ESPE2023 Poster Category 1 Fetal, Neonatal Endocrinology and Metabolism (34 abstracts)
Steroid secretion and morphological aspects of fetal adrenal before/after freezing/thawing and 14 days in organotypic culture
Lucie Renault 1,2 , Elsa Labrune 1,2 , Sandrine Giscard d'Estaing 1,2 , Valeska Bideault 1,2 , Grégoire Schneider 1 , Pierre-Yves Mure 1,2 , Enzo Lalli 3,4 , Mabrouka Doghman-Bouguerra 3,4 , Frédérique Dijoud 1,2 , Hervé Lejeune 1,2 & Ingrid Plotton 1,2
1Hospices Civils de Lyon, Lyon, France. 2Université Claude Bernard, Lyon, France. 3Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France. 4Université Côte d’Azur, Valbonne, France
Introduction: The human fetal adrenals (HFA) produce high levels of steroids. The gland is distinguishable from the 7th gestational week and can be separated in two zones: the fetal zone in the center which correspond of 80 % of the gland and the definitive zone in the periphery. At this time of the development, neural crest cells are reaching the adrenal primordium, producing catecholamines. A third zone, the transitional zone appears later in the early 2nd trimester. The objective of our study is to evaluate the functionality of adrenals after freezing, thawing and cultured in “hanging drops” for 14 days.
Tissues and Methods: Adrenal tissues from surgical elective abortions at 9-13 weeks of gestation was cut in several fragments of 1mm3 and cultured in “hanging drops” either straight after dissection or after vitrification or slow freezing and thawing in a drop of 40 µl of media containing ACTH 1ng/ml or not. Twelve steroids were measured by LC-MS/MS in medium recovered all along the culture and the expression of CYP17 was explored by immuno-histo-chemistry (IHC) after 2 weeks of 3D culture. Histology was compared between fresh tissue, cultured tissue for 14 days after vitrification, slow freezing or without cryopreservation.
Results: Four fetuses have been used for adrenal vitrification and slow freezing. Steroidogenesis is persistent after 2 weeks in organotypic culture and ACTH stimulate cortisol and corticosterone secretion. After vitrification or slow freezing procedure, steroid secretion was similar. Analysis is in progress to compare the IHC results between the first and the second week of culture and between the tissues that have been frozen and those that have not. The histological architecture is similar after 14 days of culture between the different techniques and is similar to those cultured without cryopreservation. Nevertheless, we observe an alteration of the tissues in comparison with the fresh tissues.
Conclusion: We showed that 1er trimester fetal adrenals can maintain functional activities and produce steroids comparable to fresh tissue after vitrification or slow freezing and 2 weeks in organotypic culture. Adrenals, as gonads, derivate from the mesoderm. Indeed, the optimization of these techniques could help us to enhance the freezing/thawing protocols of immature testicular tissue in the context of fertility preservation of prepubescent youth.
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