Abstract 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. human embryo, implantation, attachment, invasion, gene expression Introduction In the mid-luteal phase of the cycle, the endometrium becomes transiently receptive to embryo implantation. The exact mechanisms that enable competent human blastocysts to interact with receptive luminal endometrial epithelial cells during the implantation window have remained largely elusive. Studies in other species, i.e. primates and rodents, suggest that implantation involves a series of discrete steps (Bazer et al., 2009; Aplin and Ruane, 2017). Around 7 days post-fertilization (7dpf), the hatched embryo attaches with its epithelium-like trophectoderm (TE) to the endometrial surface epithelium. Attachment is short in duration and evolves from apposition and loose binding to stable and firm adhesion. Next, the embryo invades the underlying stroma (James et al., 2012). During implantation, TE differentiates into the proliferative cytotrophoblast (CTB) and the hCG-producing syncytiotrophoblast (STB). The inner cell mass (ICM), which gives rise to the embryo proper, consists of epiblast (EPI) cells and primitive endoderm cells (Kuijk et al., 2012; Roode et al., 2012). Various in vitro models of human implantation have been developed, including 2D co-cultures with embryos and either endometrial epithelial (Galán et al., 2000) or stromal cells (Grewal et al., 2010; Teklenburg et al., 2012), and more complex 3D models (Lindenberg, 1991; Bentin-Ley et al., 2000; Boggavarapu et al., 2016). Co-cultures with Ishikawa cells, derived from a human endometrial adenocarcinoma (Nishida, 2002) with characteristics of receptive endometrial epithelium (Hannan et al., 2010), have been validated for both human (Kang et al., 2014) and mouse embryos (Singh et al., 2010; Ruane et al., 2017). Primary trophoblast (TB) has been used to mimic human embryos (Hannan et al., 2010), although arguably this model is more relevant to study invasion upon breaching of the luminal epithelium (Sullivan, 2004). Studies on endometrial cells have yielded important insights into the repertoire of molecules expressed during the receptive period (Van Vaerenbergh et al., 2010; von Grothusen et al., 2014) and have highlighted the role of decidualizing endometrial stromal cells as natural biosensors of embryo quality (Teklenburg et al., 2010; Brosens et al., 2014; Durairaj et al., 2017). Nevertheless, there is a dearth of information on the molecular mechanisms underpinning human embryo attachment and initial invasion. Adhesion molecules and extracellular matrix (ECM) proteins are inferred to mediate the initial embryo implantation steps (van Mourik et al., 2008; Singh and Aplin, 2009). E-selectin (SELE), L-selectin (SELL) and P-selectin (SELP) are cell adhesion molecules and are known for their role in mediating the binding of different blood cells to endothelial cells (Tedder et al., 1995; Dominguez et al., 2005). L-selectin is shown to be present in the trophectoderm of hatched human blastocysts and is hypothesized to mediate the initial attachment of the embryo to the maternal epithelium (Genbacev et al., 2003; Zhang et al., 2013). Integrins, heterodimeric transmembrane glycoproteins composed of α and β subunits, act as linkers between various components of the ECM (e.g. laminins and collagens) and the cytoskeleton (Harburger and Calderwood, 2009). Different integrins have been extensively studied for their role in murine embryo implantation (Illera et al., 2000; Aplin and Ruane, 2017). Integrin αvβ3 is present in both human and mouse embryos (Kang et al., 2014), and functional blocking of the integrin reduces the implantation rate in mice (Zhang et al., 2013). Research on human embryo implantation has largely been focussed on factors involved in murine reproduction (Aplin and Ruane, 2017). Few studies to date have reported possible implantation modulators in the human, and most of these studies have used pre-implantation stage embryos (Altmäe et al., 2012; Kirkegaard et al., 2015). The aim of this study was to investigate the changes in morphology and gene expression in human embryos during the initial implantation steps. For this we co-cultured 6dpf human embryo for up to 48 h in a 2D in vitro model that uses Ishikawa cells. We followed embryo development and analysed the expression patterns of 84 adhesion and ECM genes in embryos that were either loosely or not attached to Ishikawa cells. This set of genes was chosen because of our interest in the initial embryo attachment steps. We selected the three most highly induced genes, LAMA3, THBS1 and TNC, and examined the expression of the corresponding proteins in outgrowing embryos. Our data showed that our analysis can be extrapolated to wider gene expression profiling to understand pathways that underpin the early steps of human embryo implantation. Materials and methods Human pre-implantation embryos This study was approved by both the Institutional Ethical Committee (B.U.N. 143201629028) and the Federal Committee for Scientific Research on Human Embryos in vitro (AdV045). Only cryopreserved human embryos were used; they were donated for research with written informed consent from patients who had been treated at the Centre for Reproductive Medicine (CRG, UZ Brussel) after the legally determined cryopreservation period of 5 years. Patients underwent ovarian stimulation followed by oocyte retrieval (Platteau et al., 2003), oocytes denudation and intracytoplasmic