Conceived and designed the experiments: SA SH. Performed the experiments: SA IB. Analyzed the data: SA. Contributed reagents/materials/analysis tools: SA IB DH CM HD IJK SH. Wrote the paper: SA SH. Final approval: SH.
The authors have read the journal’s policy and have the following conflicts: This study was funded by Ferring and Genevrier pharmaceutical companies. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our
Pre-implantation development of mammalian embryos encompasses a series of critical dynamic events, such as the transition from a single-cell zygote to a multicellular blastocyst and the first segregation of cells within the embryo with the formation of the inner cell mass (ICM) surrounded by trophectoderm (TE) cells. ICM retains pluripotency and gives rise to the embryo proper, whereas TE cells play an important role in embryonic implantation in the uterine endometrium and placental formation. In humans, the embryonic genome activation (EGA) program is functional by day 3 after fertilization
In order to determine the global gene expression variation in the different samples, we established the gene expression profile of mature MII oocytes (n = 3), day 3 single embryos (n = 6), TE samples from day 5 blastocysts (n = 5) and hESCs (n = 4) (to represent the ICM) by using high-density oligonucleotide Affymetrix HG-U133P microarray chips. A non-supervised analysis using the principal components analysis (PCA) showed that samples from the same group clustered together very tightly (
(A) PCA two-dimensional scatter plots represent the differential gene expression patterns of the different human samples. Each dot represents a sample and the color its origin: oocytes (green dots), day 3 embryos (blue dots), TE from day 5 embryos (black dots) and hESCs (red dots). Samples can be divided in four distinct areas based on their gene expression. (B) Average-link hierarchical clustering of 15,000 genes delineated four major gene clusters: (a) genes specifically detected in mature MII oocytes; (b) genes over-expressed in hESCs; (c) genes up-regulated in TE and (d) genes specifically over-expressed in day 3 embryos.
We then compared the expression profiles of day 3 embryos and TE cells, by using the significance analysis of microarrays (SAM) software with a 2-fold change cut-off and false discovery rate (FDR) <1%. We found that 2,196 transcripts were up-regulated in human TE cells (“TE molecular signature”) and 1,714 in day 3 embryos (“day 3 embryo molecular signature”) (
Heat map of the molecular signatures in six day 3 embryos and five TE samples. Each horizontal line represents a gene and each column represents a single sample. The color intensity indicates the level of gene expression (red for up-regulation and blue for down-regulation) “see also
We compared the GO annotations of genes specifically over-expressed in day 3 embryos and in TE cells by using the Babelomics web tool (
Probesets | Gene Name | Gene Title | UniGene | ChromosomalLocation | Fold change | FDR (%) |
1552531_a_at | NALP11 | NLR family, pyrin domain containing 11 | Hs.375039 | chr19q13.42 | 1893 | 0 |
242334_at | NALP4 | NLR family, pyrin domain containing 4 | Hs.631533 | chr19q13.42 | 892 | 0 |
214957_at | ACTL8 | actin-like 8 | Hs.2149 | chr1p36.2-p35 | 755 | 0 |
1556096_s_at | UNC13C | unc-13 homolog C | Hs.443456 | chr15q21.3 | 663 | 0 |
207443_at | NR2E1 | nuclear receptor subfamily 2, group E, member 1 | Hs.157688 | chr6q21 | 625 | 0 |
1553619_a_at | TRIM43 | tripartite motif-containing 43 | Hs.589730 | chr2q11.1 | 519 | 0 |
1552405_at | NALP5 | NLR family, pyrin domain containing 5 | Hs.356872 | chr19q13.42 | 448 | 0 |
