Here we show for the first time that decidualized H-EnSCs from fertile women are able to adjust their migratory activity in response to the quality of an embryo. In addition, decidualized H-EnSCs from RM women fail to discriminate between high- and low-quality embryos as the cells do not regulate their migratory response in response to the quality of the embryo. This inability to discriminate with respect to migration between low- and high-quality embryos may be regarded as a failure of nature’s quality control and is consistent with the “Selection Failure” hypothesis for women with RM 
. This hypothesis proposes that super-receptive endometrium may not be able to select high- from low-quality embryos leading to the implantation of low-quality embryos and -as their development fails- may cause a subsequent miscarriage.
The ability to distinguish between high- and low embryo qualities may have evolved as a response to the pressure of the high percentage of chromosomally abnormal (poor-quality) embryos present in fertile women 
. A preference for the implantation of high-quality embryos may offer a reproductive advantage; higher monthly fecundity rate and larger offspring size thereby ensuring a more effective gene pool spread. A situation where the endometrial bio-sensor does not function properly may then result in the implantation of low-quality embryos which are rejected later, presenting as a clinical miscarriage.
Consistent with this concept, we have recently shown that decidualized human endometrium responds to the presence of a low-quality embryo by downregulating the production of key pro-implantation cytokines 
. Moreover, bovine endometrium gene profiles have been shown to alter in response to embryos of low versus high development potential 
Several studies have investigated the migratory role of H-EnSCs in regulating implantation. Decidualized H-EnSCs from fertile women display profound motility around a high-quality human blastocyst, as was illustrated by 24-hour timelapse pictures of a blastocyst co-culture with CellTracker stained H-EnSCs 
. However, this study did not provide information about net migratory activity because the embryo was placed on top of the endometrial monolayer. Furthermore it has been shown that the migratory activity of H-EnSCs is increased in the secretory phase, since decidualized H-EnSCs of premenopausal women migrate significantly more than undifferentiated ESCs in the presence of extra-villous trophoblast secretory products 
In this paper we confirm the migratory nature of decidualized H-EnSCs. In addition, a series of key novel findings shed light on the mechanisms which underlie the ‘selective’ phenotype of H-EnSCs. For decidualized H-EnSCs originating from both control and RM women, migration in the presence and absence (basal migration) of a high-quality embryo is comparable. Since the presence of a high-quality embryo did not appear to result in a difference in migration compared to the basal migration observed when no embryo was present, we further hypothesized that high-quality embryos do not elicit a migratory response. The comparable basal migration and migration in the presence of a high-quality embryo is also not due to a ceiling effect as on average only 20–30% of the migration zone of the cultures with high-quality embryos was closed after 18 hours. Moreover, we observed an inhibition of the migration of normal fertile ESCs in the presence of a low-quality embryo. This implies that the distance between the embryo and the H-EnSC monolayer is not a limiting factor in regulating H-EnSC migration. It also suggests that normal fertile H-EnSCs are able to sense and respond differently to low-quality embryos.
The proposed mechanism that may underlie the ‘super-receptive’ phenotype seen in women with RM is supported by their inability to discriminate high- from low-quality embryos. This makes these RM women more receptive for low-quality embryos than normally fertile women. The ‘super-receptive’ phenotype of women with RM is also supported by the observation that these cells are more sensitive to extravillous trophoblast cell stimuli, as the migration of H-EnSCs from RM women is also enhanced by exposure to trophoblast spheroids containing fewer cells than normally present in the day 5 human embryo.
The difference in response pattern of H-EnSCs from women with RM to trophoblast spheroids and embryos is likely to reflect major differences in signalling from trophoblast spheroids and from embryos, with the former producing a stronger pro-migration signal unmodulated by signals from the other cell types represented in an embryo. It may be that H-EnSCs from women with RM are more sensitive to a stimulatory signal coming from the trophoblast spheroid or it may be that the H-EnSCs from women with RM are not able to sense an inhibitory signal coming from the trophoblast cells. In the cultures with human day 5 embryos, the failure of H-EnSCs from women with RM to respond to inhibitory signals coming from the 3PN embryo may underlie the difference between RM and fertile control women.