sperm injection (time set at 0 days post-fertilization or 0dpf) using ejaculated sperm (Van de Velde et al., 1997). Normally fertilized supernumerary blastocysts were vitrified as described previously (Van Landuyt et al., 2011, 2015). Good quality vitrified 5dpf blastocysts (full and expanding blastocysts with A or B scoring for both ICM and TE according to Gardner and Schoolcraft criteria) (Gardner and Schoolcraft 1999) were warmed using the Vitrification Thaw Kit (Vit Kit-Thaw; Irvine Scientific, USA) following the manufacturer’s instructions. Blastocysts were left to recover for 3 h in 25 μl droplets of Origio blastocyst medium (Origio, The Netherlands) in an incubator at 37°C with 5% O2, 6% CO2 and 89% N2. Blastocysts were scored prior to performing zona pellucida (ZP) thinning in 30 μl pronase droplets (1 mg/mL; Sigma-Aldrich, USA) for 8–10 minutes at 37°C. The ZP-free blastocysts were washed three times in human tubal fluid (HTF) medium containing HEPES and human serum albumin (Belgian Red Cross), put back in the 25 μl droplets of Origio blastocyst medium and were incubated overnight at 37°C with 5% O2, 6% CO2 and 89% N2. Ishikawa cell culture Ishikawa cells (The European Collection of Authenticated Cell Cultures, UK) were kept in Dulbecco’s modified Eagle’s medium (DMEM) without phenol red (Life Technologies, USA) with the addition of 10% v/v foetal bovine serum (Gibco, UK), 2 mM ʟ-glutamine (Gibco, UK), 100 μg/ml streptomycin and 100 IU/ml penicillin (Gibco, UK) in an incubator at 37°C in an atmosphere of 95% air and 5% CO2. Ishikawa co-culture model Ishikawa cells were grown on 14 mm glass coverslips (VWR, USA) at a density of 400 000 cells per well of a four-well plate (VWR, USA) and were cultured to confluence over 2 days. Glass coverslips were used for confocal imaging purposes. Based on morphological assessment, only good quality 6dpf embryos that survived the pronase treatment were used for further culture experiments. These embryos were either cultured further in blastocyst medium or co-cultured individually with confluent Ishikawa cells for up to 48 h. All embryos, whether cultured in blastocyst medium or co-cultured with Ishikawa cells, were maintained at 37°C in atmospheric air and 5% CO2, i.e. standard cell culture conditions (Singh et al., 2010; Kang et al., 2014). Ishikawa maintenance medium was used during co-culture, and medium was changed every 24 h. EVOS FL Cell Imaging System (Thermo Fisher Scientific, USA) was used for phase contrast images. Attachment of the embryo to the Ishikawa cells was evaluated under a stereo light microscope (Nikon, Japan) while the medium was gently pipetted 3–4 times using a 200 μl tip. All of the free-floating embryos were considered as unattached embryos (UNATT). Attached embryos were divided into two groups: loosely attached (L-ATT) embryos which detached from the Ishikawa cells after repeated medium pipetting and firmly attached (F-ATT) embryos that remained attached. Attachment rate was assessed for each experiment on different time points of co-culture. As a control for attachment, embryos were also kept in culture on glass coverslips without Ishikawa cells. To collection of 6dpf L-ATT embryos following their detachment from the Ishikawa cells, each embryo was recovered individually using a P2.5 precision pipette (Eppendorf, Germany) set to 0.8 μl. The same collection procedure was applied for the UNATT embryos. To ensure maximal recovery, embryos were not washed before lysis. Quantitative PCR analysis Individual embryos, were collected in 2 μl 140 μg/μl TCEP (tris(2-carboxyethyl)phosphine) (dissolved in RNAase-free H2O) (Macherey-Nagel, Germany) and snap-frozen in liquid nitrogen. RNA was extracted using the Nucleospin® RNA XS kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. The transcriptome of single embryos was reverse transcribed and amplified using a whole transcriptome amplification kit following the manufacturer’s instructions (WTA2, Sigma-Aldrich, USA). cDNA was then purified with the GenElute™ PCR Clean-Up Kit (Sigma-Aldrich, USA). The cDNA concentration was measured by the Qubit Fluorometric Quantification (Thermo Fisher Scientific, USA), using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). cDNA was eluted in a total volume of 250 μl and all the embryos used had a cDNA concentration between 20.0 and 71.2 ng/μl. Total RNA of the Ishikawa cells was extracted using the RNeasy Minikit (Qiagen, Germany). DNase treatment by RNase-Free DNase Set (Invitrogen, USA) was performed and High Capacity RNA-to-cDNA Synthesis Kit (Life Technologies, USA) was used to reverse-transcribe the isolated RNA. Transcripts from embryos and Ishikawa cells were quantified by real-time RT-PCR (ViiATM7, Thermo Fisher Scientific, USA). TaqMan® Array Human Extracellular Matrix & Adhesion Molecules 96-well plate (Fast) (Product number: 4418778; Thermo Fisher Scientific, USA) was used to detect expression levels of 96 transcripts: 18S, GAPDH, GUSB, HPRT1, ACTB, B2M, RPLP0, HMBS, TBP, PGK1, UBC, PPIA, ADAMTS1, ADAMTS13, ADAMTS8, CD44, CDH1, CLEC3B, CNTN1, COL11A1, COL12A1, COL14A1, COL15A1, COL16A1, COL1A1, COL4A2, COL5A1, COL6A1, COL6A2, COL7A1, COL8A1, CTGF, CTNNA1, CTNNB1, CTNND1, CTNND2, ECM1, FN1, HAS1, ICAM1, ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGAL, ITGAM, ITGAV, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, KAL1, LAMA1, LAMA2, LAMA3, LAMB1, LAMB3, LAMC1, MMP1, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP2, MMP3, MMP7, MMP8, MMP9, NCAM1, PECAM1, SELE, SELL, SELP, SGCE, SPARC, SPG7, SPP1, TGFBI, THBS1, THBS2, THBS3, TIMP1, TIMP2, TIMP3, TNC, VCAM1, VCAN