209160_at | AKR1C3 | aldo-keto reductase family 1, member C3 | Hs.78183 | chr10p15-p14 | 431 | 0 |
1552456_a_at | MBD3L2 | methyl-CpG binding domain protein 3-like 2 | Hs.567667 | chr19p13.2 | 394 | 0 |
1557085_at | TMEM122 | placenta-specific 1-like | Hs.132310 | chr11q12.1 | 387 | 0 |
39318_at | TCL1A | T-cell leukemia/lymphoma 1A | Hs.2484 | chr14q32.1 | 342 | 0 |
234393_at | HDAC9 | histone deacetylase 9 | Hs.196054 | chr7p21.1 | 315 | 0 |
1552912_a_at | IL23R | interleukin 23 receptor | Hs.200929 | chr1p31.3 | 306 | 0 |
1552852_a_at | ZSCAN4 | zinc finger and SCAN domain containing 4 | Hs.469663 | chr19q13.43 | 282 | 0 |
226117_at | TIFA | TRAF-interacting protein with a forkhead-associated domain | Hs.310640 | chr4q25 | 275 | 0 |
222361_at | LOC643224 | similar to tubulin, beta 8 | Hs.551805 | chr9q34.3 | 273 | 0 |
229105_at | GPR39 | G protein-coupled receptor 39 | Hs.432395 | chr2q21-q22 | 255 | 0 |
225626_at | PAG1 | phosphoprotein associated with glycosphingolipid microdomains 1 | Hs.266175 | chr8q21.13 | 230 | 0 |
1557544_at | C10orf80 | chromosome 10 open reading frame 80 | Hs.253576 | chr10q25.1 | 209 | 0 |
210634_at | KLHL20 | kelch-like 20 (Drosophila) | Hs.495035 | chr1q24.1-q24.3 | 206 | 0 |
206343_s_at | NRG1 | neuregulin 1 | Hs.453951 | chr8p21-p12 | 184 | 0 |
207213_s_at | USP2 | ubiquitin specific peptidase 2 | Hs.524085 | chr11q23.3 | 182 | 0 |
1563120_at | Hs.623820 | Homo sapiens, clone IMAGE:5528155, mRNA | Hs.630724 | 175 | 0 | |
237131_at | LOC645469 | hypothetical protein FLJ36032 | Hs.297967 | chr1q21.3 | 172 | 0 |
221630_s_at | DDX4 | DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 | Hs.223581 | chr5p15.2-p13.1 | 171 | 0 |
241550_at | DPPA5 | developmental pluripotency associated 5 | Hs.125331 | chr6q13 | 167 | 0 |
217365_at | PRAMEF5 | similar to PRAME family member 6 | chr1p36.21 | 157 | 0 | |
1570337_at | FIGLA | folliculogenesis specific basic helix-loop-helix | Hs.407636 | chr2p13.3 | 157 | 0 |
206140_at | LHX2 | LIM homeobox 2 | Hs.445265 | chr9q33-q34.1 | 154 | 0 |
229738_at | DNAH10 | dynein, axonemal, heavy chain 10 | Hs.622654 | chr12q24.31 | 154 | 0 |
209785_s_at | PLA2G4C | phospholipase A2, group IVC (cytosolic, calcium-independent) | Hs.631562 | chr19q13.3 | 149 | 0 |
237613_at | FOXR1 | forkhead box R1 | Hs.116679 | chr11q23.3 | 147 | 0 |
236914_at | AW080028 | 137 | 0 | |||
210467_x_at | MAGEA12 | melanoma antigen family A, 12 | chrXq28 | 137 | 0 | |
242128_at | OTX2 | orthodenticle homolog 2 (Drosophila) | Hs.288655 | chr14q21-q22 | 128 | 0 |
220535_at | FAM90A1 | family with sequence similarity 90, member A1 | Hs.196086 | chr12p13.31 | 128 | 0 |
215048_at | SUHW2 | suppressor of hairy wing homolog 2 (Drosophila) | Hs.43834 | chr22q11.22 | 127 | 0 |
207934_at | RFPL1 | ret finger protein-like 1 | Hs.648249 | chr22q12.2 | 127 | 0 |
209994_s_at | ABCB1 | ATP-binding cassette, sub-family B (MDR/TAP), member 1 | Hs.489033 | chr7q21.1 | 125 | 0 |
207227_x_at | RFPL2 | ret finger protein-like 2 | Hs.157427 | chr22q12.3 | 116 | 0 |
238218_at | LOC648473 | hypothetical protein LOC648473 | 112 | 0 | ||
214603_at | MAGEA2 | melanoma antigen family A, 2 | Hs.169246 | chrXq28 | 111 | 0 |
217590_s_at | TRPA1 | transient receptor potential cation channel, subfamily A, member 1 | Hs.137674 | chr8q13 | 110 | 0 |
208312_s_at | PRAMEF1 | PRAME family member 1 | Hs.104991 | chr1p36.21 | 109 | 0 |
223866_at | ARMC2 | armadillo repeat containing 2 | chr6q21 | 106 | 0 | |
216001_at | LOC390999 | PRAME family member 12 | Hs.156406 | chr1p36.21 | 106 | 0 |
213228_at | PDE8B | phosphodiesterase 8B | Hs.584830 | chr5q14.1 | 104 | 0 |
