Dolmans, Marie-MadeleineCamboni, AlessandraStratopoulou, Christina AnnaEl Grari, InesInesEl Grari2025-05-142025-05-142025-05-142024https://hdl.handle.net/2078.2/36686The enigmatic pathogenesis and elusive mechanisms of endometriosis and adenomyosis remain unresolved challenges, affecting millions of women worldwide. The absence of definitive treatments highlights the need to unravel the complexities of these diseases. In this study, we explored various hypotheses using endometrial samples and innovative 3D cell culture models. Our focus was mostly on adenomyosis-related infertility, looking to identify proteins and structures (pinopodes) associated with the window of implantation (WOI), which are impacted in both endometrial samples and assembloid cultures. Organoids and assembloids share comparable properties, both mimicking physiological conditions. These models therefore form the foundation for the ongoing part of the infertility investigation, examining whether endometrial assembloids can serve as an advanced, robust preclinical model for adenomyosis-related impaired endometrial receptivity. Firstly, endometrial specimens were collected from patients diagnosed with adenomyosis and healthy subjects. IHC was applied to assess various receptivity-associated proteins in endometrial samples, including glycodelin, OPN, HOXA10, LIF, PR and ITGB3. They were then used to generate organoids representing endometrial epithelial glands and stromal cells in culture, contributing to the formation of novel structures known as assembloids. The primary challenge at this stage was to develop an optimized protocol for endometrial assembloid generation. Thereafter, some of these 3D structures were treated with E2, P4 and cAMP to replicate the targeted mid-secretory phase inspected under a SEM. A significant reduction in stromal expression was observed in 4 receptivity-associated proteins in adenomyosis patients compared to disease-free subjects upon analysis of endometrial samples: OPN (p=0.0301), HOXA10 (p=0.0198), LIF (p=0.0381), and PR (p=0.004). In terms of their maturation stage, pinopodes exhibited notable differences in shape and ciliation status between a healthy 31-year-old woman and a 40-year-old adenomyosis patient, despite being in the same menstrual cycle phase. Dysregulation of specific signaling pathways observed in case of adenomyosis appears to result in reduced expression of proteins, particularly HOXA10 and PR, potentially contributing to infertility. In the same context, variations in pinopode morphology between adenomyosis and healthy subjects might be correlated with expression of HOXA10, a biomarker associated with their development. In adenomyosis patients, HOXA10 showed weaker expression, in line with the regressive nature and reduced ciliation of pinopodes. Further analyses of assembloids are needed to establish whether they are a suitable model to mimic the infertile endometrial environment. In the future, an evaluation of expression and secretion of protein-associated endometrial receptivity will also be conducted using ELISA. Upon full optimization of our assembloid protocol, we began scrutinizing a potential in vivo murine model that can replicate the physiopathological development of both endometriosis and adenomyosis using organoids and assembloids.AdenomyosisInfertilityAssembloidsImmunohistochemistryScanning electron microscopytext::thesis::master thesisthesis:44258