and VTN. Full names of the genes and references of the assays is given in Supplementary Table I. To the predefined and dried down assays, a total volume of 20 μl reaction mix consisting of 2× Eurogentec quantitative PCR (qPCR) Mastermix and 20 ng cDNA was added to each well. The thermal cycling conditions were: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, 60°C for 1 min for 40 cycles. GAPDH, GUSB and PGK1 were selected by BestKeeper (Excel-based tool using pair-wise correlations) (Pfaffl et al., 2004) as housekeeping genes. The baseline expression of the housekeeping genes is given in Supplementary Table. All those with cycle thresholds ≥35.0 were omitted. Genes detected in only one embryo of the experimental group were considered as not expressed (Kimber et al., 2008). Relative gene expression is expressed as fold change (FC), using the 2−ΔΔCt method (Berger et al., 2015). Immunofluorescence microscopy Human embryos and Ishikawa cells were fixed in 4% paraformaldehyde (Alfa Aesar, USA) for 10 min at room temperature. The samples were subsequently washed three times for 5 min with PBS supplemented with 2% BSA (Sigma-Aldrich, USA) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA) for 10 min at room temperature. After permeabilization, the samples were incubated overnight at 4°C with the primary antibodies (Table I). Control reactions for the non-specific binding of the primary antibodies were included in each experiment by replacing the primary antibodies with the normal IgGs from the same species under the same conditions as the primary antibodies (Supplementary Table). Alexa Fluor-conjugated IgGs (Supplementary Table) were used as secondary antibodies. Samples were incubated at a concentration of 10 μg/ml for 2 h at 4°C in the dark. All antibody solutions were prepared in PBS (Thermofisher, USA) supplemented with 2% BSA (Sigma-Aldrich, USA). After extensive washing in PBS supplemented with 2% BSA, the samples were put between two glass coverslips (VWR) in SlowFade™ Gold antifade reagent with DAPI (S36942, Thermofisher, USA) and Glycergel Mounting Medium (Dako, Belgium). Zeiss LSM 510 Meta confocal microscopy (Zeiss, Germany) was performed to record the fluorescent images. Control and test images were captured using identical settings. Table I Primary antibodies used for immunofluorescence microscopy. Antibody Host species Clonality Concentration (μg/ml) Product Number Company Anti-NANOG Rabbit Monoclonal 2.5 ab109250 Abcam (UK) Anti-Cytokeratin 7 (KRT7) Mouse Monoclonal 3.3 M7018 Dako (Belgium) Anti-human chorionic gonadotrophin (hCG) Rabbit Polyclonal 10 A 0231 Dako (Belgium) Anti-Thrombospondin 1 (THBS1) Mouse Monoclonal 1.3 ab1823 Abcam (UK) Anti-Tenascin C (TNC)a Rat Monoclonal 16.6 MAB2138 Novus Biologicals (USA) Anti-laminin subunit alpha 3 (LAMA3) Mouse Monoclonal 15 MAB2144 R&D Systems (USA) Antibody Host species Clonality Concentration (μg/ml) Product Number Company Anti-NANOG Rabbit Monoclonal 2.5 ab109250 Abcam (UK) Anti-Cytokeratin 7 (KRT7) Mouse Monoclonal 3.3 M7018 Dako (Belgium) Anti-human chorionic gonadotrophin (hCG) Rabbit Polyclonal 10 A 0231 Dako (Belgium) Anti-Thrombospondin 1 (THBS1) Mouse Monoclonal 1.3 ab1823 Abcam (UK) Anti-Tenascin C (TNC)a Rat Monoclonal 16.6 MAB2138 Novus Biologicals (USA) Anti-laminin subunit alpha 3 (LAMA3) Mouse Monoclonal 15 MAB2144 R&D Systems (USA) aAntibody used for both immunofluorescence microscopy and functional blockage experiments. Table I Primary antibodies used for immunofluorescence microscopy. Antibody Host species Clonality Concentration (μg/ml) Product Number Company Anti-NANOG Rabbit Monoclonal 2.5 ab109250 Abcam (UK) Anti-Cytokeratin 7 (KRT7) Mouse Monoclonal 3.3 M7018 Dako (Belgium) Anti-human chorionic gonadotrophin (hCG) Rabbit Polyclonal 10 A 0231 Dako (Belgium) Anti-Thrombospondin 1 (THBS1) Mouse Monoclonal 1.3 ab1823 Abcam (UK) Anti-Tenascin C (TNC)a Rat Monoclonal 16.6 MAB2138 Novus Biologicals (USA) Anti-laminin subunit alpha 3 (LAMA3) Mouse Monoclonal 15 MAB2144 R&D Systems (USA) Antibody Host species Clonality Concentration (μg/ml) Product Number Company Anti-NANOG Rabbit Monoclonal 2.5 ab109250 Abcam (UK) Anti-Cytokeratin 7 (KRT7) Mouse Monoclonal 3.3 M7018 Dako (Belgium) Anti-human chorionic gonadotrophin (hCG) Rabbit Polyclonal 10 A 0231 Dako (Belgium) Anti-Thrombospondin 1 (THBS1) Mouse Monoclonal 1.3 ab1823 Abcam (UK) Anti-Tenascin C (TNC)a Rat Monoclonal 16.6 MAB2138 Novus Biologicals (USA) Anti-laminin subunit alpha 3 (LAMA3) Mouse Monoclonal 15 MAB2144 R&D Systems (USA) aAntibody used for both immunofluorescence microscopy and functional blockage experiments. Functional blockage of TNC Anti-TNC antibody (Table I) with neutralizing activity was used to bind to the TNC protein thereby inhibiting the binding to its receptor. The neutralizing activity of the antibody was validated in a mouse arthritis model (Harada et al., 2015). The antibody was diluted in the Ishikawa culture medium to 5 or 10 μg/ml. 6dpf blastocysts were pre-incubated for 1 h in 25 μl droplets with either of the two anti-TNC antibody concentrations, where after the blastocysts were put for 48 h in co-culture with Ishikawa cells with the respective anti-TNC antibody concentration in a total volume of 250 μl. Embryos and Ishikawa cells were treated with a rat IgG2a (MAB006—Novus Biologicals, USA) in the same