During morphogenesis 2D epithelial tissue undergo architectural changes to form 3D structures called folds. Epithelial folding is essentially origami where a 2D sheet of paper can be transformed via folding and sculpting into a 3D structure. Folding is a key phenomenon during embryogenesis and organogenesis and is essential for several physiological functions. For example, folds in the stomach (rugae) and intestine (crypts) increase surface area for nutrient absorption and in the brain increase cortical surface area for neural processing. The uterine luminal epithelium in mammals including humans, horses and rodents undergoes structural changes to form folds. Although improper uterine folding in horses results in pregnancy failure, the precise role of folds in embryo implantation remains unknown. Using a focused time course around implantation in the mouse model, we uncover dynamic changes in the 3D uterine structure with the entire lumen forming pre-implantation transverse folds along the mesometrial-antimesometrial axis. During embryo-implantation the mouse uterine lumen forms a second structure called an implantation chamber. Whether uterine folds transform into implantation chambers has not been investigated. Using quantitative 3D methods, we show that uterine transverse folds are formed prior to spacing of embryo clusters whereas chambers form once an embryo begins to attach to the uterus and thus are two distinct structures. We also show that transverse folds resolve to form a flat implantation region before an embryo arrives in the center of this region (the implantation site) to form a post-implantation chamber. Although, it is known that the mouse embryo aligns itself along the uterine mesometrial-antimesometrial axis, our data suggests that the implantation chamber facilitates embryo rotation to enable embryo-uterine axes alignment. Further, using mice deficient in WNT5A and RBPJ as models of aberrant uterine folding, we show that embryos get trapped in longitudinal folds leading to embryo-uterine axes misalignment, abnormal chamber formation and defective post-implantation embryo morphogenesis. Further, we show that increased estrogen signaling and reduced progesterone signaling lead to aberrant longitudinal folds. Finally, we extend our findings to examine the effects of excess estrogen signaling on folding during hyperstimulation - a clinical procedure performed during In Vitro Fertilization (IVF) to increase egg numbers for higher success rate of implantation and pregnancy. In women, pregnancies following hyperstimulation often lead to preterm birth, placental abnormalities, and other complications. Our findings suggest that hyperstimulation in mice leads to longitudinal folds and downstream pregnancy loss due to embryo trapping. Such research can be potentially used to improve pregnancy outcomes following IVF and fresh embryo transfer. Our mouse models with disrupted uterine folds provide an opportunity to understand uterine structure-based mechanisms crucial for implantation and pregnancy success. In addition to fueling future research on endometrial folds in humans, our research will open up new avenues for the treatment of infertility and provide new targets for diagnosis based on uterine 3D structure.
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