hCG modulates the IL1B-mediated mRNA expression of immune modulating, adhesion, growth, angiogenic and tissue remodeling factors in ESCs.

<p>Confluent ESC cultures were incubated with minimal medium (MM) (control) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed, and mRNA levels were then quantified by qRT-PCR. VCAM1 (A), IL6 (B), CCL5 (C), PTGS2 (D), VEGFC (E), MMP9 (F), TIMP3 (G) and KRT19 (H) mRNA ratio was then determined following normalization to GAPDH mRNA (internal control). Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of VCAM1, IL6, CCL5, PTGS2, VEGFC, MMP9, TIMP3 or KRT19 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P<0.05, **P<0.01, *** P<0.001 relative to MM; †P<0.05, ††P<0.01, †††P<0.001 relative to cells stimulated with an equivalent concentration of hCG; +P<0.05 relative to cells stimulated with an equivalent concentration of IL1B. Data were obtained with ESC cultures issued from 7 different subjects (the 3 cultures used for microarray analysis and 4 additional cultures).</p

Enrichment score of biological processes.

<p>A) GO analysis was used to identify the main biological processes targeted by gene lists and significantly modulated by each treatment. Each functional group was assigned with a GO enrichment score that was calculated using a chi2 test. B) A forest plot using the same gene list was also generated to show the percentage of differentially expressed genes that were up-regulated (red) or down-regulated (blue) for each biological process. Light color: gene populations with a fold change ranging from 1.5 to 2. Dark color: gene populations with a fold change above 2. Data were obtained with ESC cultures issued from 3 different subjects.</p

Name of up-regulated genes.

<p>Name of up-regulated genes.</p

hCG modulates the expression of immune, angiogenic and tissue remodeling factors in ESCs at the protein level.

<p>Confluent ESC cultures were incubated with minimal medium (MM) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Supernatant was collected and soluble proteins CCL2 (A) CCL5 (B) VEGFC (C) TIMP3 (D) MMP9 (E) were then quantified by ELISA. Data were from ESC cultures issued from 4 different subjects and expressed in pg/mL. *P<0.05, *** P<0.001 relative to MM; ††P<0.01, †††P<0.001 relative to cells stimulated with an equivalent concentration of hCG; +P<0.05, +++P<0.001 relative to cells stimulated with an equivalent concentration of IL1B.</p

Genes down-regulated after in vitro treatment of human endometrium stromal cells.

á/â<p>Real-time PCR/ ELISA validation performed</p><p>Pf, proliferation; Im, immune functions; Rm, tissue remodeling; CS, cell signaling; Ap, apoptosis; Ag, angiogenesis</p

Name of down-regulated genes.

<p>Name of down-regulated genes.</p

Analysis of genes significantly modulated by each treatment.

<p>A) Headmap of probe sets corresponding to genes significantly modulated (P<0.05) in each group. Increased signal intensities are displayed in red, whereas lower signal intensities are shown in blue. Cluster distances were evaluated by Spearman correlation on average linkage (Partek Genomics Suite). B) PCA scatter plot of all samples was generated to assess the variability of micro-array data. Each sphere represents a whole chip data. As shown in the legend, samples are colored by treatment and grouped by an ellipsoid that considers two standard deviations from the center of each group. C) Venn diagram of the respective gene lists showing the overlap of action between hCG/MM, MM/IL1B and hCG/IL1B. Data were obtained with ESC cultures issued from 3 different subjects.</p

List of PCR primers.

<p>List of PCR primers.</p

hCG modulates IL1B effects on the expression of IL1 family members in ESCs.

<p>Confluent ESC cultures were incubated with minimal medium (MM) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed, and mRNA levels were then quantified by qRT-PCR. IL1A (A), IL1B (D), IL1RL1 (B), IL18R1 (E), IL33 (C) and IL18 (F) mRNA ratio was then determined following normalization to GAPDH mRNA (internal control). Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of IL1A, IL1B, IL1RL1, IL18R1, IL33 or IL18 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P<0.05, **P<0.01, *** P<0.001 relative to MM; †P<0.05, †††P<0.001 relative to cells stimulated with an equivalent concentration of hCG; ++P<0.01 relative to cells stimulated with an equivalent concentration of IL1B. Data were obtained with ESC cultures issued from 7 different subjects (the 3 cultures used for microarray analysis and 4 additional cultures).</p

Genes up-regulated after in vitro treatment of human endometrium stromal cells.

<p>Pf, proliferation; Im, immune functions; Rm, tissue remodeling; CS, cell signaling; Ap, apoptosis; Ag, angiogenesis</p>á/â<p>Real-time PCR/ELISA validation performed</p><p>Genes up-regulated after in vitro treatment of human endometrium stromal cells</p