1552807_a_at | SIGLEC10 | sialic acid binding Ig-like lectin 10 | Hs.284813 | chr19q13.3 | 104 | 0 |
236205_at | Hs.13188 | similar to ATP-binding cassette, sub-family C, member 6 | Hs.13188 | chr16p12.3 | 104 | 0 |
209942_x_at | MAGEA3 | melanoma antigen family A, 3 | Hs.417816 | chrXq28 | 100 | 0 |
226271_at | GDAP1 | ganglioside-induced differentiation-associated protein 1 | Hs.168950 | chr8q21.11 | 98 | 0 |
240031_at | AA994467 | Baculoviral IAP repeat-containing 2 | Hs.503704 | chr11q22 | 98 | 0 |
209570_s_at | D4S234E | DNA segment on chromosome 4 (unique) 234 expressed sequence | Hs.518595 | chr4p16.3 | 98 | 0 |
206207_at | CLC | Charcot-Leyden crystal protein | Hs.889 | chr19q13.1 | 96 | 0 |
230626_at | TSPAN12 | tetraspanin 12 | Hs.16529 | chr7q31.31 | 93 | 0 |
216034_at | SUHW1 | suppressor of hairy wing homolog 1 (Drosophila) | Hs.178665 | chr22q11.22 | 89 | 0 |
231756_at | ZP4 | zona pellucida glycoprotein 4 | Hs.136241 | chr1q43 | 89 | 0 |
202388_at | RGS2 | regulator of G-protein signalling 2, 24 kDa | Hs.78944 | chr1q31 | 85 | 0 |
205747_at | CBLN1 | cerebellin 1 precursor | Hs.458423 | chr16q12.1 | 84 | 0 |
230753_at | LOC197135 | hypothetical LOC197135 | Hs.11594 | chr15q21.1 | 83 | 0 |
236117_at | Hs.42747 | Transcribed locus | Hs.42747 | 83 | 0 | |
1556834_at | Hs.562766 | CDNA clone IMAGE:5296106 | Hs.562766 | 83 | 0 | |
209278_s_at | TFPI2 | tissue factor pathway inhibitor 2 | Hs.438231 | chr7q22 | 81 | 0 |
240318_at | AFMID | Arylformamidase | Hs.558614 | chr17q25.3 | 80 | 0 |
1557257_at | BCL10 | B-cell CLL/lymphoma 10 | Hs.193516 | chr1p22 | 80 | 0 |
236504_x_at | C6orf52 | chromosome 6 open reading frame 52 | Hs.61389 | chr6p24.1 | 80 | 0 |
204438_at | MRC1 | mannose receptor, C type 1 | Hs.75182 | chr10p12.33 | 80 | 0 |
1559108_at | VPS53 | Vacuolar protein sorting 53 (S. cerevisiae) | Hs.461819 | chr17p13.3 | 79 | 0 |
210180_s_at | SFRS10 | splicing factor, arginine/serine-rich 10 (transformer 2 homolog, Drosophila) | Hs.533122 | chr3q26.2-q27 | 77 | 0 |
214960_at | API5 | apoptosis inhibitor 5 | Hs.435771 | chr11p12-q12 | 77 | 0 |
232692_at | TDRD6 | tudor domain containing 6 | Hs.40510 | chr6p12.3 | 76 | 0 |
240731_at | LOC441316 | 76 | 0 | |||
230697_at | BBS5 | Bardet-Biedl syndrome 5 | Hs.233398 | chr2q31.1 | 75 | 0 |
244206_at | ANUBL1 | AN1, ubiquitin-like, homolog (Xenopus laevis) | Hs.89029 | chr10q11.21 | 75 | 0 |
222921_s_at | HEY2 | hairy/enhancer-of-split related with YRPW motif 2 | Hs.144287 | chr6q21 | 74 | 0 |
1557146_a_at | FLJ32252 | hypothetical protein FLJ32252 | Hs.250557 | chr16p13.3 | 73 | 0 |
241382_at | PCP4L1 | Purkinje cell protein 4 like 1 | Hs.433150 | chr1q23.3 | 73 | 0 |
226811_at | FAM46C | family with sequence similarity 46, member C | Hs.356216 | chr1p12 | 73 | 0 |
44783_s_at | HEY1 | hairy/enhancer-of-split related with YRPW motif 1 | Hs.234434 | chr8q21 | 73 | 0 |
239061_at | TPRXL | tetra-peptide repeat homeobox-like | Hs.638296 | chr3p25.1 | 72 | 0 |
223562_at | PARVG | parvin, gamma | Hs.565777 | chr22q13.2-q13 | 69 | 0 |
219352_at | HERC6 | hect domain and RLD 6 | Hs.529317 | chr4q22.1 | 69 | 0 |
1553697_at | C1orf96 | chromosome 1 open reading frame 96 | Hs.585011 | chr1q42.13 | 68 | 0 |
1568924_a_at | FLJ35834 | hypothetical protein FLJ35834 | Hs.159650 | chr7q31.32 | 68 | 0 |
221314_at | GDF9 | growth differentiation factor 9 | Hs.25022 | chr5q31.1 | 67 | 0 |
228737_at | C20orf100 | chromosome 20 open reading frame 100 | Hs.26608 | chr20q13.12 | 66 | 0 |
240070_at | VSIG9 | V-set and immunoglobulin domain containing 9 | Hs.421750 | chr3q13.31 | 66 | 0 |
231448_at | Tenr | testis nuclear RNA-binding protein | Hs.518957 | chr4q27 | 65 | 0 |
