Endometrial gene expression reveals compromised progesterone signaling in women refractory to embryo implantation.

This study was approved by the Ethics Review Committee for Investigations in Human Beings of Faculty of Medicine, University of Chile: protocol No. 093–2008, approved 12-29-2008, initiated 01-05-2009 finished 03-31-2014. Each volunteer participating read and signed the informed consent approved by the respective Ethics Review Committee. Three groups of women were recruited as has been described elsewhere [ 12 ]. Group A (n = 5) consisted of women that had never been pregnant and had previously participated in two or more cycles as recipients in an oocyte donation program with no evidence of embryo implantation. Male partners had normal seminal parameters and transferred embryos had good morphology, at least equivalent to embryos transferred to the oocyte donor who became pregnant. Since good quality embryos with the ability to implant and develop normally derive from good quality oocytes, it was required that the oocyte donor had become pregnant from the same oocyte pool. Women from group A were recruited within 3 years following the last failed cycle. Group B (n = 6) comprised of women who became pregnant as recipients in previous oocyte donation cycles and delivered live infants. Group C (n = 6) included normal fertile women who conceived in natural cycles and had three or more live births and had elective tubal ligation at least 1 year prior to their participation for reasons unrelated to this study. Women from groups B and C were recruited within 5 years following the last successful pregnancy. The general exclusion criteria for all volunteers included: metabolic or endocrine diseases other than those leading to ovarian failure, chronic use of medication other than HRT, polycystic ovary syndrome, drug abuse, obesity, endometriosis, pelvic inflammatory disease and current genital tract infection. Age and body mass index from recruited women as well as the plasma P and endometrial thickness measured on the day of the endometrial collection are shown in Table  1 . Table 1 Characteristics of women participating in the study and parameters evaluated during the hormonal replacement cycle Group A (n = 5) Group B (n = 6) Group C (n = 6) P value Age (years) 35.4 (26–43) 41.9 (34–46) 41.3 (36–47) 0.1117 Body mass index 25.2 (22.6-29.4) 25.6 (22.5-27.4) 25.4 (23.3-28.1) 0.573 Plasma progesterone* (nmol/L) 75.7 (38–122) 88.1 (36–192) 63.7 (43.3-75) 0.7601 Endometrial thickness* (mm) 10.8 (9–12) 9.1 (8–10) 11.2 (8.5-12.5) 0.6162 *On the day of endometrial collection. Characteristics of women participating in the study and parameters evaluated during the hormonal replacement cycle *On the day of endometrial collection. All subjects underwent the induction of an artificial endometrial cycle with exogenous ethinyl E2 for 20 days and for the last 7 days, this treatment was administered concomitantly with micronized P as described previously [ 12 ]. An endometrial sample was obtained on 20th day of the endometrial cycle. One part of the specimen was snap frozen in liquid nitrogen and kept at -80 ° C until use and the remaining portion was fixed in paraformaldehyde for histological dating, according to the criteria of Noyes et al. , [ 14 ] and for immunohistochemistry (IHC) studies. All biopsies were classified as normal secretory endometrium with no signs of inflammatory processes. Total RNA was extracted from frozen endometrial tissue samples using Trizol (Invitrogen, Gaithersburg, MD, USA) as directed by the manufacturer and then checked for yield and quality as described before [ 12 ]. The Human Genome U133 plus 2.0 GeneChip oligonucleotide microarrays (Affymetrix, Sunnyvale, CA, USA); corresponding to 47,000 transcripts and variants, including 38,500 well-characterized human genes, was used for gene expression analysis according to the manufacturer’s instruction. Microarrays Data Analysis: Replicate hybridizations were performed for each RNA sample and raw data obtained from the GeneChip Microarray Suite v 1.4 was subsequently analyzed using the National Cancer Institute’s Microarrays Data Base webtool (mAdb) ( http://nciarray.nci.nih.gov ). Significant genes were defined as ≥2, p -value < 0.001 and a false discovery rate (FDR) < 0.1 [ 15 ]. T -test was performed to determine statistical differences and from the significant genes identified, Venn diagrams were constructed to identify coincident transcripts. Was performed based on uncentered correlations with average linkage clustering using mAdb. The resulting dendogram allows data structure visualization of endometrial samples according to total gene expression, revealing samples with similar patterns of gene expression and relationships between the specimens. Was performed for simplifying the large amount of data derived from microarray analysis [ 16 ]. We applied the unbiased PCA algorithm to all samples using all transcripts analyzed with the microarray chip to look for expression patterns and underlying cluster