DEVELOPMENT OF SECRETOR AND GESTATIONAL PATIENT-DERIVED ORGANOIDS FROM ADENOMYOSIS DISEASE AS AN IN VITRO MODEL TO STUDY ADENOMYOSIS-RELATED INFERTILITY AND PERSONALIZED DRUG SCREENING

To develop organoids from human endometrium of adenomyosis patients and differentiate them to secretory phase and gestational endometrium for studying mechanisms involved in infertility and pregnancy disorders associated with adenomyosis. This in vitro model would allow the identification of therapeutic targets to improve implantation and effectively avoid miscarriages and consequently, improve ongoing pregnancy and live birth rates in these patients. Human endometrial biopsies from adenomyosis patients (n=6) were digested to isolate epithelial glandular fraction, which was embedded into Matrigel droplets and cultured with Expansion Medium (ExM) containing supplements to achieve the formation of proliferative endometrial organoids. To recapitulate secretory phase, ExM was supplemented with E2, P4 and cAMP, while human pregnancy hormones (hPL and PRL) were added to supplemented ExM to promote differentiation to gestational endometrium. To characterize patient-derived organoids, PanCytokeratin, Vimentin, Laminin and Ki67 expression, PAS staining and Muc-1 secretion were evaluated by immunohistochemistry (IHC). To confirm organoid differentiation, PAEP secretion, acetylated α-tubulin and SOX9 expression were evaluated by IHC and SPP1, PAEP, LIF and 17HSDß2 by qRT-PCR. Proliferative, secretory and gestational organoids recapitulated in vivo glandular epithelial phenotype characterized by PanCytokeratin presence, absence of stromal marker Vimentin and epithelial endometrial secretions (Muc-1 and PAS), epithelial polarity (laminin presence at the basolateral side) and cell proliferation (Ki67 expression). Distinctive features of secretory and gestational endometrium, such as PAEP secretion and cilia presence (acetylated α-tubulin expression) were identified in secretory and gestational organoids but not in proliferative organoids. The expression of the progenitor cell marker SOX9 was lower in secretory and gestational organoids compared to proliferative organoids, while differentiation markers (SPP1, PAEP, LIF and 17HSDß2) expression was higher. Here, we established for the first time organoids derived from endometrium of adenomyosis patients, which recapitulate in vivo endometrial tissue features, as well as respond to hormones reproducing secretory phase and gestational endometrium.