conditions and concentrations as a control. Every 24 h the embryo attachment rate was assessed and medium was collected and changed. Medium was also collected from Ishikawa cells treated with the anti-TNC antibody or rat IgG2a for 48 h but to which no embryo was added. All collected media were stored at −80°C until analysis. HCG hormone was detected by Electrochemiluminescence immunoassay (ECLIA) (Elecsys® HCG + b, Cobas 6000, Roche Diagnostics, Belgium) at the Department of Clinical Biology (University Hospital Brussels, Belgium). Statistical analysis Graphpad Prism 6 (Graphpad Software Inc., CA, USA) was used for statistical analysis. Due to the small sample size, unpaired Mann–Whitney U test was used to compare two groups. The Chi-square test was used to determine differences in attachment rate. P < 0.05 was considered significant. Results Co-culture of human blastocysts with Ishikawa cells Initially 28 6dpf blastocysts were co-cultured on a monolayer of Ishikawa cells (Fig.1A) and the attachment rate was assessed after 24 and 48 h (Supplementary Table). After 24 h of co-culture (i.e. 7dpf), a single embryo was loosely attached (L-ATT) and 24 embryos (86%) were F-ATT to the Ishikawa cells, giving a cumulative attachment rate of 89%. The rate of firm attachment increased to 100% after 48 h (8dpf). Blastocysts (n = 10) cultured on glass coverslips did not attach and were degenerating by 8dpf. Out of the 24 F-ATT embryos after 24 h of co-culture, 20 (83%) were attached on the polar TE, also termed the embryonic pole, (Fig. 1B) and three were attached on the mural TE (Fig. 1C). The orientation of attachment was indeterminable for one embryo because its ICM was not visible. Figure 1 View largeDownload slide Developmental stages of 6 days post-fertilization (dpf) human embryos co-cultured to 8dpf (48 h) in the 2D in vitro model. (A–C, F, G) Phase contrast images taken by EVOS FL Cell Imaging System; all images were taken on 10× magnification and scale bar is indicated in each image. (A) 6Dpf human blastocysts prior to starting co-culture; (B) 7dpf embryo attached on polar trophectoderm (TE), arrow points to ICM (focus point); (C) 7dpf embryo attached on mural TE, arrow points to ICM (focus point); (F) 8dpf embryo attached on polar TE, arrow points to ICM and white dotted line encircles attachment site (focus point); (G) 8dpf flattened embryo. (D, E, H). Immunostaining of the EPI marker NANOG (orange); nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Dotted line encircles the embryo. Scale bar is indicated in each image. (D) 7dpf F-ATT embryos (n = 4) embryo attached on the polar TE; Z-stack section Attachment (ATT) site = section 3. (E) 7dpf F-ATT embryo attached on the mural TE; Z-stack section TE midsection = section 4. (H) 8dpf F-ATT embryo (n = 2) attached on the polar TE. (I) Negative immunostaining by replacing primary antibodies with rabbit IgGs (NANOG; orange) on a human embryo. Figure 1 View largeDownload slide Developmental stages of 6 days post-fertilization (dpf) human embryos co-cultured to 8dpf (48 h) in the 2D in vitro model. (A–C, F, G) Phase contrast images taken by EVOS FL Cell Imaging System; all images were taken on 10× magnification and scale bar is indicated in each image. (A) 6Dpf human blastocysts prior to starting co-culture; (B) 7dpf embryo attached on polar trophectoderm (TE), arrow points to ICM (focus point); (C) 7dpf embryo attached on mural TE, arrow points to ICM (focus point); (F) 8dpf embryo attached on polar TE, arrow points to ICM and white dotted line encircles attachment site (focus point); (G) 8dpf flattened embryo. (D, E, H). Immunostaining of the EPI marker NANOG (orange); nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Dotted line encircles the embryo. Scale bar is indicated in each image. (D) 7dpf F-ATT embryos (n = 4) embryo attached on the polar TE; Z-stack section Attachment (ATT) site = section 3. (E) 7dpf F-ATT embryo attached on the mural TE; Z-stack section TE midsection = section 4. (H) 8dpf F-ATT embryo (n = 2) attached on the polar TE. (I) Negative immunostaining by replacing primary antibodies with rabbit IgGs (NANOG; orange) on a human embryo. Immunofluorescence microscopy for the EPI marker NANOG (Roode et al., 2012) in the F-ATT embryos confirmed attachment on the polar (Fig. 1D) and mural (Fig. 1E) TE. An increase in TE was observed towards 7dpf, progressing in time until the attached embryo spread itself and a clear attachment site was apparent between the Ishikawa cells (Fig. 1C and F). As embryos continued to spread, they progressively lost their spherical shape and by 8dpf acquired a flattened morphology (Fig. 1G). The ICM was barely visible by the light microscope due to the embryonic shape transformation, however positive immunostaining for the NANOG in 8dpf F-ATT embryos confirmed expansion of the ICM in the in vitro model (Fig. 1H). In parallel, immunostaining for the TB marker cytokeratin 7 (KRT7) (Hannan et al., 2010) in 6dpf, 7dpf and 8dpf F-ATT embryos, showed a time-dependent increase in positive cells (Fig. 2A (arrow), B and C). Three embryos degenerated following attachment. Figure 2 View largeDownload slide Immunostaining for TB markers KRT7 and hCG; nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (grey or blue). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Scale bar is indicated in each image. Dotted line encircles the embryo. Full Z-stack images of KRT7 (violet) staining in (A) 6dpf blastocyst (n = 3), arrow points to a few KRT7-positive TE cells; (B) 7dpf F-ATT embryo (n = 3) and (C) 8dpf F-ATT embryo (n = 3). (D) HCG hormone staining (green) in 8dpf F-ATT embryo (n = 3). Z-stack section attachment (ATT) site = section 7; the arrows point to hCG-positive outgrowth formation. (E) Negative immunostaining by replacing primary antibodies with mouse (KRT7; violet) or rabbit (hCG; green) IgGs on a human embryo. Figure 2 View largeDownload slide Immunostaining for TB markers KRT7 and hCG; nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (grey or blue). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Scale bar is indicated in each image. Dotted line encircles the embryo. Full Z-stack images of KRT7 (violet) staining in (A) 6dpf blastocyst (n = 3), arrow points to a few KRT7-positive TE cells; (B) 7dpf F-ATT embryo (n = 3) and (C) 8dpf F-ATT embryo (n = 3). (D) HCG hormone staining (green) in 8dpf F-ATT embryo (n = 3). Z-stack section attachment (ATT) site = section 7; the arrows point to hCG-positive outgrowth formation. (E) Negative immunostaining by replacing primary antibodies with mouse (KRT7; violet) or rabbit (hCG; green) IgGs on a human embryo. We stained co-cultured embryos for the STB marker hCG to assess the onset of invasion in 8dpf F-ATT embryos (n = 4). Although hCG was present in some TB cells, immunofluorescence was much more prominent in outgrowing cells at the attachment site (Fig. 2D, arrows). Thus, the sequence of events, i.e. progression from loose to firm embryo attachment followed by initiation of invasion, indicates that early human implantation events were recapitulated in our co-culture model. As the majority of the 7dpf embryos were irreversibly attached, we set out to examine the expression of ECM and adhesion genes in L-ATT human blastocysts recovered after 12 h in co-culture to determine their involvement in the initial implantation process. Embryonic gene expression Of 37 6dpf human embryos cultured for 12 h on Ishikawa cells, 15 embryos were L-ATT (41%) and 10 were F-ATT (27%), giving a cumulative attachment rate of 68%. Twelve blastocysts remained UNATT. Expanded and good quality UNATT (n = 8) and L-ATT (n = 10) embryos were collected for gene expression analysis. Out of the 10 L-ATT embryos, three were excluded because of low cDNA yield (<10 ng/μl). Co-culture of the remaining UNATT, L-ATT and F-ATT embryos were extended to 7dpf and all the F-ATT were fixed for immunofluorescence microscopy. The expression of 84 adhesion and ECM genes was analysed in eight UNATT, seven L-ATT embryos, eight 6pdf blastocysts cultured for 12 h in blastocyst culture medium, and four samples of Ishikawa cells. All of the results are shown in the heatmap in Fig. 3. Eleven genes were not expressed in any of the samples. The 6-dpf and UNATT groups of embryos expressed 47 genes, the L-ATT group of embryos expressed 51 and Ishikawa cells expressed 65 genes. Figure 3 View largeDownload slide Heatmap representing average 2−ΔCt values of the 84 adhesion and ECM genes analysed by qPCR in 6 days post-fertilization (dpf) human embryos and in Ishikawa cells co-cultured unattached (UNATT) and loosely attached (L-ATT) embryos. The colour scale represents the relative abundance of the transcript as compared to the endogenous control and in comparison to the expression levels of all other transcripts. Figure 3 View largeDownload slide Heatmap representing average 2−ΔCt values of the 84 adhesion and ECM genes analysed by qPCR in 6 days post-fertilization (dpf) human embryos and in Ishikawa cells co-cultured unattached (UNATT) and loosely attached (L-ATT) embryos. The colour scale represents the relative abundance of the transcript as compared to the endogenous control and in comparison to the expression levels of all other transcripts. First, to examine if we detected embryo-specific gene expression and not contamination with Ishikawa transcripts, we compared the gene expression profile of Ishikawa cells to that of the co-cultured embryos. Of the 65 genes expressed by the Ishikawa cells, 50 were in common with the co-cultured embryos (Fig. 3), while 15 transcripts were uniquely expressed by Ishikawa cells, such as COL7A1, LAMA2 and MMP10. The exclusive detection of genes in Ishikawa cells and not in the co-cultured embryos suggests that contamination of the co-cultured embryos by RNA originating from Ishikawa cells or extracellular vesicles (Pavani et al., 2017) was low and will not interfere with the detection of an embryo-specific gene expression profile. Next, we focussed on those genes expressed in ≥70% of the embryos per experimental group. L-ATT embryos not only expressed all 30 genes present in UNATT group but also COL12A1, CTNND2, ITGA3, ITGAV, LAMA3, THBS1 and TNC (Fig. 4). Genes with statistically significant different expression or with a high FC between the UNATT and L-ATT group of embryos are shown in Fig. 5. Despite the inter-embryo variability in gene expression, five genes were statistically differentially expressed: CD44 (P = 0.014, FC = 50.3), LAMA3 (P = 0.021, FC = 160.8), CTNNB1 (P = 0.029, FC = 1.7), TIMP1 (P = 0.029, FC = 14.2) and COL12A1 (P = 0.040, FC = 121.2). The results for CTNNB1 should be interpreted with caution as the FC falls within the technical limit of the qPCR analysis (i.e. <2-fold difference). Although not statistically different, two genes were highly induced in L-ATT embryos: THBS1 (P = 0.094, FC = 905.7) and TNC (P = 0.112, FC = 143.8). A single embryo in the UNATT group showed a high