Endometrium On-a-Chip Reveals Insulin- and Glucose-induced Alterations in the Transcriptome and Proteomic Secretome

Abstract

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.

The Roles of Extracellular Vesicles and Organoid Models in Female Reproductive Physiology

“…These organs-on-a-chip also have been reported in bovine and canine species, utilizing an ‘oviduct-on-a-chip’ model [ 17 , 18 ] and in bovine, utilizing an ‘endometrium-on-a-chip’ model [ 19 ]. The cow oviduct-on-a-chip was used to support in vitro fertilization (IVF) and early embryo development [ 17 ].…”

“…This model demonstrated biomimicry of this condition, with appropriate changes in cellular morphology and genetic expression. A bovine ‘endometrium-on-a-chip’ was designed to study how insulin and glucose affect endometrial function [ 19 ], and the authors found that high glucose altered gene and protein expression of the stromal and epithelial endometrial cells. Thus, ‘organ-on-a-chip’ systems can be a powerful tool to study female reproductive physiology and recapitulate the dynamic pathological changes that occur in vivo.…”

The importance of optimal body condition to maximise reproductive health and perinatal outcomes in pigs

“…Metabolic disorders associated with obesity such as hyperleptinemia and insulin resistance may lead to a harmful uterine environment during early pregnancy. Glucose and insulin play a pivotal role as mediators in the expression of genes that modulates uterine environment, and it is crucial to assure their optimum concentration to support embryo development (85) . Early pregnancy of obese Iberian sows is characterised by substantial changes in the availability of triacylglycerol and cholesterol and in the steroidogenic activity of their conceptuses (86) .…”

Insulin exposed endometrial epithelial cells cultured in a microfluidic device alters transcripts involved in translation that may contribute to reduced implantation capacity of the endometrium

“…Each treatment was loaded into a 5mL syringe and put in 37°C incubator overnight and loaded onto the syringe pump on the day of microfluidics run. The syringe pump and microfluidics system were placed into a 37°C incubator and the syringe pump was set to flow at the rate of 1µL/min in order to mimic secretion in vivo (as previously described; [18]).…”

“…Chen et al subsequently used insulin-resistant animal models driven by high-fat diet and Ishikawa cells (human endometrial adenocarcinoma cells) to create in vitro implantation models and reported that insulin resistance reduces receptivity through mitochondrial dysfunction and consequent oxidative stress [17]. Additionally, the use of a microfluidics endometrium-on-a-chip in bovine demonstrated cellular transcriptional and secretome differences if the endometrium following exposure to physiological extremes of metabolic factors glucose and insulin in microfluidics [18]. However, both models used animal endometrial tissue, and these findings can only be applied to humans to some extent.…”