Interaction between insulin and androgen signalling in decidualization, cell migration and trophoblast invasion in vitro

Abstract

Finely tuned decidualization of endometrial stromal fibroblasts into decidual cells is crucial for successful implantation and a healthy pregnancy. Both insulin and androgens are known to modulate decidualization, however, their complex effect on this process has not been fully elucidated. As hyperinsulinemia and hyperandrogenism are associated in clinical conditions, we aimed to investigate the interaction between insulin and androgens on decidualization. Primary human endometrial stromal cells were decidualized in vitro in the presence of insulin and/or androgens (dihydrotestosterone (DHT), testosterone). Gene or protein expressions of decidualization markers were measured, and cells size characteristics were determined. Migration of decidualizing endometrial stromal cells and invasion of HTR-8/SVneo trophoblast spheroids were assessed. We found that insulin and androgens in combination enhanced the upregulation of several decidualization markers including prolactin, tissue factor, tissue inhibitor of matrix metalloproteinase 3 and connexin-43, and also interacted in modulating cell size characteristics resulting in enlarged decidualizing cells. However, insulin and DHT together restricted the migration of decidualizing cells and invasion of trophoblast spheroids. Our findings suggest that insulin and androgens interact to potentiate the process of decidualization. On the other hand, inhibited cell migration and trophoblast invasion might negatively impact the function of decidualizing endometrial stromal cells.

Keywords: androgens; decidualization; endometrium; insulin; interaction.

FIGURE 1

(A) Relative levels of PRL gene expression after 6 days in response to insulin and/or DHT in in vitro decidualized (treated with MPA and db‐cAMP) human endometrial stromal cells of healthy volunteers. Interaction effect between insulin and DHT: p < 0.05. (B) Relative levels of PRL gene expression after 6 days in response to insulin and/or testosterone in in vitro decidualized (treated with MPA and db‐cAMP) human endometrial stromal cells of healthy volunteers. Main effect of insulin: p < 0.05; main effect of testosterone: p < 0.001

FIGURE 2

(A–H) Representative microphotographs (40x magnification) of wound‐healing assay of stromal cells decidualized (treated with MPA and db‐cAMP) for 6 days after wounding at 0h (A) and 24h (B), with insulin treatment at 0h (C) and 24h (D), with DHT treatment at 0h (E) and 24h (F) and with insulin and DHT treatment at 0h (G) and 24h (H) were taken with IncuCyte S3 Live‐Cell Analysis System. (I) Closure of the wound using in vitro decidualized (treated with MPA and db‐cAMP) human endometrial stromal cells of healthy volunteers in response to insulin and/or DHT after 6 days, at 24 h. Interaction effect between insulin and DHT: p = 0.10

FIGURE 3

(A‐L) Representative microphotographs (40x magnification) of co‐culture invasion assay using HTR‐8/SVneo spheroids and stromal cells decidualized (treated with MPA and db‐cAMP) for 6 days at 0h (A), 12h (B) and 16h (C), with insulin treatment at 0h (D), 12h (E) and 16h (F), with DHT treatment at 0h (G), 12h (H) and 16h (I), and with insulin and DHT treatment at 0h (J), 12h (K) and 16h (L) were taken with IncuCyte S3 Live‐Cell Analysis System. (M) Co‐culture invasion assay using HTR‐8/SVneo spheroids on in vitro decidualized (treated with MPA and db‐cAMP) human endometrial stromal cells of healthy volunteers in response to insulin and/or DHT after 6 days, at 12h. Interaction effect between insulin and DHT: p = 0.11. (N) Co‐culture invasion assay using HTR‐8/SVneo spheroids on in vitro decidualized (treated with MPA and db‐cAMP) human endometrial stromal cells of healthy volunteers in response to insulin and/or DHT after 6 days, at 16h. Main effect of insulin: p < 0.01