Supplementary Tables and Figures

Characterization of the Endometrial Transcriptome in Early Diestrus Influencing Pregnancy Establishment in Dairy Cattle after Transfer of In-Vitro Produced Embryos

Gianluca Mazzoni, Hanne S Pedersen, Maria B Rabaglino, Poul Hyttel, Henrik Callesen, Haja N Kadarmideen**

Corresponding author: hajak@ dtu.dk

Supplementary Tables: SM1, SM2, SM3, SM4, SM5, SM6 and SM7

Supplementary Figures: SM1, SM2, SM3 and SM4

Supplementary Tables

Table SM 1. List of differentially expressed genes between pregnant and non-pregnant cows

Gene IPA Gene ID Name Description FC P-adj classification

ENSBTAG0000000 7.91E- 0575 TNC Tenascin C 2.51 05 Not annotated

ENSBTAG0000001 1.13E- Transmembra 1381 SLC30A3 Zinc transporter 3 0.47 02 ne receptor ENSBTAG0000001 2.80E- 7677 SCG3 Secretogranin-3 1.96 02 Enzyme ENSBTAG0000001 Collagen type VIII 2.80E- 3662 COL8A1 alpha 1 chain 1.96 02 Other

Transforming growth factor-beta-induced ENSBTAG0000000 protein ig-h3 7.50E- 9513 TGFBI precursor 1.95 03 Other

ENSBTAG0000001 3.06E- 3990 WNT6 Wnt family member 6 1.91 02 Other

ENSBTAG0000000 3.27E- 8708 GPC5 Glypican 5 0.53 02 Other

ENSBTAG0000003 Glutamate receptor, 1.88E- 3153 GRIK2 ionotropic kainate 2 1.89 02 Other

Prostaglandin- ENSBTAG0000001 endoperoxide 3.14E- 4127 PTGS2 synthase 2 1.88 02 Enzyme

Cytochrome P450, ENSBTAG0000004 CYP4F3 family 4, subfamily F, 4.01E- 8257 (Uniprot) polypeptide 3 0.54 02 Other

ENSBTAG0000000 3.92E- 2092 PI16 Peptidase inhibitor 16 0.54 02 Transporter

Potassium voltage- gated channel ENSBTAG0000002 subfamily B member 2.80E- 7320 KCNB1 1 0.54 02 Other

ENSBTAG0000001 2.80E- Other 7020 S100G Protein S100-G 0.55 02 (cadherin)

ENSBTAG0000000 Marker of 3.92E- 2444 MKI67 proliferation Ki-67 1.83 02 Enzyme

LYZ ENSBTAG0000000 (homology 4.26E- 0198 ) Lysozyme 0.55 02 Ion channel

ENSBTAG0000000 4.98E- 3920 TGM1 Transglutaminase 1 0.55 02 Enzyme

ENSBTAG0000000 Natriuretic peptide 4.98E- 7665 NPR3 receptor 3 1.80 02 Not annotated

Signal peptide, CUB ENSBTAG0000000 domain and EGF like 4.01E- 9226 SCUBE3 domain containing 3 1.79 02 Other Pyroglutamylated ENSBTAG0000000 rfamide peptide 4.14E- Enzyme 8255 QRFPR receptor 1.79 02

ENSBTAG0000001 1.82E- Other 4169 PCDH19 Protocadherin 19 1.78 02 (cadherin)

ENSBTAG0000000 3.20E- 9230 FBLN7 Fibulin 7 1.78 02 Enzyme

ENSBTAG0000000 Lysyl oxidase 2.33E- 6214 LOXL2 homolog 2 precursor 1.75 02 Other

ENSBTAG0000001 C-type lectin domain 1.28E- 2815 CLEC12A family 12 member A 1.75 02 Enzyme

Not annotated ENSBTAG0000001 STEAP family 4.98E- (immunoglobu 5749 STEAP1 member 1 1.74 02 lin)

KCNQ1potassium KCNQ1 voltage-gated ENSBTAG0000001 (homology channel subfamily Q 3.81E- 0986 ) member 1 0.58 02 Other

ENSBTAG0000002 Matrix remodeling 3.69E- 2150 MXRA5 associated 5 1.73 02 Ion channel

ENSBTAG0000001 3.28E- 9761 MANBA Mannosidase beta 0.58 02 Phospatase

ENSBTAG0000003 NDUFA4L NADH 3.89E- 1503 2 dehydrogenase 1.73 02 Not annotated

ENSBTAG0000001 X-linked Kx blood 1.88E- 2718 XK group 0.58 02 Other

ENSBTAG0000001 Chondroitin sulfate 4.26E- 4661 CHSY3 synthase 3 1.71 02 Peptidase

Sosondowah ankyrin ENSBTAG0000000 repeat domain family 2.41E- 3671 SOWAHA member A 0.59 02 Other

Testis-specific ENSBTAG0000002 serine/threonine- 3.90E- 0652 TSSK3 protein kinase 3 0.59 02 Other

ENSBTAG0000002 4.01E- 2028 DERL3 Derlin 3 0.59 02 Enzyme ENSBTAG0000000 3.81E- Enzyme 9076 ADD2 Adducin 2 1.70 02 ENSBTAG0000002 Arachidonate 5- 3.06E- 0319 ALOX5 lipoxygenase 1.67 02 Other

G-protein ENSBTAG0000001 4.26E- coupled 2409 POSTN Periostin 1.66 02 receptor

ENSBTAG0000001 1.88E- Transcription 1545 GDA Guanine deaminase 1.65 02 regulator

Transmembra ENSBTAG0000000 Protein sprouty 2.80E- ne receptor 8250 SPRY4 homolog 4 1.64 02 (integrin)

ENSBTAG0000000 TSSK6 activating 3.89E- 6373 TSACC cochaperone 0.61 02 Enzyme

ENSBTAG0000004 Integrin subunit alpha 3.06E- Transporter 7238 ITGAM M 1.63 02 (integrin)

ENSBTAG0000001 4.01E- Not annotated 5549 PCDH18 Protocadherin 18 1.62 02 (cadherin)

Macrophage ENSBTAG0000000 scavenger receptor 6.52E- 2885 MSR1 types I and II 1.62 03 Kinase

Procollagen C- ENSBTAG0000002 endopeptidase 2.33E- 0528 PCOLCE enhancer 1.61 02 Other

ENSBTAG0000001 Retinoic acid receptor 4.98E- 4713 RARRES1 responder 1 1.61 02 Not annotated

ENSBTAG0000000 Calcium regulated 7.50E- 1441 CARHSP1 heat stable protein 1 0.62 03 Other

ENSBTAG0000001 Transmembrane 3.06E- Other 6429 TMEM205 protein 205 0.62 02

ENSBTAG0000003 Transmembrane 2.80E- 1737 TMEM102 protein 102 0.63 02 Other

ER membrane ENSBTAG0000002 protein complex 4.01E- 5778 EMC9 subunit 9 0.64 02 Other

ENSBTAG0000000 Retinoic acid induced 4.01E- 7071 RAI14 14 1.57 02 Enzyme ENSBTAG0000000 3.27E- 5250 BGN Biglycan 1.57 02 Other ENSBTAG0000003 Mannose receptor, C 4.01E- 8048 MRC1 type 1 1.57 02 Other

ENSBTAG0000000 Solute carrier family 2 4.98E- 2942 SLC2A10 member 10 1.57 02 Other

Arfgap with gtpase domain, ankyrin ENSBTAG0000000 repeat and PH 7.50E- Transcription 7156 AGAP2 domain 2 1.55 03 regulator Other (immunoglobu ENSBTAG0000002 G protein-coupled 3.92E- lin) 0144 GPR34 receptor 34 1.55 02

Low density lipoprotein receptor ENSBTAG0000000 class A domain 4.98E- Transcription 9139 LDLRAD4 containing 4 0.65 02 regulator

ENSBTAG0000002 Phosphodiesterase 1.88E- 1899 PDE9A 9A 1.54 02 Enzyme

ENSBTAG0000000 3.81E- 7228 CBX7 Chromobox 7 0.65 02 Other

Platelet endothelial ENSBTAG0000001 aggregation receptor 3.06E- 2405 PEAR1 1 1.54 02 Other

Immunoglobulin Other ENSBTAG0000000 superfamily member 3.28E- (immunoglobu 2029 IGSF5 5 0.65 02 lin)

Transmembra ENSBTAG0000001 Ras-related GTP- 1.08E- ne receptor 8438 RRAGD binding protein D 0.65 02 (integrin)

Other ENSBTAG0000000 Growth hormone 3.92E- (immunoglobu 1335 GHR receptor 1.53 02 lin)

Inverted formin, FH2 ENSBTAG0000000 and WH2 domain 4.01E- 7187 INF2 containing 1.52 02 Kinase

ENSBTAG0000000 Neutrophil cytosolic 4.48E- Other 8004 NCF2 factor 2 1.52 02

ENSBTAG0000001 Integrin subunit alpha 3.34E- 3745 ITGA5 5 1.51 02 Enzyme ENSBTAG0000000 Fascin actin-bundling 4.73E- 3191 FSCN1 protein 1 1.51 02 Other

ENSBTAG0000001 Hepatocyte growth 4.31E- 7664 HGF factor 1.51 02 Other

ENSBTAG0000001 PDZ domain 3.06E- Not annotated 4455 PDZD2 containing 2 1.51 02

ENSBTAG0000001 Zinc finger protein 3.06E- 5604 ZNF385A 385A 1.50 02 Not annotated

Ras association ENSBTAG0000004 domain family 3.28E- 5834 RASSF7 member 7 0.67 02 Peptidase

ENSBTAG0000000 Serine protease 3.89E- 8389 HTRA1 HTRA1 1.49 02 Enzyme

ENSBTAG0000002 4.26E- 1746 ANXA5 Annexin A5 1.48 02 Other

ENSBTAG0000003 RAB3IP RAB3A interacting 3.92E- 1950 (Uniprot) protein (Rabin3) 0.68 02 Kinase ENSBTAG0000001 3-oxo-5-alpha-steroid 4.14E- 5478 SRD5A1 4-dehydrogenase 1 0.68 02 Enzyme

ENSBTAG0000002 4.98E- Transmembra 6913 HHIPL1 HHIP like 1 1.46 02 ne receptor

Pseudopodium G-protein ENSBTAG0000000 enriched atypical 3.06E- coupled 5145 PEAK1 kinase 1 1.46 02 receptor

ENSBTAG0000000 ARHGAP4 Rho gtpase activating 2.01E- 4423 2 protein 42 1.45 02 Enzyme

Coiled-coil-helix- coiled-coil-helix ENSBTAG0000003 domain-containing 1.08E- 3284 CHCHD7 protein 7 0.69 02 Not annotated

ENSBTAG0000001 Transforming growth 3.69E- 9832 TGFBR2 factor beta receptor 2 1.45 02 Other

ENSBTAG0000001 4.26E- 8463 VIM Vimentin 1.45 02 Other

Serine/threonine- protein phosphatase 2A 55 kda regulatory ENSBTAG0000000 subunit B beta 2.80E- 1862 PPP2R2B isoform 1.45 02 Transporter

ENSBTAG0000000 3.28E- 5546 HOXB7 Homeobox B7 0.69 02 Other ENSBTAG0000000 MAGE family 3.28E- 8733 MAGED1 member D1 1.43 02 Enzyme

ENSBTAG0000003 FZD3 Uncharacterized 3.06E- 5084 (Uniprot) protein 0.70 02 Other

ENSBTAG0000000 4.98E- 3629 ASPA Aspartoacylase 1.43 02 Other

ENSBTAG0000001 PLA2G16 Uncharacterized 2.53E- 5248 (Uniprot) protein 0.70 02 Not annotated

ENSBTAG0000001 Tetratricopeptide 3.92E- Transmembra 5587 TTC32 repeat domain 32 0.70 02 ne receptor

ENSBTAG0000000 TLC domain 3.28E- Other 6777 TLCD1 containing 1 0.71 02 (cadherin)

ENSBTAG0000001 3.79E- Transcription 0347 EZR Ezrin 0.71 02 regulator

Histidine triad ENSBTAG0000000 nucleotide-binding 7.50E- 1956 HINT3 protein 3 0.71 03 Other

SEC11 homolog C, ENSBTAG0000001 signal peptidase 2.80E- 1954 SEC11C complex subunit 0.71 02 Other

G-protein ENSBTAG0000001 MARCH8 E3 ubiquitin-protein 3.06E- coupled 9537 (Uniprot) ligase MARCH8 1.41 02 receptor

O-acetyl-ADP-ribose ENSBTAG0000001 MACROD deacetylase 3.92E- 7181 1 MACROD1 0.71 02 Enzyme

ENSBTAG0000000 AXL receptor tyrosine 2.80E- 3166 AXL kinase 1.39 02 Transporter

Glioma tumor ENSBTAG0000000 suppressor candidate 3.89E- 4138 GLTSCR1 region gene 1 1.39 02 Enzyme

ENSBTAG0000000 Antigen peptide 3.92E- 8953 TAP1 transporter 1 0.72 02 Not annotated

ENSBTAG0000002 NTF2-related export 3.06E- 0739 NXT2 protein 2 0.73 02 Not annotated

ENSBTAG0000001 HIV-1 Tat interactive 3.06E- 3419 HTATIP2 protein 2 0.74 02 Other ENSBTAG0000004 Chromosome 4 open 3.06E- 4159 C4orf33 reading frame 33 0.74 02 Other

ENSBTAG0000001 Cytochrome b-245 3.92E- 9953 CYBB heavy chain 1.35 02 Growth factor

Bos taurus dynein, light chain, Tctex- ENSBTAG0000000 type 1 (DYNLT1), 3.32E- 4472 DYNLT1 mrna. 0.74 02 Other

ENSBTAG0000001 7.50E- 0351 SNX25 Sorting nexin-25 0.75 03 Enzyme

Sterol-C5-desaturase (ERG3 delta-5- ENSBTAG0000001 SC5DL desaturase homolog, 3.06E- 9246 (Uniprot) S. Cerevisiae)-like 0.77 02 Other

ENSBTAG0000002 Ribonuclease P 3.79E- 6248 POP7 protein subunit p20 0.77 02 Enzyme

Microsomal ENSBTAG0000002 glutathione S- 4.98E- 1779 MGST2 transferase 2 0.77 02 Other

ENSBTAG0000000 4.01E- 9127 TSPYL4 TSPY like 4 0.78 02 Other

ENSBTAG0000000 Ubiquitin-conjugating 2.33E- 4790 UBE2T enzyme E2 T 0.78 02 Enzyme

Heterogeneous G-protein ENSBTAG0000001 nuclear 4.26E- coupled 3952 HNRNPD ribonucleoprotein D 1.28 02 receptor

Transmembrane BAX ENSBTAG0000000 inhibitor motif 1.08E- 3935 TMBIM1 containing 1 0.79 02 Other

ENSBTAG0000001 3.06E- 8588 TMBIM6 Bax inhibitor 1 0.80 02 Transporter

Acetyl-coa ENSBTAG0000001 acetyltransferase, 3.06E- Transporter 2885 ACAT1 mitochondrial 0.81 02

ENSBTAG0000000 Germ cell-less 4.33E- 1108 GMCL1 protein-like 1 0.84 02 Enzyme List of differentially expressed genes between pregnant and non-pregnant cows. The gene name and description are obtained with Biomart using Ensembl release 87. Where indicated the gene names were retrieved from Uniprot annotation or by homology with human genes. FC = Fold Change; P-adj = Benjamini-Hochberg adjusted P-value. IPA annotation = classification of the proteins encoded by the DE genes. Between parentesis the domain information from PFam.

Table SM 2. GSEA enriched KEGG pathways for the genes (A) up-regulated and (B) downregulated in pregnant cows compared to non-pregnant cows. A)

KEGG gene set Size NES FDR Core enrichment ECM receptor interaction 73 2.26 ~0 TNC, ITGA5, COL5A2, HSPG2, COL5A3, COL4A2, SDC3, FN1, COL6A2, ITGA4, SDC2, COL6A1, THBS3, COL6A3, ITGA1, COL5A1, ITGA9, THBS2, TNXB, LAMA4, COL4A1, COL3A1, COL2A1, ITGA10, TNR, THBS1, SPP1, VWF, COL1A2, LAMC1, COL1A1, COL4A4, ITGA2B, LAMA2, SV2A, LAMB1, LAMC3, Glycosaminoglycan 18 1.97 6.97E-03 CHSY3, CHST3, CHST7, CHST11, biosynthesis chondroitin XYLT1, CHST15, DSE, CHST14, sulfate CHSY1, CSGALNACT2, CHPF2, B4GALT7, Cell adhesion molecules 88 1.91 1.10E-02 CNTN2, ITGAM, NFASC, L1CAM, cams SIGLEC1, CDH2, NCAM1, CNTNAP1, SDC3, NRXN2, CADM3, ITGA4, SDC2, CNTN1, ITGA9, ITGB2, NLGN1, SPN, SELPLG, CD86, CDH5, CD276, SELP, PDCD1, SELL, ESAM, ITGAL, VCAN, CD22, CD4, NCAM2, HLA- DOB, NLGN2, Focal adhesion 174 1.78 4.10E-02 TNC, ITGA5, HGF, COL5A2, COL5A3, COL4A2, FN1, COL6A2, ITGA4, COL6A1, THBS3, COL6A3, ITGA1, PAK3, COL5A1, ITGA9, THBS2, FLT1, TNXB, LAMA4, COL4A1, COL3A1, KDR, COL2A1, ITGA10, TNR, PDGFRB, PARVB, PIK3CG, THBS1, SRC, TLN2, SPP1, VWF, ACTB, ZYX, COL1A2, PARVG, SHC2, PIK3R1, LAMC1, COL1A1, PXN, COL4A4, ITGA2B, SHC1, ACTN2, LAMA2, VEGFC, PRKCA, LAMB1, LAMC3, SOS1 Basal cell carcinoma 35 1.77 3.49E-02 WNT6, WNT5A, WNT5B, WNT2, GLI3, TCF7, TP53, SMO, BMP2, DVL3, WNT7A, LEF1, WNT16, SUFU Hedgehog signaling 33 1.69 4.59E-02 WNT6, WNT5A, WNT5B, WNT2, pathway GLI3, BMP6, SMO, BMP2, PRKACA, WNT7A, WNT16, SUFU Size = number of genes in the gene set used for the analysis, NES = Normalized Enrichment Score, FDR = False Discovery Rate, Core enrichment =set of genes that contribute most to the enrichment result.

B) KEGG gene set Size NES FDR Core enrichment Ribosome 47 -2.27 ~0 RPL24, RPS27A, RPL15, FAU, RPS9, RPL18A, RPSA, RPL29, RPS8, RPLP0, RPS20, RPL19, RPL30, RPS2, RPS6, UBA52, RPL14, RPS28, RPS4X, RPL28, RPS3, RPL18, RPL38, RPS17, RPL7A, RPL37A, RPS7, RPL36A, RPS5, RPL34, RPS18, RPL37, RPL13, RPS16, RPL11, RPS11, RPL26, RPLP1, RPL10A, RPL36, RPS29 Valine leucine and 40 -1.97 5.59E- DLD, HADH, HMGCL, ACAA2, isoleucine degradation 03 OXCT1, ACADM, ALDH6A1, MCCC1, ACAD8, HMGCS1, PCCB, ACADS, HSD17B10, MUT, BCKDHA, IL4I1, IVD, HIBADH, HIBCH, ALDH3A2, ACAT1, EHHADH, AOX1, BCKDHB, ABAT, DBT Peroxisome 64 -1.86 1.71E- PEX19, PEX10, HMGCL, 02 ACOT8, PEX5, PEX7, CRAT, CAT, ACSL3, MVK, ACSL4, PEX14, PEX11G, ACSL5, GSTK1, DECR2, ABCD3, HACL1, ACSL1, PRDX5, HSD17B4, ACOX3, SCP2, ACOX2, PRDX1, DDO, PEX1, EHHADH, PMVK, IDH1, CROT, PXMP2, NUDT12, SLC27A2, EPHX2 Butanoate metabolism 23 -1.82 2.06E- HADH, HMGCL, OXCT1, 02 HMGCS1, ACADS, ALDH5A1, L2HGDH, ALDH3A2, ACAT1, EHHADH, ABAT, ACSM1, ACSM3 Size = dimension of the gene set used for the analysis, NES = Normalized Enrichment Score, FDR = False Discovery Rate, Core enrichment = set of genes that contribute most to the enrichment result.

Table SM 3. NET-GE GO terms enrichment results BH GO correcte clas d P- s GO Term N1 N2 value Description GO:00226 229 7.40E- BP 10 30 4 03 Biological adhesion GO:00974 113 9.03E- BP 85 19 6 03 Neuron projection guidance GO:00902 9.69E- Regulation of cellular response to growth factor BP 87 12 493 03 stimulus GO:00512 191 1.10E- Positive regulation of multicellular organismal BP 40 25 6 02 process GO:00074 113 1.13E- BP 11 19 6 02 Axon guidance GO:00424 1.17E- BP 76 9 294 02 Odontogenesis GO:00170 1.20E- Regulation of transforming growth factor beta BP 15 8 241 02 receptor signaling pathway GO:19025 210 1.23E- Positive regulation of intracellular signal BP 33 26 6 02 transduction GO:00430 1.23E- BP 62 17 991 02 Extracellular structure organization GO:00068 102 1.25E- BP 97 17 5 02 Endocytosis GO:19015 1.25E- BP 68 8 240 02 Fatty acid derivative metabolic process GO:00305 1.26E- Negative regulation of transforming growth BP 12 7 165 02 factor beta receptor signaling pathway GO:00986 1.29E- BP 02 15 846 02 Single organism cell adhesion GO:00301 1.32E- BP 98 17 986 02 Extracellular matrix organization GO:00015 1.33E- BP 03 11 397 02 Ossification GO:00066 1.35E- BP 90 8 240 02 Icosanoid metabolic process GO:00071 228 1.39E- BP 55 30 7 02 Cell adhesion GO:00315 1.62E- BP 89 10 410 02 Cell-substrate adhesion GO:00311 1.74E- BP 00 6 136 02 Organ regeneration GO:00335 1.89E- BP 59 8 268 02 Unsaturated fatty acid metabolic process GO:00424 2.30E- BP 75 7 211 02 Odontogenesis of dentin-containing tooth GO:00325 2.39E- BP 70 5 94 02 Response to progesterone GO:00071 2.41E- BP 59 5 96 02 Leukocyte cell-cell adhesion GO:00305 2.43E- BP 74 6 152 02 Collagen catabolic process GO:00460 2.47E- BP 98 2 4 02 Guanine metabolic process GO:00486 2.64E- BP 60 7 223 02 Regulation of smooth muscle cell proliferation GO:00455 214 2.68E- BP 97 25 1 02 Positive regulation of cell differentiation GO:00442 2.70E- BP 36 7 221 02 Multicellular organismal metabolic process GO:00603 2.71E- Negative regulation of pathway-restricted SMAD BP 94 3 23 02 protein phosphorylation GO:00706 2.75E- BP 61 7 227 02 Leukocyte proliferation GO:00019 2.81E- BP 52 7 229 02 Regulation of cell-matrix adhesion GO:00099 313 2.83E- BP 67 32 2 02 Positive regulation of signal transduction GO:00075 2.84E- BP 68 12 659 02 Aging GO:00329 3.03E- BP 63 6 176 02 Collagen metabolic process GO:00068 3.06E- BP 98 10 495 02 Receptor-mediated endocytosis GO:00310 3.10E- BP 99 8 322 02 Regeneration GO:00061 3.14E- BP 45 2 6 02 Purine nucleobase catabolic process GO:00486 3.14E- Positive regulation of smooth muscle cell BP 61 5 117 02 proliferation GO:00442 3.15E- BP 43 6 178 02 Multicellular organismal catabolic process GO:00719 3.16E- Positive regulation of protein serine/threonine BP 02 13 799 02 kinase activity GO:00303 170 3.21E- BP 34 21 8 02 Regulation of cell migration GO:00075 3.27E- BP 67 3 27 02 Parturition GO:00507 3.28E- BP 30 11 583 02 Regulation of peptidyl-tyrosine phosphorylation GO:00486 248 3.31E- Anatomical structure formation involved in BP 46 27 7 02 morphogenesis GO:00434 3.37E- BP 06 11 583 02 Positive regulation of MAP kinase activity GO:00163 3.40E- BP 37 13 778 02 Single organismal cell-cell adhesion GO:00082 221 3.44E- BP 83 25 3 02 Cell proliferation GO:00513 175 3.72E- BP 47 21 4 02 Positive regulation of transferase activity GO:00018 3.78E- BP 37 5 123 02 Epithelial to mesenchymal transition GO:00091 3.88E- BP 00 6 189 02 Glycoprotein metabolic process GO:00314 259 3.97E- Positive regulation of protein modification BP 01 27 0 02 process GO:00902 4.01E- Negative regulation of cellular response to BP 88 7 271 02 growth factor stimulus GO:00083 4.03E- BP 66 6 199 02 Axon ensheathment GO:20001 179 4.04E- BP 45 21 5 02 Regulation of cell motility GO:00442 4.04E- Multicellular organismal macromolecule BP 59 6 196 02 metabolic process GO:00108 4.08E- BP 11 7 266 02 Positive regulation of cell-substrate adhesion GO:00072 4.09E- BP 72 6 199 02 Ensheathment of neurons GO:00442 4.09E- Regulation of multicellular organismal metabolic BP 46 5 130 02 process GO:00015 4.11E- BP 25 13 829 02 Angiogenesis GO:00072 4.15E- BP 29 6 195 02 Integrin-mediated signaling pathway GO:00336 153 4.16E- BP 74 19 1 02 Positive regulation of kinase activity GO:00436 4.16E- BP 27 10 533 02 Response to estrogen GO:00442 4.19E- BP 72 7 268 02 Sulfur compound biosynthetic process GO:00423 234 4.49E- BP 27 25 9 02 Positive regulation of phosphorylation GO:00019 207 4.52E- BP 34 23 9 02 Positive regulation of protein phosphorylation GO:00066 4.54E- BP 91 4 80 02 Leukotriene metabolic process GO:00603 4.56E- Regulation of pathway-restricted SMAD protein BP 93 4 82 02 phosphorylation GO:00026 4.60E- BP 88 6 209 02 Regulation of leukocyte chemotaxis GO:00108 4.62E- BP 10 9 458 02 Regulation of cell-substrate adhesion GO:00508 171 4.63E- BP 78 20 1 02 Regulation of body fluid levels GO:00069 120 4.63E- BP 54 16 9 02 Inflammatory response GO:00434 109 4.64E- BP 10 15 4 02 Positive regulation of MAPK cascade GO:00193 4.65E- BP 69 5 140 02 Arachidonic acid metabolic process GO:00458 146 4.87E- BP 60 18 8 02 Positive regulation of protein kinase activity GO:00001 4.91E- BP 87 8 378 02 Activation of MAPK activity GO:00044 1.64E- MF 64 3 6 03 Leukotriene-C4 synthase activity GO:00055 2.06E- MF 39 12 607 02 Glycosaminoglycan binding GO:00347 2.21E- Type I transforming growth factor beta receptor MF 13 3 16 02 binding GO:19016 3.54E- MF 81 12 664 02 Sulfur compound binding GO:00192 3.86E- MF 39 4 62 02 Deaminase activity GO:00099 147 2.02E- CC 86 26 4 04 Cell surface GO:00444 501 1.66E- CC 59 48 2 02 Plasma membrane part GO:00058 288 1.85E- CC 87 33 4 02 Integral component of plasma membrane GO:00444 1.94E- CC 20 9 340 02 Extracellular matrix part GO:00310 1.97E- CC 12 14 799 02 Extracellular matrix GO:00055 2.31E- CC 78 13 639 02 Proteinaceous extracellular matrix GO:00701 107 3.70E- CC 61 16 2 02 Anchoring junction GO Class = GO term category: Biological Process (BP), Cellular Component (CC) or Molecular Function (MF); N1 = N1 - The number of input genes associated to the GO term, N2 = The number of genes associated to the GO term; BH P-value = Benjamini-Hochberg adjusted P-value.

Table SM 4. Functional classification of the genes by IPA® Biological BH P-value Molecules category Cellular 6.3E-05-2.88E-02 HTATIP2, MSR1, FZD3, RARRES1, Movement SCUBE3, GPR34, TMBIM6, LOXL2, SPRY4, TGFBR2, TMEM102, ANXA5, HGF, EZR, CYBB, POSTN, PLA2G16, PEAK1, TGFBI, BGN, VIM, ITGA5, AGAP2, ITGAM, LDLRAD4, FSCN1, NCF2, ACAT1, PTGS2, ALOX5, HTRA1 Cellular Function 1.45E-04-2.88E-02 MRC1, PEAK1, MSR1, VIM, TGFBR2, and Maintenance GHR, ITGAM, HGF, ANXA5, EZR, CYBB, PEAR1, GRIK2, PTGS2 Free Radical 3.99E-04-2.88E-02 MRC1, ITGAM, MSR1, HGF, NCF2, CYBB, Scavenging ITGA5, TMBIM6, PTGS2, ALOX5 Small Molecule 3.99E-04-2.88E-02 MRC1, PLA2G16, HTATIP2, MSR1, Biochemistry QRFPR, SRD5A1, CYP4F3, ITGA5, VIM, ASPA, TGFBR2, TGM1, ITGAM, GHR, TMEM102, MGST2, ANXA5, HGF, NCF2, CYBB, ACAT1, PTGS2, ALOX5 Cell Morphology 3.99E-04-2.88E-02 ZNF385A, GDA, MSR1, COL8A1, GPR34, TMBIM6, DYNLT1, TAP1, LOXL2, PCOLCE, SPRY4, TGFBR2, HGF, EZR, ANXA5, CYBB, POSTN, PEAK1, ADD2, TGFBI, PDE9A, BGN, ITGA5, VIM, MANBA, AGAP2, ITGAM, GHR, NPR3, FSCN1, GMCL1, PTGS2, HTRA1 Cell-To-Cell 1.78E-03-2.88E-02 MRC1, PEAK1, ADD2, MSR1, TGFBI, Signaling and BGN, VIM, ITGA5, TAP1, TGFBR2, GHR, Interaction ITGAM, EZR, HGF, ANXA5, CYBB, POSTN, PEAR1, GRIK2, PTGS2 Cellular 2.53E-03-2.88E-02 CBX7, MAGED1, ZNF385A, BGN, ITGA5, Development VIM, SCUBE3, AGAP2, LOXL2, GPC5, TGFBR2, TGM1, NPR3, HOXB7, EZR, FSCN1, HGF, POSTN, PTGS2, ALOX5, HTRA1 Cellular Growth 2.53E-03-2.88E-02 CBX7, MAGED1, BGN, COL8A1, VIM, and Proliferation ITGA5, SCUBE3, AGAP2, GPC5, TGFBR2, GHR, TGM1, NPR3, HOXB7, FSCN1, EZR, HGF, POSTN, PTGS2, ALOX5, HTRA1 Cell Death and 4.04E-03-2.88E-02 CBX7, HTATIP2, MAGED1, ZNF385A, Survival PCDH18, GDA, MSR1, FZD3, WNT6, TMBIM6, TAP1, LOXL2, TGFBR2, MARCH8, HGF, EZR, ANXA5, POSTN, CYBB, GRIK2, RAI14, PLA2G16, ADD2, TGFBI, PDE9A, BGN, VIM, ITGA5, AGAP2, ASPA, KCNB1, TGM1, ITGAM, GHR, PPP2R2B, NCF2, GMCL1, ACAT1, PTGS2, ALOX5, HTRA1 Cellular 5.05E-03-2.88E-02 TGFBR2, ITGAM, TGM1, ADD2, ANXA5, Compromise CYBB, VIM, ASPA Lipid Metabolism 5.05E-03-2.88E-02 MRC1, PLA2G16, MSR1, QRFPR, SRD5A1, CYP4F3, VIM, ASPA, TGFBR2, GHR, MGST2, ANXA5, HGF, ACAT1, PTGS2, ALOX5 Cellular Assembly 7.66E-03-2.88E-02 SCG3, PEAK1, MSR1, ADD2, BGN, VIM, and Organization TMBIM6, PCOLCE, GHR, EZR, HGF, ANXA5, FSCN1, PTGS2 Cell Cycle 8.7E-03-2.88E-02 TGFBR2, CBX7, GHR, HGF Molecular 8.79E-03-2.88E-02 MRC1, TGFBR2, GHR, MSR1, SRD5A1, Transport HGF, NCF2, ACAT1, CYBB, PTGS2, ALOX5 Protein Synthesis 1.26E-02-2.88E-02 MRC1, GHR, NPR3, HGF, PTGS2, TMBIM1 Carbohydrate 1.93E-02-2.88E-02 HTATIP2, ANXA5 Metabolism Vitamin and 2.88E-02-2.88E-02 GHR, QRFPR, SRD5A1, BGN, HGF, Mineral ACAT1, VIM, ASPA Metabolism Amino Acid 2.88E-02-2.88E-02 TGFBR2, GHR, TGM1, TMEM102, HGF, Metabolism ITGA5 Post-Translational 2.88E-02-2.88E-02 TGFBR2, GHR, TGM1, TMEM102, HGF, Modification ITGA5 Drug Metabolism 2.88E-02-2.88E-02 SRD5A1, HGF, PTGS2 DNA Replication, 2.88E-02-2.88E-02 VIM Recombination, and Repair Protein Trafficking 2.88E-02-2.88E-02 HGF BH P-value: (P-values after multiple testing correction by using Benjamini–Hochberg method).

Table SM 5. Upstream regulator analysis by IPA® Fold Upstream Molecule P-value of Chan Target molecules in dataset Regulator Type overlap ge chemical - ACAT1, EZR, GHR, HGF, ITGA5, endogenou LDLRAD4, MRC1, POSTN, PTGS2, progestero s SRD5A1, STEAP1, TGFBI, TGM1, VIM, ne mammalian 1.08E-07 WNT6 BGN, CYBB, GHR, HGF, ITGA5, NCF2, growth PCOLCE, PTGS2, SRD5A1, TAP1, TGFBI, IGF1 0.34 factor 4.03E-07 VIM ALOX5, COL8A1, EZR, GHR, HGF, ITGAM, transcriptio MRC1, NCF2, PTGS2, TGFBR2, TGM1, SP1 0.02 n regulator 1.03E-05 VIM ACAT1, BGN, CYBB, ITGA5, ITGAM, APOE 0.46 transporter 1.28E-05 MSR1, NCF2, PTGS2 transmemb rane ALOX5, CLEC12A, GDA, GHR, HGF, IL10RA 0.20 receptor 1.48E-05 PCOLCE, PLA2G16, POSTN, TAP1 transcriptio BGN, CYP4F3, EMC9, FSCN1, GHR, HIF1A -0.09 n regulator 1.60E-05 ITGA5, LOXL2, PTGS2, SRD5A1, VIM HNRNPAB -0.04 enzyme 2.11E-05 HTRA1, PTGS2, VIM CBP- ICSBP- IRF-1-PU.1 complex 2.16E-05 CYBB, NCF2 chemical - ALOX5, ANXA5, CYBB, CYP4F3, GHR, tretinoin endogenou 2.39E-05 GRIK2, HOXB7, ITGAM, LOXL2, NCF2, s POSTN, PTGS2, RAI14, RARRES1, TAP1, mammalian TGFBI, TGFBR2, TGM1, VIM, WNT6 ANXA5, CYBB, EZR, ITGA5, ITGAM, EDN1 -0.11 cytokine 4.01E-05 PTGS2, VIM growth BGN, CYBB, HGF, ITGA5, MSR1, NCF2, AGT factor 4.58E-05 NPR3, POSTN, PTGS2, TGFBR2 ADD2, EZR, FZD3, GRIK2, HTATIP2, transcriptio NCF2, PCDH19, PTGS2, TGFBI, VIM, FOS -0.09 n regulator 4.62E-05 WNT6 ANXA5, BGN, CYBB, CYP4F3, HTRA1, P38 MAPK group 4.79E-05 ITGAM, KCNB1, POSTN, PTGS2 transmemb rane OLR1 -0.12 receptor 6.27E-05 CYBB, ITGAM, NCF2, TGFBR2 transcriptio GHR, HGF, NCF2, PTGS2, TGFBR2, SP3 0.026 n regulator 6.75E-05 TGM1, VIM transcriptio CYBB, ITGA5, ITGAM, MRC1, NCF2, SPI1 0.559 n regulator 7.15E-05 PTGS2, VIM - PTGES 0.352 enzyme 1.10E-04 CYBB, EZR, PTGS2, VIM BIRC5 0.139 other 1.27E-04 BGN, COL8A1, ITGA5, VIM BGN, FSCN1, ITGA5, POSTN, PTGS2, Tgf beta group 1.31E-04 TGFBI, VIM GDA, GHR, ITGA5, MGST2, NPR3, PDGF BB complex 1.31E-04 PCOLCE, POSTN, PTGS2 FABP4 transporter 1.42E-04 ACAT1, MSR1, PTGS2 AMPK complex 2.31E-04 CYBB, FSCN1, KCNB1, PTGS2, VIM Alpha catenin group 2.40E-04 BGN, ITGA5, ITGAM, PTGS2, VIM IL27 cytokine 3.49E-04 ITGAM, MRC1, PTGS2, TAP1, VIM ALOX5, BGN, CHSY3, CYBB, FSCN1, GHR, HGF, ITGA5, ITGAM, MGST2, MSR1, - NCF2, PLA2G16, POSTN, PTGS2, TNF 0.279 cytokine 4.03E-04 RASSF7, TAP1, TGFBR2, VIM Top 25 upstream regulators obtained for the differential expression profile between pregnant and non-pregnant cows.

Table SM 6 Composition of media used for sperm holding- and washing (Sperm TALP), in-vitro maturation (IVM), in-vitro fertilization (IVF) and in-vitro embryo culture (IVC) Sigma Ingredients Sperm IVM IVF IVC cat. no. TALP NaCl 5.8 g/L 6.7 g/L 6.3 g/L S5886 KCl 0.2 g/L 0.2 g/L 0.5 g/L S5405 NaH2PO4 0.04 g/L 0.04 g/L S5011 CaCl2, 2H2O 0.3 g/L 0.3 g/L 0.3 g/L C7902 MgCl2 0.2 g/L 0.1 g/L M2393 Lactic acid 3.7 mL/L 1.9 mL/L L4263

Hepes 1.4 g/L H3784 Hepes-acid 1.1 g/L H4034 NaHCO3 2.1 g/L 2.1 g/L 2.1 g/L S4019 Pyruvic acid 110 mg/L 0.02 g/L P3662 Gentamycin 100 mg/L 0.04 0.05 g/L G1264 mg/mL Heparin 5 IU/mL H3149 Sodium 0.04 µg/mL S9000 disulfite Penicillamine 4.4 µg/mL P4875 Hypotaurine 0.16 µg/mL H1384 Epinephrine 0.27 µg/mL E4250 KH2PO4 0.2 g/L P5655 MgSO4 0.2 g/L M2643 Sodium lactate 0.6 mL/mL L4263 solution Sodium 0.08 g/L P3662 pyruvate Citric acid 0.1 g/L C3434 Myo-inositol 0.5 g/L I7508 BME amino 30 µL /mL B6766 acids MEM amino 10 µL /mL M7145 acids L-glutamine 0.10 0.03 g/L G8540 mg/mL PMSG 8.7 IU/mL hCG 4.3 IU/mL Cattle Serum 13% 5% Dissolved in X M2154 Medium 199 Dissolved in X X X W1503 H2O Osmolarity 280 ± 8 280 ± 8 280 ± 8 280 ± 8 mOsm mOsm mOsm mOsm BME: Basal Medium Eagle, MEM: Minimum Essential Medium, PMSG: Pregnant Mare Serum Gonadotropin, hCG: Human Chorionic Gonadotropin

Supplementary Figures

Fig. SM 1: Quality Control after alignment Percentages of reads unmapped, mapped to multiple position and uniquely mapped reads classified as ambiguous exonic intronic and intergenic for each sample. Numbers in parentheses correspond to the animals’ IDs.

Fig. SM 2: Library fragmentation profile Library fragmentation profiles. The samples G6 and F2 show a different fragmentation profile.

Fig. SM 3: GC content and length bias effect across samples Plots of the estimated systematic GC bias and length bias effect (cqn R package) across samples. To be noted there is no relevant difference of both the GC and the length bias effect among samples.

Fig. SM 4: Length bias effect on the proportion of DE genes Proportion of DE genes (in a gene bin) plotted as a function of the gene length (average length of the genes in the bin). To be noted, in our results there is no relevant length bias effect on the proportion of DE genes.