The use of CAM to observe early changes after ovarian tissue transplantation has a significant advantage over in vitro models, since it allows the investigation of mechanism affecting the early vascularization and follicle loss process. This technique is inexpensive, and can be performed without any risk of graft rejection due to the natural embryo immunodeficiency for the first 17 days. Moreover, this approach does not raise any ethical or legal concerns in terms of European law.
The best strategy to develop the manual skills needed for egg manipulation may be reached by training with non-embryonated eggs before the actual experiment. To begin, label the day zero eggs received from Louvain selected white leghorn chicks using a marker. Incubate the eggs in an incubator set at 37 degrees celsius, with the pointed ends facing down.
Rotate the eggs by 180 degrees, two to three times daily manually. In the dark, place an egg candler against the eggshell and pinpoint the center of the air pocket on the eggshell using a marker. Turn on the lights and rotate a sterile straight pin at the marked position to make an opening of around one millimeter.
Locate the yolk sack in the dark using the egg candler. Puncture the egg with a sterile 19 gauge needle, angled at 45 degrees toward the bottom of the egg, without disrupting the yolk sack. Aspirate 1.5 to two milliliters of the albumin to detach the CAM from the shell.
Then close the hole with adhesive tape. Turn on the lights and draw a rectangular window measuring one by 1.5 centimeters on the horizontally placed egg. Hold the egg in one hand, and gently cut a window into the eggshell, using a scroll saw blade, without cracking the shell.
Blow regularly to remove shell dust and debris. Slide sterile forceps under the rectangular piece of shell, and remove it cleanly without damaging the CAM. Then discard the white outer shell membrane to see the embryo and the CAM.
In viable embryonated eggs, clear albumin, a vascular ring around the embryo, and sometimes a beating heart, are seen on day three of chick embryo development. Under the laminar flow hood, place the egg on the egg rack with the window facing up and peel off the tape. Gently place and immediately remove a one centimeter square strip of sterile ether-extracted lens paper onto the epithelial surface, to traumatize a small area of the CAM.
Then grasp one frozen thawed ovarian cortical strip with microsurgical forceps, and place it onto the traumatized CAM with the medullary side against the CAM. Evaluate the graft macroscopically, and pay particular attention to the vascular reaction of the CAM toward the graft before capturing digital photographs or videos. Once done, harvest the graft by grasping the tissue or the surrounding CAM with forceps, and carefully excising the graft from the CAM using scissors.
The grafts were viable in 100%of cases after the first day of the grafting. They were already partially adherent to the CAM, and showed the wheel spoke pattern of blood vessels needed for their vascularization. After six days of grafting, the implants were adhered to the CAM.
Around day three post-transplantation, the grafts were covered with a second layer of CAM and became encapsulated. The graft showed good vascularization, with 80%of the transplants penetrating the egg. Two grafts did not attach to the CAM and took on a necrotic appearance, resulting in a grafted tissue survival rate of 83%after six days.
In the histological evaluation of the transplant, healthy follicles were observed at all time points. After the transplantation, an insignificant decrease was observed in the follicle densities. The follicle survival rates were maintained during the grafting period at 95%on day one, and 83%on day six.
The most challenging part is making the small hole required to aspirate the albumin, to detach the CAM from the eggshell, before creating a window applying. Too much pressure can result in overstimulation, or may even crack and destroy the egg, causing irrevocable damage to the CAM or its vasculature. This model is ideal for testing several aspects of the early ovarian tissue post-grafting period, including the impact of pro-angiogenic factors, protective antioxidant agents, cytokines, hormones, and other factor controlling follicle activation and early growth.
This model indicated the link between early vascularization and modulation of follicle activation, which are the two main events resulting in the ovarian reserve decline after ovarian tissue transplantation.
