The authors thank Mira Hryniuk, BA, for reviewing the English language of the article.

The use of human tissue was approved by the Institutional Review Board of the Catholic University of Louvain. The patients gave their written informed consent for the use of their ovarian tissue for research purposes.
1. Ordering day 0 eggs that are highly likely to be embryonated
<ol>
	<li>Find a certified laboratory-grade Lohman-selected white Leghorn egg supplier that reports high rates of embryonated eggs, which is primarily dependent on the age of the chicks.</li>
</ol>...

The most challenging part of the protocol described here is making the small hole required to aspirate the albumen in order to detach the CAM from the eggshell prior to creating a window. Applying too much pressure can result in overpenetration or may even crack and destroy the egg, causing irrevocable damage to the CAM and its vasculature. To keep mistakes to a minimum during initial attempts to separate the CAM, it is strongly advised to practice making small holes in the eggshell of non-fertilized, grocery-bought eggs...

The demand for fertility preservation for oncological and benign indications, as well as social reasons, has dramatically increased over recent decades. However, various treatments used to cure malignant and non-malignant diseases are highly toxic to the gonads and can result in iatrogenic premature ovarian insufficiency, ultimately leading to infertility1. Established techniques for fertility preservation include embryo cryopreservation, immature or mature oocyte vitrification, and ovarian tissue cryopreservation2,3,4. Ovarian tissue freezing is the only available option for preserving fertility in prepubertal girls or women who require immediate cancer therapy. The restoration of endocrine function following ovarian tissue transplantation occurs in over 95% of subjects, with live birth rates ranging from 18% to 42%5,6,7,8,9.
Although the transplantation of frozen-thawed ovarian tissue has proven successful, there is still room for improvement. Indeed, as ovarian cortical fragments are transplanted without vascular anastomosis, they experience a period of hypoxia during which graft revascularization takes place10,11,12. The vast majority of studies investigating human ovarian tissue transplantation have used a xenografting model, in which ovarian tissue is transplanted to immunodeficient mice. The complete revascularization of the xenografts takes around 10 days, with both the host and graft vessels contributing to the formation of functional vessels12,13,14. Around 50%-90% of the follicle reserve is lost during this hypoxic window before the completion of graft revascularization10,15,16. It has been strongly suggested that this massive follicle loss occurs through both direct follicle death, as demonstrated by a decrease in the absolute follicle numbers left after grafting, and the activation of primordial follicle growth, as indicated by changes in follicle proportions towards increased rates of growing follicles17,18.
Interestingly, previous research works using various animal ovarian tissues grafted to chick chorioallantoic membrane (CAM), which has a constitution mimicking the typical grafting site of the peritoneum, have reported the inhibition of spontaneous follicle activation, with the primordial follicle reserve staying intact for up to 10 days19,20,21,22. Our team&#160;previously demonstrated that the grafting of frozen-thawed human ovarian tissue to CAM constituted a reliable approach for investigating human ovarian tissue transplantation in its first ischemic stages23&#160;and recently showed that this grafting method was able to counteract follicle activation24.
The CAM model is especially appealing not only because eggs are much cheaper than mice but also because of the highly vascularized nature of CAM, allowing scrutiny of the association between follicle activation and ovarian graft revascularization. The avian system is, indeed, one of the most common and versatile ways of studying angiogenesis25. Chick embryo development (ED) takes 21 days until hatching, and the CAM is formed within the first 4-5 days through the fusion of the allantois and chorion26. Notably, the chick embryo is a naturally immunodeficient host until day 17 of ED, so xenografting experiments can be performed without any risk of graft rejection27,28. Moreover, the CAM model approach does not raise any ethical or legal concerns in terms of European law29, making it an attractive alternative to other animal models. With regard to breeding conditions, chick embryos only need an incubator set at 37 &#176;C with a relative air humidity of 40%-60%. These limited experimentation requirements significantly reduce the research costs compared to use of immunodeficient mice.
The protocol presented herein aims to describe the development of a CAM xenografting model for human ovarian tissue and provide specific insights into the effectiveness of the technique, the time frame of graft revascularization, and the tissue viability over a 6 day grafting period. This protocol could be of great interest for investigating the mechanisms behind early post-grafting follicle loss and studying the impact of several agents (growth factors, hormones, etc.) on this phenomenon.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agani hypodermic needle, 19 G</td>
