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

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

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

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 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