RESUMEN
STUDY QUESTION: What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh? SUMMARY ANSWER: Ovarian cells from fresh medullary tissue, which can be isolated in larger numbers, show higher viability and are able to improve graft vascularization. WHAT IS KNOWN ALREADY: In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells. STUDY DESIGN, SIZE, DURATION: Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay. MAIN RESULTS AND THE ROLE OF CHANCE: Cryopreservation decreased ovarian cell yield (-2804 cells/mg, P = 0.015) and viability (-9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P < 0.001) with higher viability (+18.23%, P < 0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2.47-fold increase in human and total vascular surface area, respectively. P-values were adjusted for multiple testing using the Benjamini-Hochberg method to achieve a 10% false discovery rate and adjusted P-values < 0.1 were therefore considered significant. LIMITATIONS, REASONS FOR CAUTION: Pilot study involving a limited number of experiments. WIDER IMPLICATIONS OF THE FINDINGS: Knowing that fresh medullary tissue is the best source of stromal cells is important for construction of the artificial ovary, as isolated follicles require structural support and a rich vascular network for their survival and development. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (5/4/150/5 and 7.4518.12F), Fonds Spéciaux de Recherche, Fondation Saint Luc and Foundation Against Cancer, and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any conflicting interests to declare.