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To date, 13 live births have been reported worldwide that testify the promising future of cryopreservation and transplantation of human ovarian tissue. However, ischaemic stress post transplantation could affect the follicular population. It is therefore essential to ameliorate the angiogenic process following grafting with the aim of improving the experimental procedures. Xenografting of human ovarian tissue in immunodeficient mice is currently one of the best models to study vascularisation of ovarian tissue.
Recent studies in our laboratory has shown the presence of both murine host blood vessels and preexisting human vessels contributing to the progressive reperfusion of the graft following xenotransplantation of frozen-thawed human ovarian tissue.
The objective if this work is to characterize the human-murine vascular network established after xenografting of frozen-thawed human ovarian cortex. Our study therefore, aims to evaluate the relative contribution of the human and murine vessels to the formation of new vascular network, and their implication in the vascularisation process and follicular growth.
The kinetic analysis of the early revascularization of the graft shows that vascular density of the graft gradually increases from day 3 to day 21 post-transplantation. Double immunostaining of CD-34 specific for human and murine blood vessels showed a significant contribution of the human vessels following 21 days of ovarian xenotransplantation, implicating the presence of human angiogenic process. This specific study of human vessels indicates an increase of the proliferative index of human endothelial cells from day 5 till day 9 post-transplantation and a turnover of basal state by day 21. We observed a linear increase in pericytes from day 9 till day 21, indicating a stabilization of the human blood vessels.
Also after 6 months of grafting, the human-murine vascular network still persists and vascular density of the ovarian stroma is equivalent to that of a native ovary. Analysis of vascularisation in the theca layer of late secondary follicles showed the presence of both human and murine blood vessels from the stroma recruited by these growing follicles to ensure their vascularisation.
In conclusion, this study shows that vessels from the host but especiaaly preexisting vessels of the fragment are able to from a vascular network similar to that observed in an ungrafted ovary and this, un spite of the cryopreservation of the fragment and the hypoxia generated by the avascular graft. And also vascular network persists up to 6 months following grafting, recruiting by the late secondary follicles Les 13 naissances mondiales reportées à ce jour témoignent de l’avenir prometteur de la technique de cryopréservation et de greffe du cortex ovarien. Cependant, le stress ischémique lié à la greffe avasculaire présente un effet délétère sur la population folliculaire. Il est donc indispensable d’approfondir la compréhension des processus angiogéniques post-greffe pour le tissu ovarien dans le but d’améliorer les protocoles opératoires. Ces études de vascularisation utilisent classiquement la souris comme modèle de xénogreffe de tissu ovarien humain.
Les travaux récents du laboratoire de gynécologie ont démontré qu’après xénogreffe du cortex ovarien humain cryopréservé, les vaisseaux murins de l’hôte et les vaisseaux humains préexistants du tissu greffé prennent part tous deux au phénomène de reperfusion progressif du greffon.
L’objectif de ce mémoire est de caractériser le réseau vasculaire humain-murin établi après xénogreffe de cortex ovarien humain cryopréservé. Notre étude vise à évaluer la contribution relative des vaisseaux humains et murins à la formation du réseau vasculaire post-greffe, à étudier leur devenir et leur implication dans la vascularisation des follicules en croissance.
L’analyse cinétique de la revascularisation précoce du greffon montre que la densité vasculaire du greffon augmente progressivement du jour 3 au jour 21 post-transplantation. Le double marquage immunohistochimique anti-CD34 spécifique humain au 21ème jour post-greffe, suggérant dès lors l’implication d’un processus angiogénique humain. L’étude spécifique des vaisseaux humains indique une augmentation de l’index de prolifération des cellules endothéliales humaines à partir du jour 5 jusqu’au jour 9 post-transplantation pour retourner à un état basal au jour 21. A partir du jour 9 jusqu’au jour 21, une augmentation de la couverture péricytaire est observée, indiquant une stabilisation des vaisseaux humains.
Arpsè 6 mois de greffe, le réseau vasculaire humain-murin persiste et la densité vasculaire du stroma ovarien est équivalente à celle du tissu antif. L’analyse individualisée de la vascularisation thécale des follicules secondaires tardifs montre que tant les vaisseaux humains que murins du stroma peuvent être recrutés par ces follicules en développement pour assurer leur vascularisation.
En conclusion, cette étude montre que les vaisseaux de l’hôte mais surtout les vaisseaux préexistants du fragment sont capables de former un réseau vasculaire histologiquement similaire à celui de l’ovaire non greffé et ce, malgré la cryopréservation du tissu et l’hypoxie engendrée par la greffe avasculaire. De plus, ce réseau vasculaires persiste jusqu’à 6 mois de greffe et peut être recruté par les follicules secondaires tardif

Fetal Tissue Transplantation --- Transplantation, Heterologous --- Cryopreservation --- Ovarian Follicle --- Angiogenesis Inducing Agents

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Frozen sections are performed while a patient is undergoing surgery as a basis for making an immediate diagnosis that will impact treatment decisions. Frozen section diagnosis is often a highly demanding situation for the pathologist who must render a diagnosis quickly for the patient and surgeon. The Frozen Section Library series will provide concise, user-friendly, site specific handbooks that are well illustrated and highlight the pitfalls, artifacts and differential diagnosis issues that arise in the hurried frozen section scenario. Timothy Craig Allen, MD, JD, is the Chairman of the Department of Pathology, University of Texas Health Science Center at Tyler, Tyler, Texas. Philip T. Cagle, MD, is Professor of Pathology, Weill Medical College of Cornell University, New York and Director of Pulmonary Pathology, The Methodist Hospital, Houston, Texas.

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