Fig. 1: FSH treatment markedly reduces the growth, migratory, and multilineage differentiation potential of endometrial stem cells in vitro.

We hypothesized that FSH treatment would suppress various tissue repair and regeneration capacities of endometrial stem cells, such as the self-renewal, migration, and multilineage differentiation capacities (a). The inhibition of self-renewal capacity by treatment with FSH at different doses (5 IU, 10 IU, 20 IU, and 30 IU) was analyzed at 72 h after treatment by MTT assays. The cell proliferation rate (%) was analyzed as the viability of FSH-treated cells as a percentage of control cells treated with vehicle (b). The activation status (whether intermediate, inactivate, or activate) of various FSH (GSE50831)-related genes in proliferative and nonproliferative cells was assessed using the ingenuity pathway analysis (IPA) system (c). Endometrial stem cells were treated with FSH at 10 IU or 30 IU for 72 h. The inhibitory effects of FSH exposure on migration ability were then analyzed using transwell assays. FSH exposure markedly reduced cell migration across the membrane of transwells (d). Protein levels of positive regulatory factors of cell migration (MMP-2/9) in response to FSH exposure were analyzed by western blotting (e). The activation status (intermediate, inactivate, or activate) of various FSH (GSE36133)-related genes in migrating and nonmigrating cells was assessed using the IPA system (f). Cells were cultured for 14 days in adipocyte or osteoblast differentiation medium with or without FSH (10 IU or 30 IU) treatment. The inhibitory effects of FSH treatment on the adipocyte and osteoblast differentiation of endometrial stem cells were analyzed by oil red O staining and alizarin red S staining, respectively. Relative quantification of calcium deposition and lipid droplet (LD) secretion from differentiating cells was performed by measuring the absorbance of solubilized cells at 500 nm and 570 nm, respectively (g). β-Actin was used as an internal control. All experiments were performed in triplicate. Data are presented as the mean ± standard deviation (SD). *p < 0.05; **p < 0.005; and ***p < 0.001 (two-sample t test).