Migration of H-EnSCs of fertile control and RM women in response to a trophoblast spheroid.

<p>In a confluent well of a 48-well plate a migration zone was created. H-EnSCs were left to migrate in the presence or absence of a trophoblast spheroid in the migration zone. Data is shown as a reduction of the migration zone after 18 hours. Experiments were performed in triplicates. Data represent means ± SEM of 6 women with RM (grey bars) and 6 normally fertile women (white bars) and was analysed by 2-way ANOVA and Bonferroni post hoc tests, ***p<0.001.</p

F-Actin architecture in H-EnSCs in the presence and absence of a trophoblast spheroid.

<p>In a confluent well of a 48-well plate a migration zone was created. Decidualized H-EnSCs of a RM patient were left to migrate in the presence (A) or absence (B) of a trophoblast spheroid consisting of 3000 cells. The white arrow in panel A indicates the position of the trophoblast spheroid. Both micrographs were obtained from the same location in the well. Cells were fixed and stained for F-actin (red) and DNA was stained with DAPI (blue). Magnification: ×20.</p

Migration of decidualized H-EnSCs in response to a high-quality or a low-quality human embryo.

<p>In a confluent well of a 4-well plate a migration zone was created. Decidualized H-EnSCs from control women (white bars) and RM women (grey bars) were left to migrate for 18 hours in the presence or absence of a high- or low-quality embryo. Data is shown as percentage reduction of the migration zone (the percentage reduction of the migration zone in the presence of an embryo minus the percentage reduction in the absence of an embryo). Data represent means ± SEM of 3 women with RM and 3 controls in the presence of a high-quality embryo (n = 13) or a low-quality embryo (n = 12) and were analysed by 2-way ANOVA and Bonferroni post hoc tests **p<0.01.</p

The migration zone after adding a high-quality, low-quality or no embryo.

<p>The migratory response of decidualized H-EnSCs from normally fertile (A–C) and RM women (D–F) was analyzed in absence of a human embryo (A and D), in presence of a high-quality embryo (B and E) or a low-quality embryo (C and F). Phase contrast pictures were taken 18 hours after creating the migration zone. The dotted line represents the front of the migration zone directly after its creation. As a reference for the position of the embryo, the bottom of the plate was marked. The arrows indicate the position of the embryo. All pictures were taken with 25x magnification.</p

Migration of H-EnSCs in response to three different sizes of trophoblast spheroids.

<p>In a confluent well of a 48-well plate a migration zone was created. H-EnSCs were left to migrate in the presence or absence of a three different sizes of trophoblast spheroids (consisting of either 12, 40 or 120 cells as depicted by the white, grey or black bars respectively) in the migration zone. Data is shown as a reduction of the migration zone after 18 hours (the percentage reduction of the migration zone in the presence of a trophoblast spheroid minus the percentage reduction in the absence of a trophoblast spheroid). Experiments were performed in triplicates. Data represent means ± SEM of 2 women with RM and 3 normally fertile women and was analysed by 2-way ANOVA and Bonferroni post hoc tests, *** and ^###p<0.001.</p

Signaling pathways activated by PDGF-AA, PDGF-BB, HB-EGF, TCM, or VECM.

<p>(<b>A</b>) Decidualized St-T1b were starved in Opti-MEM and then treated for 5 or 30 min with PDGF-BB, HB-EGF, TCM or PDGF-AA. Levels of phosphorylated (p-) or total ERK1/2, Akt, and p38 were determined by Western blotting. (<b>B</b>) Decidualized hESC were starved in Opti-MEM and then treated for 5 min with PDGF-BB, HB-EGF, TCM, 5 individual VECM preparations, villous explant control medium (VECM-Co), or PDGF-AA. Western blotting was performed as above.</p

Chemotactic response of St-T1b cells or primary hESCs to PDGF-BB, HB-EGF and trophoblast conditioned medium.

<p>St-T1b cells (<i>upper panel</i>) or hESC (<i>lower panel</i>), non-decidualized (ND) or decidualized (D) by 5 d treatment with 8-Br-cAMP/MPA, were subjected to transwell migration assay. The bottom reservoir contained MM1-10% (controls), PDGF-BB, HB-EGF or conditioned medium (TCM) from the trophoblast cell line AC-1M88 undiluted (100%) or diluted to 80% or 20%. The motility index designates the percentage of migrated cells relative to the total cell number (non-migrated plus migrated) at the end of the 18 h migration period. Shown are results representative of 3 similar experiments (means± SD, n = 3). Data were analyzed by ANOVA and Dunnett <i>post-hoc</i> test. *, <i>P</i><0.05; **, <i>P</i><0.01; *** <i>P</i><0.001 compared to the respective control within the ND or D groups.</p

Chemotactic response of hESCs to trophoblast secretory products identified by proteome profiling.

<p>(<b>A</b>) Decidualized hESCs were analyzed in transwell migration assay in response to PDGF-BB, HB-EGF, TCM, PDGF-AA, PLGF-1 or VEGF-165. Motility indices are shown as means±SD (n = 3), and were analyzed by ANOVA and Dunnett test. ***, <i>P</i><0.001 compared to the control without chemoattractant. (<b>B, C</b>) Effect of neutralization of PDGF activity. Decidualized hESCs were subjected to transwell migration assay with two different doses of PDGF-AA (<b>B</b>) or with TCM and two individual VECM preparations (<b>C</b>) in the absence or presence of a neutralizing antibody to PDGF-AA/-AB/-BB (pan). Motility indices are shown as means±SD (n = 3) and were analyzed by ANOVA and Dunnett or Tukey test. ***, <i>P</i><0.001; **, <i>P</i><0.01 in the absence vs. presence of antibody. <i>a</i>, <i>P</i><0.001; <i>b</i>, <i>P</i><0.01; <i>c</i>, <i>P</i><0.05 compared to the respective control without stimulation or antibody (white or light grey columns).</p

Proteome profiling for cytokines and angiogenesis factors in conditioned medium from AC-1M88 human trophoblast cells, two individual first trimester villous explant cultures, and the St-T1b human endometrial stromal cell line after decidualizing treatment.

<p>Proteome profiling for cytokines and angiogenesis factors in conditioned medium from AC-1M88 human trophoblast cells, two individual first trimester villous explant cultures, and the St-T1b human endometrial stromal cell line after decidualizing treatment.</p

Chemokinetic response of St-T1b cells or primary hESCs to PDGF-BB, HB-EGF, TCM and trophoblast secretory products identified by proteome profiling.

<p>St-T1b cells (<i>upper panel</i>) or hESC (<i>lower panel</i>), non-decidualized (ND) or decidualized (D), were subjected to Oris migration assay to monitor random motility, in the presence of PDGF-BB, HB-EGF, TCM, PDGF-AA, PLGF-1, or VEGF-165 at the indicated concentrations. Numbers of cells that had migrated into the detection zone after 18 h incubation were determined and normalized to unstimulated controls within each ND or D group. Shown are the means±SEM of n = 3 independent experiments. Results were analyzed by ANOVA and Dunnett test within each group. ***, <i>P</i><0.001; **, <i>P</i><0.01 compared to untreated controls (white columns).</p