structures of endometrial samples. To increase the effectiveness of DNA microarray analysis, data sets of differentially expressed genes from the comparison between A vs. B and A vs. C were intersected to define those transcripts consistently up- or down-regulated and combined with external data sources, such as gene annotation, in order to associate the expression patterns of this particular set of genes with the biological processes that they may represent. In our analysis, we submitted our gene lists to the web-based tools DAVID (Database for Annotation, Visualization and Integrated Discovery) [ 17 ] and GATHER (Gene Annotation Tool to Help Explain Relationships) [ 18 ] for functional annotation analysis in order to gain an in-depth understanding of their biological themes, which otherwise would require laborious and somewhat subjective manual literature searches. Up- and down-regulated genes were submitted to DAVID database for systematically extracting biological meaning for them by retrieving pathway maps from the Kyoto Encyclopedia of Genes and Genomes (KEGG) [ 19 ] and Biocarta pathways database ( http://www.biocarta.com/genes/index.asp ) along with Gene Ontology (GO) functional annotations from Entrez Gene [ 20 ]. The parameters of the “Functional Annotation Clustering” (a part of the “Functional Annotation Tool”) were set to the highest level of stringency in order to obtain the smallest number of maps. The DAVID database associates each annotation to a gene group using a contingency table representation and calculates its significance. Regulated genes were submitted as well to GATHER database ( http://gather.genome.duke.edu/ ) that contains the GO annotations and KEGG pathways. The GATHER database associates each group of transcripts with the same functional annotation and calculates a Bayes factor [ 18 ] which is a measure of the strength of the evidence supporting an association of an annotation with the submitted gene list. We have selected a low Bayes factor (≥3) for presenting the preponderant evidences for associations. A portion of each endometrial sample was fixed in paraformaldehyde, included in paraffin blocks and 5 μm sections were prepared. PR-A/B, PR-B, glycodelin and Specificity protein 1 (Sp1) were evaluated by IHC in the endometrial samples using the antibodies and dilutions shown in Table  2 and the broad spectrum Histostain-SP kit (Life Technologies, Carlsbad, CA, USA) as described previously [ 21 ]. Immunoreactive PRA/B, PRB, Sp1 and glycodelin in endometrial sections was semi-quantified using the expression level score (ELS), calculated by means of Image Pro Plus software (Media Cybernetics Rockville, MD, USA) as described previously [ 21 ]. Briefly, ELS = Mean Optical Density of immunostaining x Percent Area Positively Stained x 100. Table 2 Antibodies and dilutions used for immunohistochemistry Antibody Source Dilution Progesterone receptor (PR)-A/B Santa Cruz Biotech. (sc-810) 1:50 PR-B Novocastra (NCL-PGR-B) 1:100 Glycodelin H. Koistinen [ 22 ] 1:1000 Specificity protein-1 (Sp1) Santa Cruz Biotech. (sc-14027) 1:100 Antibodies and dilutions used for immunohistochemistry Genomic DNA was isolated from leukocytes derived from peripheral blood obtained by venipuncture using the PAXgene Blood DNA Validation kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The detection of Alu insertion in intron G and restriction fragment length polymorphism (RFLP) analysis in exon 5 to confirm the presence of PROGINS mutation was performed as described by Pisarska et al. [ 23 ].

Women with implantation failure (group A, n = 5), women with 2 or more livebirths conceived either by oocyte donation (group B, n = 6) or naturally (group C, n = 6) were subjected to an oocyte donation mock cycle as recipients and on the seventh day of P administration an endometrial sample was obtained. Total RNA was extracted from each tissue sample and used to individually probe the HG_U133 plus 2.0 human gene microarray comprising of 54,675 genes and expressed sequence tags.We performed PCA for all the endometrial samples analyzed using their respective gene expression profiles for their representation on a three-dimensional graphic (Figure  1 A). Each point in a PCA graph represents the gene expression profile of an endometrial sample and the distance between two plotted points is proportional to the degree of similarity between the gene expression profiles. The PCA plot comprising of a projection on the first three principal components, which together explain 48.8% (21%, 14%, and 13%) of the total variance, showed that endometrial samples from infertile subjects clustered apart from samples belonging to the control groups. In addition, gene expression profiles from endometrial samples obtained from microarray analysis were subjected to unsupervised hierarchical clustering analysis in order to generate a dendogram, which is a tree-structured graph that illustrates the similarities in gene expression profiles between endometrial samples from all groups. The dendogram obtained displayed