Endometrium on-a-chip reveals insulin- and glucose-induced alterations in the transcriptome and proteomic secretome.

The molecular interactions between the maternal environment and the developing embryo that are key for early pregnancy success and are influenced by factors such as maternal metabolic status. Our understanding of the mechanism(s) through which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success is, however, limited. Here we report, for the first time, the use of an endometrium-on-a-chip microfluidics approach to produce a multi-cellular endometrium in vitro. Isolated endometrial cells (epithelial and stromal) from the uteri of non-pregnant cows in the early-luteal phase (Day 4-7), were seeded in the upper chamber of the device (epithelial cells; 4-6 10 4 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 10 4 cells/mL). Exposure of cells to different concentrations of glucose (0.5, 5.0 or 50 mM) or insulin (Vehicle, 1 or 10 ng/mL) were performed at a flow rate of 1µL/min for 72 hr. Quantitative differences in the cellular transcriptome and the secreted proteome of in vitro-derived uterine luminal fluid (ULF) were determined by RNA-sequencing and Tandem Mass Tagging Mass Spectrometry (TMT-MS), respectively. High glucose concentrations altered 21 and 191 protein-coding genes in epithelial and stromal cells, respectively (p<0.05), with a dose-dependent quantitative change in the protein secretome (1 and 23 proteins). Altering insulin concentrations resulted in limited transcriptional changes including transcripts for insulin-like binding proteins that were cell specific but altered the quantitative secretion of 196 proteins. These findings highlight one potential mechanism by which changes to maternal glucose and insulin alter uterine function.