			<td>Terumo Europe</td>
			<td>AN*1950R1</td>
			<td>19 G needle to aspirate albumen</td>
		</tr>
		<tr>
			<td>Terumo syringe, 5 mL concentric Luer lock</td>
			<td>Terumo Europe</td>
			<td>SS*05LE1</td>
			<td>5-mL sterile syringe</td>
		</tr>
		<tr>
			<td>Caseviewer v2.2</td>
			<td>3DHISTECH</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Merck Chemicals</td>
			<td>603-022-00-4</td>
			<td>Sterile ether to traumatize the CAM</td>
		</tr>
		<tr>
			<td>Eosin Y aqueous solution 0.5%</td>
			<td>Merck</td>
			<td>1098441000</td>
			<td>Staining solution</td>
		</tr>
		<tr>
			<td>Formaldehyde 4% aqueous solution buffered (Formalin 10%)</td>
			<td>VWR</td>
			<td>97139010</td>
			<td>Formaldehyde used for tissue fixation</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Liebherr</td>
			<td>7081260</td>
			<td>Fridge at 4 &#176;C used for paraffin-embedding</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Schott</td>
			<td>SLK2</td>
			<td>Hot plate used to dry the slides</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Forma Scientific 3111</td>
			<td>10365156</td>
			<td>Oven used for slide incubation</td>
		</tr>
		<tr>
			<td>Leica CLS 150 XE microscope cold light source</td>
			<td>Leica</td>
			<td>CLS 150 XE</td>
			<td>Focal cold light source to candle the eggs</td>
		</tr>
		<tr>
			<td>Lens cleaning tissue, grade 541</td>
			<td>VWR</td>
			<td>111-5003</td>
			<td>Tissue to soak in sterile ether to traumatize the CAM</td>
		</tr>
		<tr>
			<td>Mayer&#39;s hematoxylin</td>
			<td>MERCK</td>
			<td>1092491000</td>
			<td>Staining solution</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20847307</td>
			<td>Methanol</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>ThermoScientific-MICROM</td>
			<td>HM325-2</td>
			<td>Microtome</td>
		</tr>
		<tr>
			<td>Pannoramic P250 Flash III</td>
			<td>3DHISTECH</td>
			<td>/</td>
			<td>Slide scanner at 20x magnification</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>1,04,00,51,000</td>
			<td>Paraffin-embedding solution</td>
		</tr>
		<tr>
			<td>Paraplast Plus R</td>
			<td>Sigma</td>
			<td>P3683-1KG</td>
			<td>Paraffin</td>
		</tr>
		<tr>
			<td>Petri dish, 60x15 mm, sterile</td>
			<td>Greiner</td>
			<td>628161</td>
			<td>Sterile petri dish</td>
		</tr>
		<tr>
			<td>Pin holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td>Pin holder</td>
		</tr>
		<tr>
			<td>Polyhatch</td>
			<td>Brinsea</td>
			<td>CP01F</td>
			<td>Egg incubator with automatic rotator</td>
		</tr>
		<tr>
			<td>Scroll saw blade, 132 mm</td>
			<td>Sencys</td>
			<td>/</td>
			<td>Saw blade to create a window in the eggshell</td>
		</tr>
		<tr>
			<td>Stainless steel insert pins</td>
			<td>Fine Science Tools</td>
			<td>26007-02</td>
			<td>Straight pin to make a hole in the eggshell</td>
		</tr>
		<tr>
			<td>Steril-Helios&#160;</td>
			<td>Angelantoni Industrie</td>
			<td>ST-00275400000</td>
			<td>Laminar flow hood</td>
		</tr>
		<tr>
			<td>Superfrost Plus bords rod&#233;s 90&#176;</td>
			<td>VWR</td>
			<td>631-9483</td>
			<td>Glass slides&#160;</td>
		</tr>
		<tr>
			<td>Tissue-Tek VIP 6Al</td>
			<td>Sakura</td>
			<td>60320417-0711 VID6E3-1</td>
			<td>Automatic embedding device</td>
		</tr>
		<tr>
			<td>Titanium forceps</td>
			<td>Fine Science Tools</td>
			<td>11602-16</td>
			<td>Forceps for eggshell removal and ovarian tissue manipulation</td>
		</tr>
		<tr>
			<td>Toluene, pa</td>
			<td>VWR</td>
			<td>28701364</td>
			<td>Paraffin-embedding solution</td>
		</tr>
	</tbody>
</table>

the chick chorioallantoic membrane (cam) model as a tool to study ovarian tissue transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining&#160;natural immunodeficiency up to day 17 postfertilization, making it ideal to study&#160;short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

Chick embryo survival rates 
The embryo survival rate from windowing (day 3 of&#160;ED) to ovarian tissue grafting (day 7 of ED) was 79% (33/42). Since the percentage of embryonated day 0 eggs is unknown, supernumerary day 0 eggs from Lohman-selected white Leghorn chickens were ordered to ensure sufficient embryonated eggs would be available for grafting. A total of 23 viable day 7 eggs were used for grafting, one of which perished during the first 24 h, resulting in an overall embryo survival rate ...

Watch this Scientific Journal Video about The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation at JoVE.com

The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Here, we describe a protocol for developing a chick chorioallantoic membrane (CAM) xenografting model for human ovarian tissue and demonstrate the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle ...

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.

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551 Views.  Université Catholique de Louvain. 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 b...