a striking segregation of samples into two major clustering branches, corresponding to the implantation failure group (Group A) and the successful implantation groups (Groups B and C, Figure  1 B). Figure 1 Principal component analysis (PCA) plot of gene expression profiles from endometrial samples and Venn diagrams from differentially expressed transcripts. A , The profiles from infertile women (group A (n = 5); red dots) cluster separately to clusters representative of women with embryo implantation (groups B (n = 6) and C (n = 6); green and blue dots, respectively). B , hierarchical clustering analysis represented in a tree-like dendogram revealing the similarities on gene expression profiles of endometrial samples. A clear segregation of samples into two major clustering branches, one with samples from group A and the other with samples form groups B and C that self-cluster together. C , Venn diagrams showing the differentially expressed genes in each group, which are either 2-fold down- (panel C ) or up-regulated (panel D ) in endometrial samples from women with implantation failure (group A) compared to those from women that conceived either by IVF (Group B) or naturally (group C). Principal component analysis (PCA) plot of gene expression profiles from endometrial samples and Venn diagrams from differentially expressed transcripts. A , The profiles from infertile women (group A (n = 5); red dots) cluster separately to clusters representative of women with embryo implantation (groups B (n = 6) and C (n = 6); green and blue dots, respectively). B , hierarchical clustering analysis represented in a tree-like dendogram revealing the similarities on gene expression profiles of endometrial samples. A clear segregation of samples into two major clustering branches, one with samples from group A and the other with samples form groups B and C that self-cluster together. C , Venn diagrams showing the differentially expressed genes in each group, which are either 2-fold down- (panel C ) or up-regulated (panel D ) in endometrial samples from women with implantation failure (group A) compared to those from women that conceived either by IVF (Group B) or naturally (group C). The microarrays data analysis revealed that 747 transcripts were down-regulated in group A compared to group B; whereas 218 transcripts were up-regulated (Figure  1 C and 1 D). When group A was compared to group C, 156 and 884 transcripts were decreased and increased respectively in group A (Figure  1 C and 1 D). Only 31 and 51 transcripts down- and up-regulated respectively were common when comparing group A with the control groups B and C (Figure  1 C and 1 D). The lists of common differentially expressed genes in the comparison of gene expression profiles from group A vs. Group B and Group A vs. group C is in Table  3 for down- and Table  4 for up-regulated transcripts. An independent validation by Real Time RT-PCR for some of the differentially expressed genes found in the samples used in this study has been reported elsewhere [ 12 ], supporting our microarrays findings. Table 3 Genes whose transcript level was down-regulated in Group A (n = 5) when compared with Group B (n = 6) and Group C (n = 6) in the microarray analyses UniGene ID Gene symbol Gene title A vs. B p value A vs. C p value Average Hs.699841 IGHA1 Immunoglobulin heavy constant alpha 1 0.1 0.00086 0.08 0.00043 0.09 Hs.436657 CLU Clusterin 0.11 0.00043 0.17 0.00013 0.14 Hs.356624 NID1 Nidogen 1 0.19 0.00013 0.14 0.00022 0.16 Hs.82071 CITED2 Cbp/p300-interacting transactivator. with Glu/Asp-rich carboxy-terminal domain. 2 0.15 0.00086 0.19 0.00043 0.17 Hs.532325 PAEP Progestagen-associated endometrial protein (PAEP) 0.09 0.00086 0.29 0.00086 0.19 Hs.38972 TSPAN1 Tetraspanin 1 0.14 0.00022 0.24 0.00086 0.19 Hs.445705 RRM1 Ribonucleotide reductase M1 0.18 0.00043 0.23 0.00022 0.2 Hs.1012 C4BPA Complement component 4 binding protein. alpha 0.21 0.00043 0.22 0.00043 0.22 Hs.513261 HN1L Hematological and neurological expressed 1-like 0.18 0.00022 0.35 0.00043 0.26 Hs.80658 UCP2 Uncoupling protein 2 (mitochondrial. proton carrier) 0.1 0.00043 0.49 0.00013 0.29 Hs.502989 UNC93B1 Unc-93 homolog B1 (C. elegans) 0.23 0.00013 0.37 0.00043 0.3 Hs.414099 CNPY3 Canopy 3 homolog (zebrafish) 0.29 0.00013 0.32 0.00022 0.31 Hs.110571 GADD45B Growth arrest and DNA-damage-inducible. beta 0.2 0.00022 0.44 0.00086 0.32 Hs.320151 AGPAT2 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic acid acyltransferase. beta) 0.23 0.00086 0.43 0.00013 0.33 Hs.77422 PLP2 Proteolipid protein 2 (colonic epithelium-enriched) 0.26 0.00043 0.42 0.00043 0.34 Hs.1497 RARG Retinoic acid receptor. gamma 0.33 0.00043 0.35 0.00043 0.34 Hs.389700 MGST1 Glutathione S-transferase. microsomal 0.4 0.00022 0.29 0.00043 0.34 Hs.292078 LARP1 La ribonucleoprotein domain family. member 1 0.34 0.00043 0.36 0.00086 0.35 Hs.334587 RBPMS RNA binding protein with multiple splicing 0.29 0.00086 0.43 0.00022 0.36 Hs.5298 ADIPOR1 Adiponectin receptor 1 0.3 0.00022 0.42 0.00013 0.36 Hs.439894 CASZ1 