Endometrial stromal cells of women with recurrent miscarriage fail to discriminate between high- and low-quality human embryos

Background The aetiology of recurrent miscarriage (RM) remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs) of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women. Methodology/Principal Findings Monolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13), a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12) or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05) and compared to the migration in the presence of high-quality embryo (p<0.01). Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001). Conclusions H-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site

Cell Lineage Specific Distribution of H3K27 Trimethylation Accumulation in an In Vitro Model for Human Implantation

Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation) followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3) marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ∼25% of the trophectoderm cells and in ∼7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion

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 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

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

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

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