Development of decidualized and 3D human endometrial models for the study of human implantation and endocrine pathologies.

CARNOVALE N; OVIEDO MF; GANIEWICH D; OPPENHEIMER F; LEIROS G; OLIVARES C; BILOTAS M; MERESMAN G

Congreso; International Federation of Placenta Associations 2019 (IFPA2019), 8th Latin American Symposium on Maternal-Fetal Interaction and Placenta (VIII SLIMP); 2019

ObjectiveThe aims of this study were to develop an in-vitro 3D-system, imitating the human endometrium architecture, and to optimize an in-vitro decidualization model.Methods3D-endometrial cell system was developed by seeding human endometrial stromal cells (t-HESC) into bovine type-I collagen as scaffold. After gelation, human endometrial epithelial cells (ECC-1) were added and incubated for 7 days. 3D-systems were formalin fixed, paraffin embedded and haematoxylin-eosin stained. Cytokeratin expression was assessed by immunohistochemistry. T-HESC were grown to 80% confluence in DMEM/F12+10% charcoal-stripped Fetal Bovine Serum (FBS) or in DMEM/F12+10% FBS. Decidualization was induced with 0.5mM cAMP plus 10−6M medroxy-progesterone acetate (MPA) with or without 10−8M estradiol, for 7 or 9 days. Every third day, conditioned media were collected and culture medium and stimuli were refreshed. Experimental and control cell cultures were daily photographed. Finally, cells were harvested for RNA extraction for further analysis.ResultsIn 3D-system, t-HESC incorporated into the scaffold, elongated and spread-out. Clear contraction of collagen by stromal cells was observed as a result of its proliferation. Also, a typical stratified-like layer of epithelial cells, some of them attempted to penetrate the stromal matrix, indicated the beginning of spontaneous formation of glands. Epithelial cell origin was confirmed by cytokeratin positive immunostaining.More typical decidualized cells were observed after 7 days of stimulation of t-HESC with cAMP, MPA and estradiol in DMEM/F12+10% charcoal-stripped FBS, compared with other experimental conditions or control. The following evaluation of decidualization markers will confirm this finding.Conclusion: A novel model of endometrial 3D-system was developed: collagen scaffold provided an adequate environment for cell growth, and gland formation was stimulated.Also, a protocol of artificial decidualization of t-HESC using supplemented charcoal-stripped FBS medium was optimized. These models could be important tools for studying the mechanisms involved in the human implantation process and the impact of endocrine pathologies on endometrial health.