Castor zinc finger 1 0.24 0.00022 0.49 0.00086 0.37 Hs.371727 SCNN1G Sodium channel. nonvoltage-gated 1. gamma 0.29 0.00013 0.45 0.00043 0.37 Hs.474596 LIMK2 LIM domain kinase 2 0.26 0.00043 0.5 0.00013 0.38 Hs.459940 LITAF Lipopolysaccharide-induced TNF factor 0.37 0.00022 0.39 0.00086 0.38 Hs.442449 CHST14 Carbohydrate (N-acetylgalactosamine 4–0) sulfotransferase 14 0.42 0.00086 0.35 0.00086 0.38 Hs.518525 GLUL Glutamate-ammonia ligase 0.42 0.00043 0.48 0.00086 0.45 Hs.119177 ARF3 ADP-ribosylation factor 3 0.47 0.00013 0.45 0.00043 0.46 Hs.497417 KIAA0317 KIAA0317 0.49 0.00086 0.44 0.00013 0.46 Hs.501728 RHOG Ras homolog gene family. member G (rho G) 0.46 0.00043 0.47 0.00043 0.47 Hs.414614 SCNN1B Sodium channel. nonvoltage-gated 1. beta 0.48 0.00013 0.47 0.00022 0.47 Hs.436896 POLR3A Polymerase (RNA) III (DNA directed) polypeptide A. 155 kDa 0.48 0.00043 0.47 0.00043 0.48 Data includes genes with decreased transcript levels displaying a ≥2-fold difference in average A vs. B and A vs. C. Table 4 Genes whose transcript level was up-regulated in Group A (n = 5) when compared with Group B (n = 6) and Group C (n = 6) in the microarray analyses UniGene ID Gene symbol Gene title A vs. B p value A vs. C p value Average Hs.35086 USP1 Ubiquitin specific protease 1 (USP1), mRNA. 42.52 0.00022 44.32 0.00043 43.42 Hs.436977 SYTL3 Synaptotagmin-like 3 20.68 0.00043 26.72 0.00013 23.70 Hs.133421 LIFR Leukemia inhibitory factor receptor 43.71 0.00086 3.32 0.00022 23.52 Hs.160211 THRAP3 Thyroid hormone receptor associated protein 3 (THRAP3), mRNA. 29.04 0.00013 3.66 0.00043 16.35 Hs.532399 ZC3H11A KIAA0663 gene product (KIAA0663), mRNA. 4.38 0.00043 28.05 0.00086 16.21 Hs.652169 PLGLB2 Plasminogen-like B2 21.71 0.00086 9.45 0.00086 15.58 Hs.524809 CLIP1 Restin (Reed-Steinberg cell-expressed intermediate filament-associated protein) (RSN), transcript variant 2, mRNA. 2.19 0.00043 25.81 0.00043 14.00 Hs.16355 MYH10 Myosin, heavy polypeptide 10, non-muscle (MYH10), mRNA. 2.6 0.00013 24.08 0.00022 13.34 Hs.502829 SF1 Splicing factor 1 (SF1), transcript variant 4, mRNA. 21.86 0.00043 4.08 0.00086 12.97 Hs.517949 MAP4 Microtubule-associated protein 4 (MAP4), transcript variant 1, mRNA. 10.41 0.00043 11.96 0.00013 11.18 Hs.8118 SMCHD1 KIAA0650 protein 18.77 0.00013 2.17 0.00043 10.47 Hs.514806 GALNT1 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) (GALNT1), mRNA. 11 0.00043 9.38 0.00043 10.19 Hs.130293 LUC7L3 Cisplatin resistance-associated overexpressed protein (CROP), transcript variant 2, mRNA. 6.32 0.00043 11.88 0.00022 9.10 Hs.143728 WASL Wiskott-Aldrich syndrome-like (WASL), mRNA. 2.07 0.00022 14.32 0.00086 8.20 Hs.532082 IL6ST Interleukin 6 signal transducer (gp130, oncostatin M receptor) (IL6ST), transcript variant 2, mRNA. 2.95 0.00013 13 0.00043 7.97 Hs.2913 EPHB3 EphB3 = HEK2 = tyrosine kinase receptor = large erk kinase 11.39 0.00086 4.41 0.00086 7.90 Hs.431081 USP53 Ubiquitin specific protease 53 2.23 0.00013 13.55 0.00043 7.89 Hs.194726 BAG4 BCL2-associated athanogene 4 (BAG4), mRNA. 11.71 0.00013 3.16 0.00086 7.44 Hs.464971 PIK3C3 Phosphoinositide-3-kinase, class 3 3.92 0.00043 10.41 0.00086 7.16 Hs.9997 SECISBP2L KIAA0256 gene product (KIAA0256), mRNA. 5.43 0.00086 7.36 0.00013 6.39 Hs.497788 EPRS Glutamyl-prolyl-tRNA synthetase (EPRS), mRNA. 4.41 0.00043 8.11 0.00043 6.26 Hs.101014 CEP57 Translokin (KIAA0092), mRNA. 5.03 0.00086 7.31 0.00086 6.17 Hs.143600 GOLIM4 Golgi phosphoprotein 4 (GOLPH4), mRNA. 2.46 0.00043 9.85 0.00022 6.16 Hs.24485 SMC3 Chondroitin sulfate proteoglycan 6 (bamacan) (CSPG6), mRNA. 2.53 0.00022 9.65 0.00043 6.09 Hs.193832 GPATCH4 G patch domain containing 4 (GPATC4), transcript variant 3, mRNA. 3.32 0.00013 7.94 0.00086 5.63 Hs.406695 PRDM7 PR domain containing 7 (PRDM7), mRNA. 2.04 0.00086 8.46 0.00086 5.25 Hs.42194 SPCS3 Signal peptidase complex subunit 3 homolog (S. cerevisiae) (SPCS3), mRNA. 2.1 0.00043 8.34 0.00022 5.22 Hs.458418 KIAA1731 PREDICTED: KIAA1731 protein (KIAA1731), mRNA. 2.04 0.00013 8.4 0.00086 5.22 Hs.49853 CCAR1 Cell division cycle and apoptosis regulator 1 2.75 0.00086 7.67 0.00013 5.21 Hs.496414 ATP7A ATPase, Cu++ transporting, alpha polypeptide (Menkes syndrome) 2.89 0.00086 7.16 0.00043 5.02 Hs.481181 NEK1 NIMA (never in mitosis gene a)-related kinase 1 (NEK1), mRNA. 3.07 0.00086 5.35 0.00043 4.21 Hs.440833 PKN2 protein kinase N2 (PKN2), mRNA. 6.02 0.00022 2.14 0.00043 4.08 Hs.524009 AASDHPPT Aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase 3.89 0.00013 4.2 0.00086 4.04 Hs.26904 SEC63 SEC63 homolog (S. cerevisiae) 2.95 0.00086 3.78 0.00043 3.37 Hs.93485 SCN2A MRNA; cDNA DKFZp761D191 (from clone DKFZp761D191) 2.08 0.00043 4.5 0.00013 3.29 Hs.31082 TMEM33 Transmembrane protein 33 2.19 0.00086 4.35 0.00043 3.27 Hs.371372 CWC27 Serologically defined colon cancer antigen 10 (SDCCAG10), mRNA. 2.95 0.00043 3.51 0.00086 3.23 Hs.523299 EIF3A Eukaryotic translation initiation factor 3, subunit 10 theta, 150/170 kDa (EIF3S10), mRNA. 2.68 0.00022 3.58 0.00022 3.13 Hs.440320 CUL5 Cullin 5 (CUL5), mRNA. 2.6 0.00043 3.25 0.00043 2.93 Hs.203965 PHTF2 Putative homeodomain transcription factor 2 3.56 0.00013 2.3 0.00086 2.93 Hs.335068 TGS1 Nuclear receptor coactivator 6 interacting protein (NCOA6IP), mRNA. 3.63 0.00086 2.16 0.00013 2.89 Hs.189075 TWF1 Twinfilin, actin-binding protein, homolog 1 (Drosophila) 2.36 0.00086 2.97 0.00043 2.67 Hs.127310 UHMK1 U2AF homology motif (UHM) kinase 1 (UHMK1), mRNA. 2.85 0.00086 2.46 0.00043 2.66 Hs.430849 OSBPL8 Oxysterol binding protein-like 8 (OSBPL8), transcript variant 1, mRNA. 2.03 0.00022 3.05 0.00022 2.54 Hs.150557 KLF9 Basic transcription element binding protein 1 (BTEB1), mRNA. 2.6 0.00086 2.39 0.00043 2.50 Hs.210850 HECTD1 HECT domain containing 1 (HECTD1), mRNA. 2.35 0.00013 2.6 0.00086 2.47 Hs.142442 HP1BP3 Heterochromatin protein 1, binding protein 3 2.08 0.00086 2.41 0.00013 2.25 Hs.369284 ESF1 Chromosome 20 open reading frame 6 (C20orf6), mRNA. 2.13 0.00013 2.3 0.00086 2.21 Hs.119023 SMC2 SMC2 structural maintenance of chromosomes 2-like 1 (yeast) (SMC2L1), mRNA. 2.25 0.00022 2 0.00022 2.13 Hs.481927 NIPBL Nipped-B homolog (Drosophila) 2.11 0.00043 2.04 0.00086 2.08 Hs.374201 KIF21A kinesin family member 21A (KIF21A), mRNA. 2.13 0.00013 2 0.00013 2.06 Data includes genes with increased transcript levels displaying a ≥2-fold difference in average A vs. B and A vs. C. Genes whose transcript level was down-regulated in Group A (n = 5) when compared with Group B (n = 6) and Group C (n = 6) in the microarray analyses Data includes genes with decreased transcript levels displaying a ≥2-fold difference in average A vs. B and A vs. C. Genes whose transcript level was up-regulated in Group A (n = 5) when compared with Group B (n = 6) and Group C (n = 6) in the microarray analyses Data includes genes with increased transcript levels displaying a ≥2-fold difference in average A vs. B and A vs. C. In order to gain further understanding of the potential functional roles of dysregulated endometrial transcripts from group A, we obtained the functional annotations from each gene and determined the enriched processes associated to them from two different web-based tools. Within the down-regulated transcripts, the functional classifications immune response and complement activation, classical pathway were found to be statistically over-represented using the web based applications DAVID and GATHER respectively (p < 0.01). The Bayes factor obtained with the analysis using the GATHER database was 3, which indicates that the association of this particular function with the total of the transcripts in our gene list is weak. The up-regulated transcript list was not enriched with transcripts related to a particular function. We reasoned that the endometrium of women from group A might have a dysregulation in P-regulated transcripts as it has been described for endometriosis and also these genes might be coincident with those whose expression in the endometrium is altered upon treatment with the PR antagonist mifepristone. Since women from groups A and B only differ on the embryo implantation outcome, the list of dysregulated transcripts in group A vs. group B during the receptive phase of the endometrium was selected. Within this repertoire, we searched for those genes known to be regulated in normal cycling endometrium by P as it has been described before [ 4 ]. For that we accounted for those transcripts that, directed by P, get regulated for the acquisition of endometrial receptivity [ 24 – 32 ] and/or dysregulated in conditions that render the endometrium with an unreceptive phenotype ( i.e. , endometriosis and mifepristone treatment) and that intersected with our list of up and down regulated genes ( i.e. , A vs. B). We considered only those that had the opposite regulation compared with receptive endometrium, and same regulation in endometrium from women with compromised P signaling in the endometrium such as treated with mifepristone [ 7 ] and/or from women with endometriosis [ 4 ]. We found 14 and 86 up- and down-regulated genes respectively in the endometrium during the receptive period of women with implantation failure vs. control group B (Tables  5 and 6 ). Table 5 Genes previously described to be progesterone regulated that are down-regulated in endometrium of subjects with repeated embryo implantation failure UniGene ID Gene symbol Gene title Up regulated in window of implantation Down regulated in endometriosis or mifepristone Fold change p value Hs.386793 GPX3 Glutathione peroxidase 3 (plasma) (GPX3), mRNA. [ 25 , 27 , 29 , 32 ] 0.01 0.00013 Hs.458355 C1S Complement component 1, s subcomponent, transcript variant 1, mRNA. [ 28 , 29 ] 0.02 0.00043 Hs.647023 CLDN3 Claudin 3 [ 24 ] 0.07 0.00086 Hs.89603 MUC1 Mucin 1, transmembrane, mRNA. [ 25 ] [ 4 ] 0.11 0.00022 Hs.436657 CLU Clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J), transcript variant 1, mRNA. [ 27 , 29 , 31 , 32 ] 0.12 0.00086 Hs.276770 CD52 CD52 molecule [ 7 ] 0.14 0.00043 Hs.498173 SMPD1 Sphingomyelin phosphodiesterase 1, acid lysosomal (acid sphingomyelinase), transcript variant 1, mRNA. [ 24 , 26 ] 0.14 0.00013 Hs.523414 LOC492304 Putative insulin-like growth factor II associated protein, mRNA. [ 27 ] 0.15 0.00022 Hs.532325 PAEP Progestagen-associated endometrial protein [ 24 , 25 , 27 , 31 , 32 ] [ 39 ] 0.15 0.00086 Hs.590970 AXL AXL receptor tyrosine kinase [ 24 ] 0.15 0.00022 Hs.163893 PICALM Phosphatidylinositol binding clathrin assembly protein [ 4 ] 0.16 0.00086 Hs.525607 TNFAIP2 Tumor necrosis factor, alpha-induced protein 2, mRNA. [ 27 – 29 , 32 ] 0.18 0.00043 Hs.654439 APOE Apolipoprotein E [ 24 , 29 ] 0.18 0.00086 Hs.201978 PTGS1 Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase), transcript variant 2, mRNA. [ 29 ] [ 7 ] 0.19 0.00013 Hs.82071 CITED2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2, mRNA. [ 25 ] 0.19 0.00086 Hs.524518 STAT6 Signal transducer and activator of transcription 6, interleukin-4 induced, mRNA. [ 29 ] 0.19 0.00022 Hs.478588 BCL6 B-cell CLL/lymphoma 6 (zinc finger protein 51), transcript variant 1, mRNA. [ 25 , 27 , 29 ] 0.20 0.00086 Hs.1012 C4BPA Complement component 4 binding protein, alpha [ 24 , 25 , 27 , 29 , 31 , 32 ] [ 39 ] 0.22 0.00043 Hs.21765 FADS3 Fatty acid desaturase 3, mRNA. [ 26 ] 0.23 0.00043 Hs.4055 KLF6 Kruppel-like factor 6 [ 25 ] 0.23 0.00013 Hs.332708 FBLN5 Fibulin 5, mRNA. [ 27 , 29 ] 0.23 0.00022 Hs.25292 JUNB Jun B proto-oncogene, mRNA. [ 25 , 26 ] 0.25 0.00043 Hs.431048 ABL1 V-abl Abelson murine leukemia viral oncogene homolog 1, transcript variant b, mRNA. [ 24 ] 0.27 0.00086 Hs.190783 HAL Histidine ammonia-lyase [ 26 , 32 ] 0.27 0.00086 Hs.513984 FLII Flightless I homolog (Drosophila), mRNA. [ 24 , 32 ] 0.27 0.00043 Hs.643357 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 [ 29 ] 0.29 0.00022 Hs.44227 HPSE Heparanase [ 29 ] 0.29 0.00086 Hs.515536 RRAS Related RAS viral (r-ras) oncogene homolog, mRNA. [ 27 ] 0.29 0.00013 Hs.409578 STK38 Serine/threonine kinase 38 [ 26 ] 0.29 0.00043 Hs.549171 C1orf56 Chromosome 1 open reading frame 56 [ 7 ] 0.29 0.00022 Hs.494457 NINJ1 Ninjurin 1, mRNA. [ 26 ] 0.29 0.00013 Hs.270291 ACTN4 Actinin, alpha 4 (ACTN4), mRNA. [ 29 ] 0.29 0.00086 Hs.381099 LCP1 Lymphocyte cytosolic protein 1 (L-plastin), mRNA. [ 28 , 29 ] 0.31 0.00043 Hs.185172 GNB2 Guanine nucleotide binding protein (G protein), beta polypeptide 2, mRNA. [ 26 ] 0.31 0.00013 Hs.1497 RARG Retinoic acid receptor, gamma [ 7 ] 0.33 0.00043 Hs.474751 MYH9 Myosin, heavy polypeptide 9, non-muscle, mRNA. [ 29 ] 0.33 0.00043 Hs.255093 PFKL Phosphofructokinase, liver, transcript variant 2, mRNA. [ 24 ] 0.33 0.00086 Hs.503911 NNMT Nicotinamide N-methyltransferase [ 25 , 27 ] 0.33 0.00043 Hs.504877 ARHGDIB Rho GDP dissociation inhibitor (GDI) beta , mRNA. [ 24 , 26 , 27 , 29 ] 0.33 0.00022 Hs.210995 CA12 Carbonic anhydrase XII, transcript variant 2, mRNA. [ 27 , 32 ] 0.35 0.00043 Hs.520640 ACTB Actin, beta, mRNA. [ 7 ] 0.35 0.00086 Hs.514819 AP2B1 Adaptor-related protein complex 2, beta 1 subunit, mRNA. [ 24 ] 0.35 0.00013 Hs.511605 ANXA2 Annexin A2, transcript variant 2, mRNA. [ 29 ] 0.35 0.00086 Hs.87752 MSN Moesin, mRNA. [ 29 ] 0.35 0.00022 Hs.654958 ABCF2 ATP-binding cassette, sub-family F (GCN20), member 2 [ 7 ] 0.35 0.00013 Hs.443577 TNFRSF21 Tumor necrosis factor receptor superfamily, member 21 [ 29 ] [ 4 ] 0.35 0.00086 Hs.591868 ZBTB10 Zinc finger and BTB domain containing 10 [ 4 ] 0.35 0.00043 Hs.25348 VAMP2 Vesicle-associated membrane protein 2 (synaptobrevin 2) [ 4 ] 0.38 0.00086 Hs.159161 ARHGDIA Rho GDP dissociation inhibitor (GDI) alpha, mRNA. [ 24 ] [ 7 ] 0.38 0.00022 Hs.131269 RARRES1 Retinoic acid receptor responder (tazarotene induced) 1 [ 27 ] 0.38 0.00086 Hs.513915 CLDN7 Claudin 7, mRNA. [ 25 ] 0.38 0.00013 Hs.10326 COPE Coatomer protein complex, subunit epsilon, transcript variant 2, mRNA. [ 24 ] 0.38 0.00013 Hs.416024 NRSN2 Neurensin 2 [ 7 ] 0.38 0.00043 Hs.434248 PLEC Plectin [ 26 , 29 ] [ 39 ] 0.38 0.00086 Hs.584854 AVIL Advillin [ 26 , 29 ] 0.41 0.00022 Hs.183109 MAOA Monoamine oxidase A [ 24 , 25 , 27 , 28 , 31 , 32 ] 0.41 0.00013 Hs.365405 SELO Selenoprotein O [ 4 ] 0.41 0.00013 Hs.645228 KIR3DL1 Killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 [ 29 ] 0.41 0.00086 Hs.528299 HTATIP HIV-1 Tat interacting protein, 60 kDa, transcript variant 3, mRNA. [ 26 ] 0.41 0.00043 Hs.164226 THBS1 Thrombospondin 1, mRNA. [ 29 ] 0.41 0.00086 Hs.647078 CDK5 Cyclin-dependent kinase 5 [ 7 ] 0.41 0.00043 Hs.278573 CD59 CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, EJ16, EJ30, EL32 and G344), transcript variant 2, mRNA. [ 29 ] 0.41 0.00022 Hs.515162 CALR Calreticulin [ 7 ] 0.41 0.00043 Hs.465744 INSR Insulin receptor [ 26 ] 0.41 0.00013 Hs.274256 ELOVL7 ELOVL family member 7, elongation of long chain fatty acids (yeast) [ 4 ] 0.44 0.00086 Hs.450230 IGFBP3 Insulin-like growth factor binding protein 3 [ 27 , 29 , 32 ] 0.44 0.00086 Hs.504687 MYL9 Myosin, light polypeptide 9, regulatory [ 27 ] 0.44 0.00022 Hs.446641 ARAF V-raf murine sarcoma 3611 viral oncogene homolog, mRNA. [ 25 ] 0.44 0.00086 Hs.2030 THBD Thrombomodulin [ 25 , 27 , 29 ] 0.44 0.00013 Hs.104672 FILIP1L Filamin A interacting protein 1-like [ 27 ] 0.44 0.00086 Hs.75862 SMAD4 SMAD family member 4 [ 4 ] 0.44 0.00022 Hs.520757 TBXAS1 Thromboxane A synthase 1 (platelet, cytochrome P450, family 5, subfamily A), transcript variant TXS-II, mRNA. [ 29 ] 0.47 0.00013 Hs.283741 EXOSC5 Exosome component 5 [ 7 ] 0.47 0.00086 Hs.174312 TLR4 Toll-like receptor 4, transcript variant 2, mRNA. [ 29 ] 0.47 0.00043 Hs.24601 FBLN1 Fibulin 1 [ 31 ] [ 7 ] 0.47 0.00086 Hs.501728 RHOG Ras homolog gene family, member G (rho G) [ 7 ] 0.47 0.00043 Hs.220864 CHD2 Chromodomain helicase DNA binding protein 2 [ 4 ] 0.47 0.00086 Hs.524809 CLIP1 CAP-GLY domain containing linker protein 1 [ 29 ] 0.47 0.00043 Hs.92236 MLL4 Myeloid/lymphoid or mixed-lineage leukemia 4 [ 7 ] 0.47 0.00086 Hs.654688 MKL1 Megakaryoblastic leukemia (translocation) 1 [ 26 ] 0.47 0.00043 Hs.279837 GSTM2 Glutathione S-transferase mu 2 (muscle) [ 26 ] 0.47 0.00086 Hs.645227 TGFB1 Transforming growth factor, beta 1 [ 7 ] 0.50 0.00086 Hs.149261 RUNX1 Runt-related transcription factor 1 [ 27 ] 0.50 0.00013 Hs.522818 L1CAM L1 cell adhesion molecule (hydrocephalus, stenosis of aqueduct of Sylvius 1, MASA (mental retardation, aphasia, shuffling gait and adducted thumbs) syndrome, spastic paraplegia 1) [ 26 ] 0.50 0.00043 Hs.840 IDO1 Indoleamine 2,3-dioxygenase 1 [ 24 , 25 , 29 ] 0.50 0.00086 Hs.2256 MMP7 Matrix metalloproteinase 7 (matrilysin, uterine) [ 27 ] 0.50 0.00043 Data is expressed as fold change for endometrial genes down-regulated ≥2-fold in group A vs. group B that have been shown either up-regulated during the window of implantation or down-regulated in women with endometriosis or treated with mifepristone. Bolded transcripts are decreased also in group A vs. Group C. Table 6 Genes previously described to be progesterone regulated that are up-regulated in endometrium of subjects with repeated embryo implantation failure UniGene ID Gene symbol Gene title Down regulated in window of implantation Up regulated in endometriosis or RU486 Fold change p value Hs.208854 CD69 CD69 antigen (p60, early T-cell activation antigen) [ 7 ] 2,3 0.00022 Hs.406515 NQO1 NAD(P)H dehydrogenase, quinone 1 [ 29 ] 2,2 0.00043 Hs.335614 SEC14L2 SEC14-like 2 (S. cerevisiae), mRNA. [ 4 ] 1,9 0.00013 Hs.481181 NEK1 NIMA (never in mitosis gene a)-related kinase 1 (NEK1), mRNA. [ 29 ] 1,6 0.00043 Hs.86368 CLGN Calmegin, mRNA. [ 7 ] 1,5 0.00086 Hs.189075 TWF1 Twinfilin, actin-binding protein, homolog 1 (Drosophila) [ 4 ] 1,5 0.00043 Hs.127680 LOC389332 PREDICTED: hypothetical LOC389332 (LOC389332), mRNA. [ 4 ] 1,4 0.00013 Hs.369430 PAM Peptidylglycine alpha-amidating monooxygenase, transcript variant 3, mRNA. [ 24 , 28 ] 1,1 0.00022 Hs.514806 GALNT1 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1), mRNA. [ 7 ] 1,1 0.00043 Hs.509447 GRLF1 Glucocorticoid receptor DNA binding factor 1 [ 7 ] 1,1 0.00086 Hs.481927 NIPBL Nipped-B homolog (Drosophila) [ 7 ] 1,1 0.00086 Hs.444558 KHDRBS3 KH domain containing, RNA binding, signal transduction associated 3, mRNA. [ 24 , 29 , 32 ] [ 4 ] 1,1 0.00043 Hs.495710 GPM6B Glycoprotein M6B (GPM6B), transcript variant 4, mRNA. [ 29 ] 1,0 0.00022 Hs.496414 ATP7A ATPase, Cu++ transporting, alpha polypeptide (Menkes syndrome) [ 29 ] 1,0 0.00043 Data is expressed as fold change for endometrial genes up-regulated ≥2-fold in group A vs. group B that have been shown either down-regulated during the window of implantation or up-regulated in women with endometriosis or treated with mifepristone. Bolded transcripts are increased also in group A vs. Group C. Genes previously described to be progesterone regulated that are down-regulated in endometrium of subjects with repeated embryo implantation failure Data is expressed as fold change for endometrial genes down-regulated ≥2-fold in group A vs. group B that have been shown either up-regulated during the window of implantation or down-regulated in women with endometriosis or treated with mifepristone. Bolded transcripts are decreased also in group A vs. Group C. Genes previously described to be progesterone regulated that are up-regulated in endometrium of subjects with repeated embryo implantation failure Data is expressed as fold change for endometrial genes up-regulated ≥2-fold in group A vs. group B that have been shown either down-regulated during the window of implantation or up-regulated in women with endometriosis or treated with mifepristone. Bolded transcripts are increased also in group A vs. Group C. Since the comparative gene expression analysis of P-regulated genes in endometrial samples from group A, suggested an altered P response, we determined the presence of the Alu insertion in intron G of the PR gene ( PROGINS ) in women from groups A, B and C. Restriction fragment length polymorphism (RFLP) analysis was also carried out on exon 5 of PR gene for confirmation. We found 4 heterozygous subjects for pgr (Figures  2 A and B). Two were from group B and two from group C, whereas no PROGINS alleles were detected in women from group A. Figure 2 Screening for PROGINS allele. A , identification