A tissue-engineered human endometrial stroma that responds to cues for secretory differentiation, decidualization, and menstruation

Abstract

Objective: To show the responsiveness of tissue-engineered human endometrial stroma to combinations of hormones that mimic the secretory and menstrual phases of the cycle.

Design: In vitro experimental study.

Setting: University uterine biology research laboratory.

Patient(s): None.

Intervention(s): Telomerase immortalized human endometrial stromal cells cultured in monolayers (two-dimensional, 2D) or encapsulated in a collagen I hydrogel (three-dimensional, 3D) to create a simplified tissue-engineered stroma were exposed to hormone treatments mimicking early and late secretory phases, decidualization, and steroid withdrawal conditions to recapitulate menstruation.

Main outcome measure(s): Morphologic and biochemical markers of decidualization and collagenase activity.

Result(s): The 3D tissue can manifest changes in morphology and biochemical markers of decidualization similar to 2D culture and characteristic of endometrial stroma in vivo. Unlike 2D culture, the 3D tissue responded to steroid withdrawal by increased collagenase activity and tissue breakdown.

Conclusion(s): Three-dimensional tissue-engineered endometrial stroma can mimic secretory and menstrual phases of the cycle and may be useful for studying uterine receptivity and menstruation in a physiological endocrine environment.

Figure 1

A change in HESC morphology is seen in response to hormone treatment. Images of the cells in 2D with phase contrast or 3D histological sections stained with H&E are shown (A). Scale bars represent 50 μm. Only images of the early secretory (ES) and decidual (D) conditions are shown as there are no visible differences noticeable between early secretory, late secretory (LS), and menstruation/steroid withdrawal (M) conditions (n=6). In order to quantify morphological differences, cellular (2D) and nuclear (3D) shape indices were calculated from cells and nuclei in n=6 samples (B). * indicates p<0.05 compared to all other conditions within the culture condition (2D or 3D). In 2D culture, decidualization induced a rounder cell shape as compared to the other hormone treatments. In 3D culture, the early secretory phase nuclei were significantly flatter than those in the other hormone treatments, but late secretory, decidual and menstruation conditions were not significantly different.

Figure 2

ELISAs were performed to determine the secretion of decidualization biomarkers PRL (A), IGFBP-1 (B), and VEGF (C) which has been shown to increase during stromal differentiation (n=6). The results were consistent across all three proteins. In 2D and 3D cultures, cells secreted significantly higher amounts of these biomarkers when given the decidualization hormones (D). The late secretory (LS) stimulus responses were not significantly different from any of the other conditions in 3D culture.

Figure 3

Steroid withdrawal led to visible degradation of the 3D tissues. Images of the 2D and 3D cultures at day 10 are shown (A). Scale bars indicate 50 μm in the 2D frames and 20 μm in the 3D frames. Examples of late secretory (LS) and menstrual/steroid withdrawal (M) conditions are shown, the menstrual condition is the only one that showed evidence of breakdown in 3D tissues. An increase in active collagenase was seen following steroid withdrawal in 3D tissues, but was not significantly affected in 2D cultures (n=6) (B). Gelatin zymography was performed to visualize latent and active gelatinases (n=5). MMP-2 and MMP-9 were identified by their molecular weights. The lanes from left to right are 3D: ES, LS, D, M followed by 2D: ES, LS, D, M. The menstrual conditions lead to increase MMP-2 activation in 3D culture, however, no differences are seen in 2D culture (C).

Figure 4

The hormone treatments resulted in subtle differences in stromal cell numbers as indicated by DNA content. The content for 3D was normalized to dry weight in order to take into consideration differences in hydrogel size. The only significant finding was the lower DNA content detected in the 2D cultures exposed to decidualization hormones (n=6).