214612_x_at | MAGEA6 | melanoma antigen family A, 6 | Hs.441113 | chrXq28 | 64 | 0 |
206696_at | GPR143 | G protein-coupled receptor 143 | Hs.74124 | chrXp22.3 | 62 | 0 |
205551_at | SV2B | synaptic vesicle glycoprotein 2B | Hs.592018 | chr15q26.1 | 61 | 0 |
219686_at | STK32B | serine/threonine kinase 32B | Hs.133062 | chr4p16.2-p16.1 | 61 | 0 |
230645_at | FRMD3 | FERM domain containing 3 | Hs.127535 | chr9q21.32 | 60 | 0 |
1555396_s_at | LOC340602 | similar to CG32656-PA | Hs.97053 | chrXp11.22 | 59 | 0 |
237464_at | IMAA | SLC7A5 pseudogene | Hs.448808 | chr16p12.2 | 58 | 0 |
212158_at | SDC2 | syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan) | Hs.1501 | chr8q22-q23 | 57 | 0 |
220657_at | KLHL11 | kelch-like 11 (Drosophila) | Hs.592134 | chr17q21.2 | 57 | 0 |
223883_s_at | STK31 | serine/threonine kinase 31 | Hs.309767 | chr7p15.3 | 57 | 0 |
222925_at | DCDC2 | doublecortin domain containing 2 | Hs.61345 | chr6p22.1 | 56 | 0 |
210148_at | AF305239 | homeodomain interacting protein kinase 3 | Hs.201918 | chr11p13 | 56 | 0 |
Probesets | Gene Name | Gene Title | UniGene | Chromosomal Location | Fold Change | FDR (%) |
205980_s_at | ARHGAP8 | Rho GTPase activating protein 8/PRR5-ARHGAP8 fusion | chr22q13.31 | 514 | 0 | |
218237_s_at | SLC38A1 | solute carrier family 38, member 1 | Hs.533770 | chr12q13.11 | 469 | 0 |
201596_x_at | KRT18 | keratin 18 | Hs.406013 | chr12q13 | 445 | 0 |
204515_at | HSD3B1 | hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 | Hs.364941 | chr1p13.1 | 383 | 0 |
227048_at | LAMA1 | laminin, alpha 1 | Hs.270364 | chr18p11.31 | 372 | 0 |
34260_at | KIAA0683 | TEL2, telomere maintenance 2, homolog (S. cerevisiae) | Hs.271044 | chr16p13.3 | 361 | 0 |
224348_s_at | AF116709 | 341 | 0 | |||
223168_at | RHOU | ras homolog gene family, member U | Hs.647774 | chr1q42.11-q42.3 | 310 | 0 |
204158_s_at | TCIRG1 | T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3 | Hs.495985 | chr11q13.2 | 283 | 0 |
212203_x_at | IFITM3 | interferon induced transmembrane protein 3 (1–8 U) | Hs.374650 | chr11p15.5 | 279 | 0 |
242705_x_at | Hs.592928 | Full length insert cDNA clone YT86E01 | Hs.592928 | 277 | 0 | |
204351_at | S100P | S100 calcium binding protein P | Hs.2962 | chr4p16 | 262 | 0 |
201650_at | KRT19 | keratin 19 | chr17q21.2 | 260 | 0 | |
229125_at | ANKRD38 | ankyrin repeat domain 38 | Hs.283398 | chr1p31.3 | 238 | 0 |
224646_x_at | H19 | H19, imprinted maternally expressed untranslated mRNA | Hs.533566 | chr11p15.5 | 208 | 0 |
221538_s_at | PLXNA1 | plexin A1 | Hs.432329 | chr3q21.3 | 204 | 0 |
210381_s_at | CCKBR | cholecystokinin B receptor | Hs.203 | chr11p15.4 | 196 | 0 |
217853_at | TNS3 | tensin 3 | Hs.520814 | chr7p12.3 | 194 | 0 |
209771_x_at | CD24 | CD24 molecule | Hs.644105 | chr6q21 | 194 | 0 |
210201_x_at | BIN1 | bridging integrator 1 | Hs.193163 | chr2q14 | 156 | 0 |
224579_at | Hs.592612 | solute carrier family 38, member 1 | Hs.533770 | chr12q13.11 | 147 | 0 |
204720_s_at | DNAJC6 | DnaJ (Hsp40) homolog, subfamily C, member 6 | Hs.647643 | chr1pter-q31.3 | 135 | 0 |
212444_at | Hs.632997 | CDNA clone IMAGE:6025865 | Hs.632997 | 135 | 0 | |
203767_s_at | STS | steroid sulfatase (microsomal), arylsulfatase C, isozyme S | Hs.522578 | chrXp22.32 | 135 | 0 |
215729_s_at | VGLL1 | vestigial like 1 (Drosophila) | Hs.496843 | chrXq26.3 | 134 | 0 |
227241_at | MUC15 | mucin 15, cell surface associated | Hs.407152 | chr11p14.3 | 133 | 0 |
204121_at | GADD45G | growth arrest and DNA-damage-inducible, gamma | Hs.9701 | chr9q22.1-q22.2 | 125 | 0 |
212077_at | CALD1 | caldesmon 1 | Hs.490203 | chr7q33 | 122 | 0 |
