-AM and dead cells stained red with ethidium homodimer I; original magnification ×200): (A) high survival rate of stromal cells and follicles (arrow) in ovarian tissue retrieved 5 days after grafting to CAM; (B) live neovessels in ovarian tissue retrieved 3 days after grafting to CAM. (C–H) Histologic evaluation by hematoxylin-eosin staining: (C) section of an ovarian graft with CAM adherent to it: in the left inset, note CAM proliferation and invasion of the outer boundaries of the graft, compared with normal CAM thickness (right inset; original magnification ×50); (D) three zones can be seen in the ovarian graft: an outer layer of fibrosis (fib), a core of healthy tissue (h), and some inner spots of necrosis (nec); (E) neovascularization (inset) resulting from invasion of the ovarian graft stroma by avian blood vessels (D and E: original magnification ×100); (F) detail of the presence of nucleated avian erythrocytes in vessels, showing that the vessels are of avian origin; (G) healthy primordial follicle (arrow) in healthy ovarian stroma close to a fibrotic area (F and G: original magnification ×200); (H) an area with a high degree of fibrosis where the only cellular structure still present is a follicle (original magnification ×400). Fertility and Sterility 2009 91, 285-292DOI: (10.1016/j.fertnstert.2007.11.026) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions