101 Use of decellularized extracellular matrix as scaffold to create a three-dimensional endometrium

Maintaining pregnancy requires a suitable uterine environment, involving various cells such as those of epithelium, stroma, immune system, and endothelium. This complex communication is challenging to replicate in two-dimensional (2D) cultures, but can be partially recreated using a three-dimensional (3D) co-culture system. In this study, we validated the use of decellularized endometrial extracellular matrix (dECM) to produce a 3D endometrium designed to support embryo development. Bovine endometrium was decellularized with SDC 4% and DNase I to produce 20 mg mL⁻¹ bioinks. To test embryo biocompatibility, Day 3 IVF embryos were incubated with the dECM for 96 h. Proteomics (n = 5) and mechanical properties (nanoindentation; n = 3) were also performed. Epithelial glands and stroma cells were isolated by digestion (HBSS, 0.1% collagenase V, 2.5% dispase, and 1% DNase I), embedded in the dECM, and co-cultured for 14 days. Hydrogels without cells were also prepared. In the first experiment, we tested the influence of hormonal stimulation on the gland spheroid area and RNaseq. At Day 4 (D4) of culture (D0 of experiment), the culture medium was supplemented with 200 pg of E2 and 4.5 ng of P4, and on D1, with 3 pg of E2 and 4.5 ng of P4. These additions were followed by 3 pg of E2 and 7.5 ng of P4 on D2 and D3, and 3 pg of E2 and 15 ng of P4 from D3 to D9. A control group without hormones was also evaluated. Cells were imaged at each time point (n = 6 per group), and at D9, cells were fixed for immunocytochemistry (ICC) and RNaseq (n = 3). In the second experiment, five D7 blastocysts were added to culture wells, with or without cells, and cultured until D9. A control 2D well without cells was used. At D9, hatching was evaluated, and hydrogels were collected for ICC and RNaseq (n = 3). Data were analyzed in R, using a generalized linear mixed model and a Tukey post-hoc test. The dECM matrisome presented 86.7% collagens, 3.17% glycoproteins, and 6.37% proteoglycans (n = 6); it also had different isoforms of laminin. dECM had similar stiffness to native tissue (2.5 vs. 3.1 kPa, respectively; P > 0.05), with comparable elastic and viscoelastic properties. dECM was biocompatible with embryo development and supported spheroid culture for over 13 days without passaging. On D4, D7, and D9, the spheroid area of hormone-treated samples was smaller than the control, regardless of embryo presence. The endometrial co-culture model responded to hormonal stimulation, showing 541 upregulated and 238 downregulated genes, which were related to immune response modulation, secretory activity, and vascular remodeling. Embryo co-culture in the endometrial model had increased hatching rate at D9, compared with the control (67% vs. 20%; P < 0.005). Moreover, it also responded to embryo presence with 482 upregulated and 85 downregulated genes, which were part of interferon response cascades, PARP signaling, and implantation. Here, we present the use of dECM as a natural scaffold for creating a 3D endometrium. The dECM promoted cell viability and differentiation, supported long-term spheroid growth, and was biocompatible with embryo development. Notably, this endometrial co-culture model was responsive to hormonal stimulation and the presence of embryos, further validating its potential for reproductive applications.