201787_at | FBLN1 | fibulin 1 | Hs.24601 | chr22q13.31 | 121 | 0 |
202286_s_at | TACSTD2 | tumor-associated calcium signal transducer 2 | Hs.23582 | chr1p32-p31 | 109 | 0 |
218571_s_at | CHMP4A | chromatin modifying protein 4A | Hs.279761 | chr14q12 | 108 | 0 |
205829_at | HSD17B1 | hydroxysteroid (17-beta) dehydrogenase 1 | Hs.50727 | chr17q11-q21 | 108 | 0 |
205093_at | PLEKHA6 | pleckstrin homology domain containing, family A member 6 | Hs.253146 | chr1q32.1 | 105 | 0 |
209735_at | ABCG2 | ATP-binding cassette, sub-family G (WHITE), member 2 | Hs.480218 | chr4q22 | 104 | 0 |
213050_at | COBL | cordon-bleu homolog (mouse) | Hs.99141 | chr7p12.1 | 97 | 0 |
205081_at | CRIP1 | cysteine-rich protein 1 (intestinal) | Hs.122006 | chr14q32.33 | 93 | 0 |
209262_s_at | NR2F6 | nuclear receptor subfamily 2, group F, member 6 | Hs.466148 | chr19p13.1 | 91 | 0 |
203438_at | STC2 | stanniocalcin 2 | Hs.233160 | chr5q35.2 | 90 | 0 |
214285_at | FABP3 | fatty acid binding protein 3, muscle and heart | Hs.584756 | chr1p33-p32 | 89 | 0 |
209369_at | ANXA3 | annexin A3 | Hs.480042 | chr4q13-q22 | 89 | 0 |
209723_at | SERPINB9 | serpin peptidase inhibitor, clade B (ovalbumin), member 9 | Hs.104879 | chr6p25 | 88 | 0 |
209921_at | SLC7A11 | solute carrier family 7, (cationic amino acid transporter, y+ system) member 11 | Hs.390594 | chr4q28-q32 | 87 | 0 |
216604_s_at | SLC7A8 | solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 | Hs.632348 | chr14q11.2 | 86 | 0 |
228949_at | GPR177 | G protein-coupled receptor 177 | Hs.647659 | chr1p31.3 | 84 | 0 |
202007_at | NID1 | nidogen 1 | Hs.356624 | chr1q43 | 84 | 0 |
209513_s_at | HSDL2 | hydroxysteroid dehydrogenase like 2 | Hs.59486 | chr9q32 | 83 | 0 |
225520_at | MTHFD1L | methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like | Hs.591343 | chr6q25.1 | 82 | 0 |
202023_at | EFNA1 | ephrin-A1 | Hs.516664 | chr1q21-q22 | 81 | 0 |
205710_at | LRP2 | low density lipoprotein-related protein 2 | Hs.470538 | chr2q24-q31 | 78 | 0 |
217764_s_at | RAB31 | RAB31, member RAS oncogene family | Hs.99528 | chr18p11.3 | 77 | 0 |
225516_at | SLC7A2 | solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 | Hs.448520 | chr8p22-p21.3 | 77 | 0 |
200832_s_at | SCD | stearoyl-CoA desaturase (delta-9-desaturase) | Hs.558396 | chr10q23-q24 | 76 | 0 |
202418_at | YIF1A | Yip1 interacting factor homolog A (S. cerevisiae) | Hs.446445 | chr11q13 | 74 | 0 |
200872_at | S100A10 | S100 calcium binding protein A10 | Hs.143873 | chr1q21 | 74 | 0 |
209603_at | GATA3 | GATA binding protein 3 | Hs.524134 | chr10p15 | 73 | 0 |
1555832_s_at | KLF6 | Kruppel-like factor 6 | Hs.4055 | chr10p15 | 73 | 0 |
202737_s_at | LSM4 | LSM4 homolog, U6 small nuclear RNA associated (S. cerevisiae) | Hs.515255 | chr19p13.11 | 71 | 0 |
226604_at | TMTC3 | transmembrane and tetratricopeptide repeat containing 3 | Hs.331268 | chr12q21.32 | 71 | 0 |
220139_at | DNMT3L | DNA (cytosine-5-)-methyltransferase 3-like | Hs.592165 | chr21q22.3 | 70 | 0 |
206269_at | GCM1 | glial cells missing homolog 1 (Drosophila) | Hs.28346 | chr6p21-p12 | 69 | 0 |
203743_s_at | TDG | thymine-DNA glycosylase | Hs.584809 | chr12q24.1 | 69 | 0 |
219010_at | C1orf106 | chromosome 1 open reading frame 106 | Hs.518997 | chr1q32.1 | 69 | 0 |
225021_at | ZNF532 | zinc finger protein 532 | Hs.529023 | chr18q21.32 | 69 | 0 |
205524_s_at | HAPLN1 | hyaluronan and proteoglycan link protein 1 | Hs.591758 | chr5q14.3 | 68 | 0 |
206548_at | FLJ23556 | hypothetical protein FLJ23556 | chr10q26.11 | 66 | 0 | |
202800_at | SLC1A3 | solute carrier family 1 (glial high affinity glutamate transporter), member 3 | Hs.481918 | chr5p13 | 65 | 0 |
