Significance of invasion of decidualized endometrial stromal cells by human embryonic stem cells derived trophoblastic spheroids (BAP-EB) in pregnancy outcomes of in vitro fertilization (IVF)

Abstract

Study question: Does invasion of decidualized endometrial stromal cells (hEnSC) towards trophoblast-like BAP-EB relate to IVF pregnancy outcome? Summary answer: The invasion of decidualized hEnSC from patients with a live birth towards BAP-EB was significantly higher than those with negative pregnancy outcome. What is known already: The success rate of IVF remains low even after the transfer of good quality embryos. Implantation-competent blastocysts can induce migration of decidualizing hEnSC. The encapsulation of the invading blastocyst by the decidua supports subsequent trophoblast invasion. The study of the dynamic fetal–maternal interaction between the decidua and the implanting blastocyst requires a proper implantation model. We have derived trophoblastic spheroids (BAP-EB) from human embryonic stem cells that resemble human trophectoderm and trophoblast during early implantation process. They attach specifically onto receptive endometrial epithelial cells. BAP-EB can be used as human embryo surrogate for studying the early implantation process. Study design, size, duration: hEnSC were isolated from endometrial biopsies obtained from IVF patients in their natural cycle 7 days after luteinizing hormone surge (LH+7). Human embryonic stem cells were differentiated into early trophectoderm-like BAP-EB-48h or trophoblast-like BAP-EB-96h. An invasion assay using Matrigel Invasion Chamber was established with decidualized hEnSC and BAP-EB-48h or -96h. hEnSC isolated from patients with live births (n=5) and negative pregnancy outcomes (n=5) in their subsequent stimulated IVF cycle were compared. Participants/materials, setting, methods: Human endometrial stromal cell line (T-HESC) was induced to decidualize in vitro. Purity of primary hEnSC was confirmed by immunohistochemical staining of stromal (Vimentin) and epithelial (cytokeratin) markers. The 24h-invasion of decidualized hEnSC through Matrigel towards trophectoderm-like BAP-EB-48h or trophoblast-like BAP-EB96h was compared to medium alone control. The expression levels of decidualization markers (PRL, IGFBP1) and the invasion ability towards BAP-EB were compared between hEnSC isolated from patients with live births and negative pregnancy outcomes. Main results and the role of chance: After treatment with cAMP for 3, 6 and 9 days, the decidualization markers PRL and IGFBP1 were significantly induced from day 6 onwards. The isolated primary hEnSC were of high purity as demonstrated by positive vimentin staining (>95%) but not cytokeratin staining. The invasion of hEnSC through Matrigel towards medium alone without BAP-EB, BAP-EB-48h or BAP-EB-96h was quantified by the absorbance of crystal violet stained cells. It was found that significantly more hEnSC invaded towards BAP-EB-96h than BAP-EB-48h when compared to medium alone control, suggesting the specific invasion of hEnSC towards the trophoblast-like but not the trophectoderm-like BAP-EB. The decidualization and invasion abilities of hEnSC isolated from patients with live births and negative pregnancy outcomes were compared. The expression levels of PRL and IGFBP1 were similar in the two groups of patients 6 days after in vitro induction of decidualization (p>0.05). The invasion of hEnSC through Matrigel in the presence or absence of BAP-EB96h was measured. Interestingly, the percentage of BAP-EB-96h-induced invasion was significantly higher in hEnSC from patients with live births (54.3% ±11.1%) than those with negative pregnancy outcomes (20.8%±9.1%, p<0.05). The data indicated a correlation of responsiveness of hEnSC towards BAP-EB with pregnancy outcome. Limitations, reasons for caution: BAP-EB and the isolated endometrial stromal cells may not fully represent the in vivo developed human blastocysts and endometrial cells, respectively. The in vitro nature of the hEnSC experiments and the limited sample size in this study may also limit the interpretation of the data. Wider implications of the findings: The study of early implantation failure is limited by the number and the ethical concerns of human embryos donated for research. The current data demonstrated the potential use of BAP-EB as human embryo surrogate for assessing the responsiveness of hEnSC towards implanting embryos and as predictive tool for pregnancy outcome.