expression of both THBS1 and TNC (red circle, Fig. 5) and if omitted from the analysis, the results were a P-value <0.05 for both genes. Worth noting is that all significant differences between the two groups of embryos were always due to an up-regulation in expression in the L-ATT embryos. Also, none of the embryos expressed transcripts coding selectins (SELE, SELL or SELP) (Fig. 3). Finally, the gene expression analysis of the 6dpf embryos cultured in blastocyst medium shows no statistically significant differences to the UNATT embryos, but only to the L-ATT (Supplementary Fig. S1). This highlights that embryo attachment is the major modulator in the expression of these genes. Genes expressed in ≥70% of the group of blastocysts are shared with the UNATT group of embryos. All data on the differential gene expression in the embryos are shown in Supplementary Fig. S1 and Supplementary Table. Figure 4 View largeDownload slide Venn diagram representing genes expressed in ≥70% of the UNATT and L-ATT groups of embryos. The latter is marked by the additional expression of seven genes. Figure 4 View largeDownload slide Venn diagram representing genes expressed in ≥70% of the UNATT and L-ATT groups of embryos. The latter is marked by the additional expression of seven genes. Figure 5 View largeDownload slide Graphs representing expression levels of statistically significant or most upregulated adhesion and ECM genes in unattached (UNATT) (n = 8) and loosely attached (L-ATT) (n = 7) embryos following co-culture with Ishikawa cells. Gene expression analysis was performed by qPCR and data are presented as 2−ΔCt values. Normalization was performed to the geometrical mean of GAPDH, GUSB and PGK. Each dot represents an embryo and data is represented as mean with range. Red circle indicated the same embryo expressing both THBS1 and TNC at high levels. Statistical analysis between the UNATT and L-ATT group of embryos was performed with unpaired Mann–Whitney U test on the ΔCt values. Figure 5 View largeDownload slide Graphs representing expression levels of statistically significant or most upregulated adhesion and ECM genes in unattached (UNATT) (n = 8) and loosely attached (L-ATT) (n = 7) embryos following co-culture with Ishikawa cells. Gene expression analysis was performed by qPCR and data are presented as 2−ΔCt values. Normalization was performed to the geometrical mean of GAPDH, GUSB and PGK. Each dot represents an embryo and data is represented as mean with range. Red circle indicated the same embryo expressing both THBS1 and TNC at high levels. Statistical analysis between the UNATT and L-ATT group of embryos was performed with unpaired Mann–Whitney U test on the ΔCt values. Validation of LAMA3, THBS1 and TNC expressions at the protein level To validate expression at protein level, we focussed on LAMA3, THBS1 and TNC, the three most highly induced genes in L-ATT embryos. Immunostaining for the encoded proteins was performed in 6dpf blastocysts cultured in blastocyst medium (n = 3) and 7dpf F-ATT embryos (n = 3 for LAMA3; n = 4 for THBS1 and TNC) in the co-culture model. LAMA3 encodes for laminin subunit alpha 3, a secreted protein and member of the laminin family. The protein was present in the trophectoderm of both the 6dpf and 7dpf F-ATT embryos (Fig. 6). LAMA3 showed predominantly membrane staining (Fig. 6B, zoom TE midsection). Within an embryo, inter-cellular variability was observed in the LAMA3 staining, ranging from none to very bright detection. Remarkably, the protein was barely detected at the attachment site of two 7dpf F-ATT embryos (Fig. 6B, zoom TE Full Z-stack). Figure 6 View largeDownload slide Immunostaining for LAMA3 (green); nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Scale bar is indicated in each image. White dotted line encircles the embryo. Blue dotted line indicates a zoom in a region of interest. (A) 6dpf blastocyst (n = 3); Trophectoderm (TE) midsection = section 15. (B) 7dpf F-ATT embryos (n = 3); TE midsection = section 16. (C) Negative immunostaining by replacing primary antibodies with mouse IgGs (green). Figure 6 View largeDownload slide Immunostaining for LAMA3 (green); nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Scale bar is indicated in each image. White dotted line encircles the embryo. Blue dotted line indicates a zoom in a region of interest. (A) 6dpf blastocyst (n = 3); Trophectoderm (TE) midsection = section 15. (B) 7dpf F-ATT embryos (n = 3); TE midsection = section 16. (C) Negative immunostaining by replacing primary antibodies with mouse IgGs (green). THBS1 and TNC encode thrombospondin 1 (THBS1) and tenascin C (TNC), respectively, and were assessed by double staining on the same embryos. THBS1 was detected in the cytoplasm of the TE cells of both 6dpf and 7dpf F-ATT embryos (Fig. 7A and B, Supplementary Fig. S2). However, the immunofluorescent signal for THBS1 was less prominent in 7dpf F-ATT embryos, suggesting that this ECM protein is either secreted or only transiently expressed upon contact with the Ishikawa cells. Inhibition of protein secretion was attempted by treating 6dpf (n = 3) and 7dpf F-ATT (n = 3) embryos with 2 μg/ml Brefeldin A prior to fixation (Ripley et al., 1993). However, unlike 6dpf blastocysts that survived the 2 h treatment, attached embryos degenerated within 30 min of exposure to Brefeldin A, precluding further analysis. TNC protein was barely detectable in the 6dpf blastocysts (Fig. 7A) but clearly localized to the membrane of TE cells in 7dpf F-ATT embryos (Fig. 7B, zoom TE Midsection). Thus, the temporal expression of TNC at the protein level was in line with the gene expression data. Furthermore, the protein was present in different patches throughout the whole TE, also at the attachment site (Fig. 7B, Supplementary Fig. S2B). To investigate expression in the ICM, embryos were co-stained for NANOG (Supplementary Fig. S2A and B). Only THBS1 co-localized with NANOG in 7dpf F-ATT embryos. In Ishikawa cells, intense THBS1 and TNC immunofluorescent foci were observed in keeping with their localization in membrane-bound aggregates (Fig. 7C and D). Figure 7 View largeDownload slide Immunostaining for THBS1 (violet) and TNC (green); nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (grey). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Scale bar is indicated in each image. White dotted line encircles the embryo. Blue dotted line indicates a zoom in a region of interest. (A) 6dpf blastocyst (n = 3); trophectoderm (TE) midsection = section 9. (B) 7dpf F-ATT embryos (n = 4); TE midsection = section 9. (C, D) Staining on Ishikawa cells. (E) Negative immunostaining by replacing primary antibodies with mouse (THBS1; violet) or rat (TNC; green) IgGs on Ishikawa cells. Figure 7 View largeDownload slide Immunostaining for THBS1 (violet) and TNC (green); nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) (grey). Confocal microscopy was used to visualize the stainings and images are shown as full Z-stack or an indicated section. Scale bar is indicated in each image. White dotted line encircles the embryo. Blue dotted line indicates a zoom in a region of interest. (A) 6dpf blastocyst (n = 3); trophectoderm (TE) midsection = section 9. (B) 7dpf F-ATT embryos (n = 4); TE midsection = section 9. (C, D) Staining on Ishikawa cells. (E) Negative immunostaining by replacing primary antibodies with mouse (THBS1; violet) or rat (TNC; green) IgGs on Ishikawa cells. Because of the unambiguous presence of TNC in the TE cells, we examined if it plays an essential role for embryo attachment and invasion in the in vitro model. For this, 6dpf blastocysts were co-cultured with Ishikawa cells for 48 h in the presence of a neutralizing TNC antibody (TNC Ab, same antibody as used for immunofluorescence microscopy) or IgGA2 isotype control (IgG). Embryo attachment was evaluated after 24 and 48 h (7dpf and 8dpf, respectively) (Supplementary Table). Only stable attachment was observed, irrespective of the presence or absence of the TNC Ab. Secreted hCG levels were also measured to assess the impact of TNC blocking on invasion but again no significant differences were observed (Supplementary Fig. S3). Discussion Embryo implantation is the rate-liming step in reproduction. It is also a poorly understood process due to the limitations of both animal and in vitro models. Here we demonstrate that a relatively simple 2D co-culture system can be used to investigate changes in embryonic morphology and gene expression associated with the initial steps in the implantation process. When co-cultured with Ishikawa, human embryos progress from free-floating to loose attachment, firm adherence and ultimately invasion. Notably, loose attachment is a reversible process whereas subsequent firm embryo attachment is not (Aplin and Ruane, 2017). We also observed that a majority of human blastocysts (83%) attached to Ishikawa cells by the polar TE, in line with previous reports (Lindenberg, 1991; Grewal et al., 2008). By contrast, mouse embryo attachment is mediated by TE cells surrounding the blastocyst’s cavity (mural TE). Additionally, initial positioning of the murine ICM determines the developmental potential of the oocyte cylinder (Wu et al., 1981). The importance of orientation of human embryos upon attachment is not yet been established but may be linked to proper endometrial embedment and subsequent TB invasion and placenta formation. The attachment rate in our co-culture system was 100% by 8dpf, although the kinetics of this process differed markedly between individual embryos. Further, a number of embryos degraded following attachment, indicating that initiation of the implantation process does not guarantee further progression. If extrapolated to the in vivo situation, our findings suggest that most, if not all, good quality embryos will initiate attachment with receptive luminal epithelial cells, and that subsequent failure may either be due to chromosomal errors (Wells et al., 1999; Fiorentino et al., 2011) or to an aberrant decidual response (Durairaj et al., 2017). The gradual spreading of the attachment site is thought to reflect breaching of epithelial cells by the embryo (Lindenberg, 1991; Bentin-Ley et al., 2000; Ruane et al., 2017) and, combined with the flattened morphology, reflect the acquisition of an invasive embryonic phenotype (Grewal et al., 2008). These phenomena coincide with hCG secretion and the increased KRT7 protein expression (Niakan and Eggan, 2013; Deglincerti et al., 2016; Shahbazi et al., 2016). Transcriptional analysis of loosely attached versus unattached embryos strongly infer that induction of ECM and adhesion genes plays a role in initiating the implantation process. However, inter-embryo variability was marked, both in terms of gene expression and kinetics of the attachment response, which is not surprising given the intrinsic heterogeneity of pre-implantation human embryos (Shaw et al., 2013). L-selectin coding transcripts were below