229699_at | Hs.61558 | CDNA FLJ45384 fis, clone BRHIP3021987 | Hs.61558 | 65 | 0 | |
229830_at | Hs.376032 | Transcribed locus | Hs.535898 | 65 | 0 | |
202308_at | SREBF1 | sterol regulatory element binding transcription factor 1 | Hs.592123 | chr17p11.2 | 64 | 0 |
203219_s_at | APRT | adenine phosphoribosyltransferase | Hs.28914 | chr16q24 | 64 | 0 |
225078_at | EMP2 | epithelial membrane protein 2 | Hs.531561 | chr16p13.2 | 64 | 0 |
218180_s_at | EPS8L2 | EPS8-like 2 | Hs.55016 | chr11p15.5 | 63 | 0 |
201440_at | DDX23 | DEAD (Asp-Glu-Ala-Asp) box polypeptide 23 | Hs.130098 | chr12q13.12 | 62 | 0 |
201236_s_at | BTG2 | BTG family, member 2 | Hs.519162 | chr1q32 | 62 | 0 |
218721_s_at | C1orf27 | chromosome 1 open reading frame 27 | Hs.371210 | chr1q25 | 61 | 0 |
223062_s_at | PSAT1 | phosphoserine aminotransferase 1 | Hs.494261 | chr9q21.2 | 61 | 0 |
201613_s_at | AP1G2 | adaptor-related protein complex 1, gamma 2 subunit | Hs.343244 | chr14q11.2 | 60 | 0 |
211986_at | AHNAK | AHNAK nucleoprotein (desmoyokin) | Hs.502756 | chr11q12.2 | 60 | 0 |
223449_at | SEMA6A | sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A | Hs.156967 | chr5q23.1 | 60 | 0 |
1567107_s_at | TPM3 | tropomyosin 4 | Hs.631618 | chr19p13.1 | 58 | 0 |
208659_at | CLIC1 | chloride intracellular channel 1 | Hs.414565 | chr6p22.1-p21.2 | 57 | 0 |
202546_at | VAMP8 | vesicle-associated membrane protein 8 (endobrevin) | Hs.534373 | chr2p12-p11.2 | 57 | 0 |
227042_at | LOC150223 | hypothetical protein LOC150223 | Hs.355952 | chr22q11.21 | 57 | 0 |
202625_at | LYN | v-yes-1 Yamaguchi sarcoma viral related oncogene homolog | Hs.651186 | chr8q13 | 56 | 0 |
235436_at | BE503800 | 55 | 0 | |||
223839_s_at | Hs.597496 | PRO1933 | Hs.597496 | 55 | 0 | |
202830_s_at | SLC37A4 | solute carrier family 37 (glycerol-6-phosphate transporter), member 4 | Hs.132760 | chr11q23.3 | 54 | 0 |
228834_at | TOB1 | transducer of ERBB2, 1 | Hs.649528 | chr17q21 | 54 | 0 |
210589_s_at | GBA | glucosidase, beta; acid (includes glucosylceramidase) | Hs.282997 | chr1q21 | 53 | 0 |
208683_at | CAPN2 | calpain 2, (m/II) large subunit | Hs.350899 | chr1q41-q42 | 53 | 0 |
201428_at | CLDN4 | claudin 4 | Hs.647036 | chr7q11.23 | 52 | 0 |
217775_s_at | RDH11 | retinol dehydrogenase 11 (all-trans/9-cis/11-cis) | Hs.226007 | chr14q24.1 | 51 | 0 |
208613_s_at | FLNB | filamin B, beta (actin binding protein 278) | Hs.476448 | chr3p14.3 | 49 | 0 |
230204_at | AU144114 | 49 | 0 | |||
209710_at | GATA2 | GATA binding protein 2 | Hs.367725 | chr3q21.3 | 48 | 0 |
215464_s_at | TAX1BP3 | Tax1 (human T-cell leukemia virus type I) binding protein 3 | Hs.12956 | chr17p13 | 47 | 0 |
1559266_s_at | FLJ45187 | hypothetical protein LOC387640 | Hs.350848 | chr10p12.31 | 47 | 0 |
202090_s_at | UQCR | ubiquinol-cytochrome c reductase, 6.4 kDa subunit | Hs.534521 | chr19p13.3 | 47 | 0 |
209652_s_at | PGF | placental growth factor, vascular endothelial growth factor-related protein | Hs.252820 | chr14q24-q31 | 47 | 0 |
232164_s_at | EPPK1 | epiplakin 1 | Hs.200412 | chr8q24.3 | 47 | 0 |
We then investigated the expression of genes coding for proteins linked to the extrinsic and intrinsic apoptosis pathways in day 3 embryos and TE cells. The expression of genes of the
(A) Histograms show the microarray signal values for apoptosis-related genes in day 3 embryos (black) and TE samples (white). (B) The mean expression level of 48 stemness genes in six day 3 embryos and five TE samples was plotted on a logarithmic scale in a radar graph. Asterisks indicate a statistically significant difference (
The microarrays data were also used to investigate the expression of a comprehensive list of DNA repair genes
