Bioprinted Hormone-Responsive Bilayer Model of Human Endometrium for Embryo Implantation Studies

Implantation failure is a major challenge in reproductive medicine, with two-thirds of cases attributed to poor uterine receptivity. Current models have limited utility in capturing the complexities of the endometrium. This study introduces a novel bioprinted endometrial model with epithelial and stromal cells in a bilayer structure, designed to replicate the hormone-regulated endometrial environment and support embryo implantation studies. Alginate-based bioink formulations, cross-linked with calcium chloride or calcium gluconate, were optimized for 3D cell bioprinting, based on key parameters: reduced spreading ratio, lower printed line width standard deviation (SD), slower degradation rates, and enhanced cell viability. Human endometrial epithelial (RL95-2) and stromal (T HESCs) cell lines were encapsulated in the bioink and bioprinted in a bilayer structure: Clear stratification mimicking the layered architecture of native endometrium was confirmed using fluorescent microscopy. Sequential hormonal treatments with estradiol (proliferative phase), followed by estradiol and progesterone (secretory phase) highlighted the model's hormone-responsiveness. Estradiol significantly enhanced cell viability by day 2, while progesterone reduced cell viability by day 5, consistent with adaptation to the proliferative and secretory phases. Hormone-treated constructs displayed significantly lower E-cadherin expression, higher mRNA expression of various integrins and of vascular endothelial growth factor (VEGF), and reduced metalloproteinase (MMP)-2 secretion after 5 days, mirroring in vivo endometrial remodeling under progesterone influence. JAR spheroids, representing human blastocyst cells, adhered to and infiltrated the epithelial layer of the hormone-treated, bilayered model, effectively simulating embryo implantation. This bioprinted bilayer endometrial model represents a significant advancement in reproductive biology. It offers a platform for studying in vitro endometrial receptivity and implantation and paves the way for personalized treatment approaches in recurrent implantation failure.