The proliferative phase endometrium in IVF/ICSI: an in-cycle molecular analysis predictive of the outcome following fresh embryo transfer.

STUDY QUESTION: Does an early proliferative phase endometrial biopsy harvested during ovarian stimulation harbour information predictive of the outcome following fresh embryo transfer (ET) in that same cycle? SUMMARY ANSWER: Transcriptome analysis of the whole-tissue endometrium did not reveal significant differential gene expression (DGE) in relation to the outcome; however, the secretome profile of isolated, cultured and in vitro decidualized endometrial stromal cells (EnSCs) varied significantly between patients who had a live birth compared to those with an implantation failure following fresh ET in the same cycle as the biopsy. WHAT IS KNOWN ALREADY: In the majority of endometrial receptivity research protocols, biopsies are harvested during the window of implantation (WOI). This, however, precludes ET in that same cycle, which is preferable as the endometrium has been shown to adapt over time. Endometrial biopsies taken during ovarian stimulation have been reported not to harm the chances of implantation, and in such biopsies DGE has been observed between women who achieve pregnancy versus those who do not. The impact of the endometrial proliferative phase on human embryo implantation remains unclear, but deserves further attention, especially since in luteal phase endometrial biopsies, a transcriptional signature predictive for repeated implantation failure has been associated with reduced cell proliferation, possibly indicating proliferative phase involvement. Isolation, culture and in vitro decidualization (IVD) of EnSCs is a frequently applied basic research technique to assess endometrial functioning, and a disordered EnSC secretome has previously been linked with failed implantation. STUDY DESIGN, SIZE, DURATION: This study was nested in a randomized controlled trial (RCT) investigating the effect of endometrial scratching during the early follicular phase of ovarian stimulation on clinical pregnancy rates after IVF/ICSI. Of the 96 endometrial biopsies available, after eliminating those without fresh ET and after extensive matching in order to minimize the risk of potential confounding, 18 samples were retained to study two clinical groups: nine biopsies of patients with a live birth versus nine biopsies of patients with an implantation failure, both following fresh ET performed in the same cycle as the biopsy. We studied the proliferative endometrium by analysing its transcriptome and by isolating, culturing and decidualizing EnSCs in vitro. We applied this latter technique for the first time on proliferative endometrial biopsies obtained during ovarian stimulation for in-cycle outcome prediction, in an attempt to overcome inter-cycle variability. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA-sequencing was performed for 18 individual whole-tissue endometrial biopsies on an Illumina HiSeq1500 machine. DGE was analysed three times using different approaches (DESeq2, EdgeR and the Wilcoxon rank-sum test, all in R). EnSC isolation and IVD was performed (for 2 and 4 days) for a subset of nine samples, after which media from undifferentiated and decidualized cultures were harvested, stored at -80°C and later assayed for 45 cytokines using a multiplex suspension bead immunoassay. The analysis was performed by partial least squares regression modelling. MAIN RESULTS AND THE ROLE OF CHANCE: After correction for multiple hypothesis testing, DGE analysis revealed no significant differences between endometrial samples from patients who had a live birth and those with an implantation failure following fresh ET. However secretome analysis after EnSC isolation and culture, showed two distinct clusters that clearly corresponded to the two clinical groups. Upon IVD, the secretome profiles shifted from that of undifferentiated cells but the difference between the two clinical groups remained yet were muted, suggesting convergence of cytokine profiles after decidualization. LIMITATIONS, REASONS FOR CAUTION: Caution is warranted due to the limited sample size of the study and the in vitro nature of the EnSC experiment. Validation on a larger scale is necessary, however, hard to fulfil given the very limited availability of in-cycle proliferative endometrial biopsies outside a RCT setting. WIDER IMPLICATIONS OF THE FINDINGS: These data support the hypothesis that the endometrium should be assessed not only during the WOI and that certain endometrial dysfunctionalities can probably be detected early in a cycle by making use of the proliferative phase. This insight opens new horizons for the development of endometrial tests, whether diagnostic or predictive of IVF/ICSI treatment outcome. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Fonds Wetenschappelijk Onderzoek (FWO, Flanders, Belgium, 11M9415N, 1 524 417N), Wetenschappelijk Fonds Willy Gepts (WFWG G160, Universitair Ziekenhuis Brussel, Belgium) and the National Medicine Research Council (NMRC/CG/M003/2017, Singapore). There are no conflicts of interests. TRIAL REGISTRATION NUMBER: NCT02061228.

BMP4 plays a role in apoptosis during human preimplantation development.

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra-embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4-treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4-treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

STUDY QUESTION: What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? SUMMARY ANSWER: Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. WHAT IS KNOWN ALREADY: Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in vitro implantation model. STUDY DESIGN, SIZE, DURATION: Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Six days post-fertilization (6dpf) human embryos were co-cultured with Ishikawa cells for 12, 24 (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 145 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expressions were validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This in vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. WIDER IMPLICATIONS OF THE FINDINGS: Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. STUDY FUNDING AND COMPETING INTEREST(S): A.A. was supported by a grant from the 'Instituut voor Innovatie door Wetenschap en Technologie' (IWT, 121716, Flanders, Belgium). This work was supported by the 'Wetenschappelijk Fonds Willy Gepts' (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

WNT3 and membrane-associated ß-catenin regulate trophectoderm lineage differentiation in human blastocysts.

WNT/ß-catenin signaling has been described as a crucial regulator of embryonic stem cells and embryogenesis. However, little is known on its role during human preimplantation embryo development, besides the RNA expression of its multiple players. In this study, we performed ß-catenin loss- and gain-of-function studies on human preimplantation embryos by adding either Cardamonin or GSK3 inhibitor, 1-Azakenpaullone, to the embryo culture medium from the cleavage until blastocyst stages (Days 3-5/6). ß-Catenin was displayed in the cortical region underneath the membrane during all stages, but it only showed nuclear localization at cleavage stages after stabilization with 1-Azakenpaullone. We did not observe any effects on the inner cell mass markers NANOG, POU5F1, SOX2 and SALL4 in these functional experiments. However, both ß-catenin degradation and stabilization caused inhibition of the trophectoderm (TE) fate, illustrated by KRT18 and GATA3 RNA, and CDX2 protein expression. Based on the TE-specific WNT3 protein expression in blastocysts, we postulated that this protein may be an upstream regulator for the observed membrane ß-catenin function. The addition of either WNT3 or 1-Azakenpaullone to the culture medium promoted EOMES expression specific for trophoblast development. In both studies, the canonical WNT pathway target gene, TCF1, was not affected. Therefore, we conclude that WNT3 and membrane-associated ß-catenin promote progenitor trophoblast development in human blastocysts. These results have important implications in assisted reproduction and stem cell biology.