We then performed a stemness gene enrichment analysis using a previously published dataset from hESCs, in which we defined a consensus hESC stemness gene list (n = 48 genes)
(A) The interaction network was generated with the Ingenuity software and shows that many genes from the
Since specification of the TE lineage during blastocyst formation involves initiation of differentiation, it is likely that epigenetic regulators may have an important role in this first developmental decision. The majority of the epigenetic regulators that were up-regulated in TE cells are associated with a repressive epigenetic status (
In an effort to link the genes involved in the day 3 embryo-TE transition with early embryonic development, we further investigated differences and similarities in the gene expression patterns of MII oocytes, day 3 embryos, TE cells and hESCs samples (comprehensive list in
Here, we compared the transcriptomes of day 3 human embryos and TE cells from day 5 human blastocysts to identify transcripts that are differentially expressed during the embryo-to-TE transition and the specification of the TE cell lineage. Many of the genes that were up-regulated in TE cells are already known to be associated with human TE differentiation
Among the models used to study trophoblast development, hESCs have emerged as a useful tool to examine the emergence and differentiation of TE cells. Particularly, the transcriptomic analysis of TE cells derived from hESCs has provided new insights into the signaling pathways and the molecular mechanisms underlying early trophoblast development. Recently, by using a microarray approach, Marchand and colleagues investigated gene expression during differentiation of hESCs into the trophoblast lineage upon addition of Bone Morphogenetic Protein 4 (BMP4) for 10 days and identified 670 genes that were up-regulated from day 0 to day 10
The “day 3 embryo molecular signature” was enriched in genes from the
Our transcriptome analysis also shows that the TE molecular signature includes many genes that are annotated as “membrane”, demonstrating a strong bias towards genes involved in cell-to-cell communication processes. Conversely, genes specifically expressed by day 3 embryos are largely “nuclear”. Additionally, we categorized the genes that were up-regulated during the MII-day 3 transition according to their molecular and cellular function using the GO annotations and found that they were mainly associated with nuclear localization. This is in line with previously published data showing that proteins produced by the most up-regulated genes during the MII-day 2 embryo transition are mainly localized in the nucleus
Apoptotic cell death has been observed in human and other mammalian pre-implantation embryos
The expression of some DNA repair genes has been detected in mammalian embryos at different stages of development
Epigenetic mechanisms, including DNA methylation, are key elements for controlling gene expression during the embryo-TE transition. In mouse blastocysts, DNA methyltransferase expression is restricted to the ICM, in which nuclei are highly methylated
As the day 3 embryos and the day 5 embryos used to isolate TE cells were donated from infertile women who underwent IVF treatments, the gene expression profiles could be have been influenced by the controlled ovarian stimulation (COS) carried out during IVF and thus they might not completely reflect the physiological situation under natural cycles. Moreover, due to the bioethics law that regulates the research on human embryos in France, the number of embryos donated for research is smaller. In view of these limitations, we optimized our technique to obtain transcriptome data for each single embryo and trophectoderm sample, respectively.