An interesting observation from the Supporting information, Video S1 and S2 is the intense interaction between the H-EnSCs and the trophoblast spheroid or day 5 3PN human embryo. In Video S1 it is seen that H-EnSC migration is enhanced from the moment spheroid derived satellites have reached the H-EnSC monolayer. In this case extensive directed migration towards the trophoblast spheroid is also observed, which however is not observed consistently in all spheroid migration experiments. Video S2 provides some novel insights into the earliest human embryo implantation events. It shows how a 3PN blastocyst travels to one side of the H-EnSC monolayer and rolls alongside it. It also shows the fast H-EnSC response to the 3PN blastocyst by migrating towards and away from the blastocyst, retraction and repulsion. In our experience, hatched human blastocysts do not adhere to the plastic cultures dishes, which may reflect the disposable material used. Also other groups noted a similar lack of adhesion or outgrowth when a trophoblast spheroid or embryo was placed on or adjacent to decidualized H-EnSCs 
We have chosen to use 3PN embryos as we consider they are a suitable model for the ‘low-quality embryo’. The rate of aneuploid concepti in mid-late first trimester RM has been reported to be 30%, versus around 2% detected by chorion villus sampling of intact first trimester pregnancies 
. Moreover, triploidy is one of the more commonly found aneuploidies in concepti examined after RM 
. Since triploid fetuses very rarely reach term 
there is clearly a selection mechanism in place, and this is likely to occur prior to any pregnancy becoming clinically evident 
Although we should acknowledge that morphology is only part of what determines embryo quality, karyotyping of some blastomeres may not reflect the rate of mosaicism. For this, CGH would have to be done in all blastomeres, which was not feasible.
The mechanisms by which healthy H-EnSCs sense the difference between high- and low-quality embryos remain to be fully elicited. Compromised embryos are metabolically very active, producing increased ATP and reactive oxygen species and demonstrating increased amino acid turnover when compared to viable embryos 20
. Conversely, increased metabolism (as illustrated by amino acid turnover) has been shown to be associated with increased DNA damage and lower cytogenic health in the embryo 
. These metabolites might be good candidate signals of embryo quality to be sensed by H-EnSCs. Secondly, it is known that syncytiotrophoblast shed microparticles that can subsequently reach the maternal circulation 
. Early trophoblast cells might also shed microparticles. Decidualized H-EnSCs may then adjust their migration in response to these particles. Thirdly, human chorionic gonadotropin (hCG), a hormone secreted by the embryonic trophoblast, may modulate the response of H-EnSCs. It has been shown that decidualized H-EnSCs of RM women display a dysregulated response to bHCG with regard to prolactin and prokineticin1 mRNA expression 
. In line with this, it is possible that decidualized H-EnSCs from women with RM also have a dysregulated migratory response to hCG. Decidualized H-EnSCs from women with RM may be less discriminatory in response to one of the products released by low-quality embryos (e.g. byproducts of increased metabolism, trophoblast microparticles or hormones such as bHCG) and as a result show no inhibition of migration.
An interesting follow-up experiment would be to stimulate the decidualized H-EnSCs with culture medium of the human embryo or trophoblast spheroid. So far, we have not been able to identify factors that are secreted by the blastocyst. Extensive crosstalk between the H-EnSCs and the trophoblast has been reported to occur at the gene expression level in a co-culture system 
In view of the “selection failure” hypothesis we would expect a somewhat higher live-birth rate than in RM patients that conceive naturally. However, we would not expect a dramatic increase in live birth rate following PGS for patients that suffer from RM because PGS only screens for a few number of chromosomal abnormalities, while there is a high incidence of mosaicism in young couples that undergo IVF 
and the fecundity rate of RM is not low (with a live birth rate of around 35%). Furthermore, although the best available evidence suggests a similar live-birth rate in women with unexplained RM after PGS vs. natural conception (42 vs. 35% respectively), this is still under debate as no RCT or non-randomized comparative studies (directly comparing PGS vs. natural conception) have been performed in this study group 
Thus far no treatment options exist for women with unexplained recurrent miscarriage. There is insufficient evidence to support the use of immunotherapy, hCG or progesterone in women with unexplained RM to increase live birth rate.
In conclusion, we report several new findings that describe the mechanisms behind the ‘selective phenotype’ seen in fertile women and behind the ‘super-receptive’ phenotype seen in women with RM. Decidualized H-EnSCs of fertile women may actively select a high-quality embryo for implantation by inhibiting their migratory response in the presence of low-quality embryos. In contrast, the migration of H-EnSCs of RM women that is already elicited by trophoblast spheroids and by low-quality embryos highlight the super-receptive state of the endometrium of women suffering from RM. This enhanced migratory response of H-EnSCs of RM women towards a low-quality embryo or towards small spheroids may become a biomarker for identifying ‘selection failure’ as the aetiology in those patients diagnosed with otherwise unexplained RM.
Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of decidualized H-EnSCs of fertile women and how this is deregulated in the H-EnSCs of RM women. Clarification of the mechanism of non-discriminative migration of to high- and low-quality embryos of H-EnSCs of women with RM may have important clinical implications both for understanding the aetiology of this distressing condition, and for the future development of a therapeutic target for the prevention of further miscarriages.