the detection levels in 6dpf blastocysts. Further, SELL expression was also not found in earlier human pre-implantation stages (Bloor et al., 2002). However, lack of detectable SELL mRNA expression in blastocysts does not necessarily preclude the presence of the protein if transcribed from the maternal genome prior to embryonic genome activation (Braude et al., 1988; Vassena et al., 2011). Experimental data indicated that interaction between integrin αvβ3 expressed on human blastocysts and on apical endometrial epithelial cells via the bridging molecule osteopontin is involved in the attachment step (Kang et al., 2014). We confirmed the presence of ITGAV and ITGB3 transcripts in human blastocysts but also identified other adhesion genes, including ITGB5 and CD44. Further, upon attachment, human embryos express several genes involved in ECM deposition and turnover, including FN1 (fibronectin 1), LAMA3 (laminin subunit alpha 3), COL12A1 (collagen type XII alpha 1 chain), MMP14 (membrane-type matrix metallopeptidase 14) and TIMP1 (TIMP metallopeptidase inhibitor 1), in keeping with an active role for the conceptus in effecting endometrial remodelling during the implantation process (Harburger and Calderwood, 2009; Uchida et al., 2012; Macklon and Brosens, 2014). LAMA3, THB1 and TNC all bind with different integrins and play a role in cell attachment, migration and organization (Miner and Yurchenco, 2004; Domogatskaya et al., 2012; Resovi et al., 2014; Tucker and Chiquet-Ehrismann, 2015; Midwood et al., 2016). Like osteopontin, these three ECM components may be bridging mediators between the TE and the luminal epithelium. A number of integrins, α1β1, α4β1, αvβ3 and α6β1, are expressed in the receptive endometrium (Tabibzadeh, 1992; van Mourik et al., 2008; Elnaggar et al., 2017). Laminin family members are important modulators of the murine invasion step (Zhang et al., 2000; Miner et al., 2004). In the human, laminin was detected in early pre-implantation embryos (Turpeenniemi-Hujanen et al., 1992) and plays a role in TB invasion (Wang et al., 2013; Shan et al., 2015). In murine blastocysts, THBS1 is expressed on the TE surface and TE outgrowth is attenuated in the presence of a blocking antibody (O’Shea et al., 1990). In human blastocysts, THBS1 immunofluorescence was more prominent in control embryos when compared to 7dpf F-ATT embryos, suggesting active secretion of THSB1 into the ECM. TNC has been implicated in neural crest formation (Davoli et al., 2001), but its role in embryo implantation has not yet been investigated. TNC immunofluorescence was most prominent in TE of attached embryos. Functional analysis did not reveal an essential role of this ECM glycoprotein either attachment or differentiation of CTB (as reflected by hCG secretion) in our in vitro model. However, these observations do not negate an important role for embryonic TNC expression during implantation in vivo, and possibly suggest functional redundancy in the many ligands that bind different integrins. Also the neutralizing activity of the antibody was only validated in a mouse study (Harada et al., 2015). Detection of LAMA3, THBS1 and TNC in the embryo confirmed our embryo-specific gene expression analysis. Different localization patterns observed in the analysed proteins might be due to the ongoing TB lineage differentiation. Expression of different integrins and their ligands in human blastocysts highlights the importance of these adhesion molecules in processes underlying the early steps of embryo implantation. In summary, in this ‘proof of concept’ study, we demonstrate that a 2D co-culture system can be used to interrogate the morphological and gene expression changes in human embryos that progress through the apposition and adhesion steps of the implantation process upon co-culture with endometrial epithelial cells. We envisage that the current model can be refined in several aspects. For example, embryo co-cultures are commonly performed under atmospheric air (21% O2) conditions (Bentin-Ley et al., 2000; Lalitkumar et al., 2007; Grewal et al., 2008; Singh et al., 2010; Kang et al., 2014; Berger et al., 2015). However, experiments under 5% O2 may be more informative as culturing pre-implantation human embryos under low oxygen concentrations has been reported to improve life birth rates (Bontekoe et al., 2012). Further, application of emerging technologies, such as RNA-sequencing combined with CRISPR/Cas9 gene editing, provide a powerful approach to both exhaustively map and functionally investigate key embryonic genes and pathways involved in human implantation. Supplementary data Supplementary data are available at Molecular Human Reproduction online. Acknowledgements We are grateful to all the patients donating embryos for research. We are also thankful to the staff, with a special acknowledgment to the lab technicians, of the Centre for Reproductive Medicine for creating a proper environment for us to warm the cryopreserved embryos. J Schiettecatte and P. Roelandt were of great help in performing the hCG measurements. We also thank our colleagues at the Research Group REGE and Prof. G. Nie (Hudson Institute, Australia) for the critical discussions on the data. Authors’ roles A.A. and H.V.D.V designed the study. A.A. executed the experimental work. 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Molecular Human Reproduction – Oxford University Press
Published: May 26, 2018
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