Human day 3 (post-fertilization) embryos and day 5 blastocysts were donated for research by infertile couples undergoing IVF treatment. All patients signed informed consent forms and the protocol for collecting human embryos and TE was approved by the Ethical Committee of the French National Agency of Biomedicine.
9 embryos from 6 different couples were used for microarray analyses (n = 6) and qRT-PCR validation (n = 3). Day 3 embryos were all 6–8 cells with <20% fragmentation. Each embryo was individually transferred in a tube containing extraction buffer and frozen at −80°C for subsequent RNA extraction.
8 day 5 blastocysts were used for TE isolation for microarray analyses (n = 5) and qRT–PCR validation (n = 3). Blastocysts were fully expanded with a well-defined ICM and TE was scored according to Gardner
After informed consent, unfertilized MII oocytes were collected 24 or 48 hours post-insemination as previously described
The RNeasy Micro kit (Qiagen) was used to isolate total RNA from TE samples and the Picopure RNA isolation kit (Arcturus Reagents/Molecular Devices, KIT0204, USA) for day 3 embryos, according to the manufacturers’ recommended protocols. The quantity and purity of the total RNAs were determined by using a NanoDrop® ND-1000 spectrophotometer (NanoDrop ND-Thermo Fisher Scientific, Wilmington, DE, USA) and their integrity by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA,
Total day 3 embryo RNA samples (from 450 pg to 855 pg) were subjected to three rounds of linear amplification and total TE RNA samples (between 50 and 100 ng) were twice amplified to generate suitable quantity of labeled cRNA for hybridization to HG-U133 plus 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA, USA) as described in
After image processing using the Affymetrix Microarray Suite 5.0, the cell files were analyzed using the Affymetrix Expression Console software and normalized with the MAS5 algorithm by scaling each array to a target value of 100 using the global scaling method to obtain an intensity value signal for each probe set. Gene annotation was performed using NetAffx (
Gene Ontology (GO) annotation analysis was carried out using the Fatigo+ tool
Gene expression profiles derived from microarray analyses were confirmed quantitatively by real-time qRT-PCR analysis using RNAs from three TE samples, three day 3 embryos, three MII oocytes and three hESC samples. The primer sequences are shown in
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List of the 1,714 transcripts specific to the day 3 embryo molecular signature.
(XLS)
List of the 2,196 transcripts specific to the TE molecular signature.
(XLS)
Primer pairs used for validating the array data by qRT-PCR.
(DOC)
List of the transcripts included in the signatures analyzed in this manuscript. (a) Day 3 embryo/MII oocyte signature, (b) Day 3 embryo/hESC signature, (c) TE/MII oocyte signature, (d) TE/hESC signature, (e) specific day 3 embryo signature and (f) specific TE signature.
(XLS)
List of the 104 genes that were up-regulated both in TE cells obtained by hESCs differentiation in the presence of BMP4 for 10 days (transcriptome analysis by Marchand et al,
(XLS)
We thank the direction of the University-Hospital of Montpellier and the ART/PGD teams for their support and Dr. Robert Feil for insightful discussions and critical review of the manuscript. We thank Genevrier and Ferring Pharmaceutical Companies for their support.