3D human endometrium from noninvasively retrieved primary endometrial cells is a reliable model to mimic the implantation window

Abstract Study question Can human endometrial organoids (hEOs) from a noninvasive source (menstrual blood-MB) mimic the physiology of a receptive endometrium Summary answer Endometrial organoids from MB functionally respond to stimuli and express markers of receptivity and can be modulated. What is known already 3D models are considered a new step in promoting precision medicine and an advanced tool for studying endometrial biology, endometrial-associated diseases, and understanding the complex mechanisms that comprise endometrial-embryo crosstalk. So far, both 2D and 3D models are usually obtained from endometrial biopsy. The recent discovery of molecules crucial for successful embryo implantation has offered researchers valuable insights in this field. However, important questions about the molecular mechanisms that drive this process remain to be deciphered. Study design, size, duration hEOs are usually obtained from endometrial biopsy, in this case a noninvasive source(MB) was used to isolate endometrial epithelial (Ep) and stromal (hESC) cells. The hEOs were generated to study the morphological changes that occur in vivo during the menstrual cycle, particularly in the window of implantation (WOI). The hEOs obtained from MB were exposed to hormones treatments to mimic WOI and were also treated with the progesterone blocker mifepristone to reverse the decidualization process. Participants/materials, setting, methods 3D hEOs were prepared from 6 healthy volunteers and cultured for 4 days in expansion medium supplemented with 10-8 M E2 + 10-6 M P4 and 500 uM cAMP. As a control, hEOs were treated with the progesterone blocker mifepristone to reverse the decidualization process. Finally, hEOs were washed with PBS and fixed for immunofluorescent staining or for scanning-electron-microscopy (SEM) or stored at -80 °C for gene expression analysis. Main results and the role of chance In this study, we were able to establish a reliable 3D model using both epithelial and stromal cells with a less invasive approach to mimic the mid-secretory phase. Immunofluorescence staining of vimentin (marker of stromal cells) and cytokeratin 19 (marker of epithelial cells) confirmed that organoids are formed by both Ep and hESCs. Using SEM, we closely observed the luminal surface of hEOs in proliferative phase, where the inner side of Ep cells was characterized by the presence of pinopods, a specific marker of the implantation window. In addition, hEOs express PP14, an implantation marker that has been shown to be significantly up-regulated during WOI. Finally, the expression of FKBP5, regulated by cAMP, and ZBP16, regulated by progesterone, increased significantly during decidualization, while their expression was up-regulated at dawn by mifepristone, a progesterone blocker, thus confirming that hEOs are an effective model of WOI. Limitations, reasons for caution All results need to be validated in a larger cohort and further implemented with other cell types (e.g., endothelial and immune cells) to implement this model for drug testing and therapy Wider implications of the findings Effective and reliable modeling of embryonic implantation is needed to mimic the cascade of molecular events that occurs in vivo. This model can be used for personalized medicine without the invasiveness of biopsy. Trial registration number Not applicable