of Alu insertion in Intron G. The Alu insertion in the progesterone receptor gene generates a 494-bp PCR product compared to the 174-bp fragment obtained for the wild type. Samples 04, 05, 24 and 25 with bands at 494 bp and 174 bp indicate the presence of PROGINS in the heterozygous state. All the other lanes with a single fragment of 174 bp indicate the presence of the wild-type progesterone receptor in the homozygous state. B , restriction digestion of exon 5 with NlaIII. Lanes for samples 04, 05, 24 and 25 confirm the presence of PROGINS in heterozygous state; NlaIII cleaves the PCR product into two fragments, 106 and 53 bp. All the other samples displayed the uncleaved 159-bp fragment only, indicating the presence of the wild type receptor. Screening for PROGINS allele. A , identification of Alu insertion in Intron G. The Alu insertion in the progesterone receptor gene generates a 494-bp PCR product compared to the 174-bp fragment obtained for the wild type. Samples 04, 05, 24 and 25 with bands at 494 bp and 174 bp indicate the presence of PROGINS in the heterozygous state. All the other lanes with a single fragment of 174 bp indicate the presence of the wild-type progesterone receptor in the homozygous state. B , restriction digestion of exon 5 with NlaIII. Lanes for samples 04, 05, 24 and 25 confirm the presence of PROGINS in heterozygous state; NlaIII cleaves the PCR product into two fragments, 106 and 53 bp. All the other samples displayed the uncleaved 159-bp fragment only, indicating the presence of the wild type receptor. Since the levels of both isoforms of PR in human endometrium have been found to be abnormal in patients with endometriosis [ 33 , 34 ], we evaluated the immunoreactive presence of PR-A/B (Figures  3 A and 3 C), PR-B (Figures  3 D and 3 F) along with Sp1 (Figure  3 G and 3 I) and the P-regulated glycoprotein glycodelin (Figures  3  J and 3 L) in paraformaldehyde-fixed paraffin embedded endometrial tissue from groups A, B and C by IHC. Immunostaining was semi-quantified by calculating the respective ELS scores for each detected molecule in all groups of women (Figure  4 ). ELS for glycodelin in groups B and C was 10.6 and 12.1 fold from group A respectively (p = 0.00509, Figure  4 A). The presence of PR-A/B and PR-B in endometrial tissue was evaluated (Figures  3 A-C and 3 D-F, respectively), since a possible post-translational dysregulation of PR expression (not detected by transcript analysis) might explain the differential gene expression of P-regulated genes in the endometrium from women of group A such as glycodelin. The ELS scores obtained for PR-A/B and PRB did not show significant differences amongst groups (Figures  4 B and 4 C respectively). In addition, semi-quantitation of immunoreactive Sp1, a known co-activator and trans-activator of the PR that mediates P-induced glycodelin expression, did not show significant differences amongst groups A, B and C (Figure  4 D). Figure 3 Immunodetection of progesterone receptor (A and B isoforms, PR), progesterone receptor B (PRB), Specificity Protein 1 (Sp1) and glycodelin in endometrial sections. Representative photomicrographs of endometrial sections immunostained in triplicate for PR (panels A , B and C ), PRB (panels D , E and F ), Sp1 (panels G , H and I ) and glycodelin (panels J , K and L ) are shown in women from group A (panels A , D , G and J ; n = 5), group B (panels B , E , H and K ; n = 6) and group C (panels C , F , I and L ; n = 6). Figure 4 Immunohistochemistry semiquantitation. Expression Level Score (ELS) for immunostaining of glycodelin (panel A ), PRA/B (panel B ), PRB (panel C ) and Sp1 (panel D ) in endometrial sections from groups A (n = 5), B (n = 6) and C (n = 6). Data is expressed as average ELS ± SD for each group. *p < 0.05, Kruskal Wallis U-test. Immunodetection of progesterone receptor (A and B isoforms, PR), progesterone receptor B (PRB), Specificity Protein 1 (Sp1) and glycodelin in endometrial sections. Representative photomicrographs of endometrial sections immunostained in triplicate for PR (panels A , B and C ), PRB (panels D , E and F ), Sp1 (panels G , H and I ) and glycodelin (panels J , K and L ) are shown in women from group A (panels A , D , G and J ; n = 5), group B (panels B , E , H and K ; n = 6) and group C (panels C , F , I and L ; n = 6). Immunohistochemistry semiquantitation. Expression Level Score (ELS) for immunostaining of glycodelin (panel A ), PRA/B (panel B ), PRB (panel C ) and Sp1 (panel D ) in endometrial sections from groups A (n = 5), B (n = 6) and C (n = 6). Data is expressed as average ELS ± SD for each group. *p < 0.05, Kruskal Wallis U-test.

Cellular and molecular events in the uterine milieu that lead to successful blastocyst implantation are required in the endometrium to become receptive and ready for implantation. Acquisition of receptivity is driven by estradiol (E2) and progesterone (P), which acting through their receptors, change the transcription rate of target genes [ 1 ]. Particularly, the postovulatory rise in P triggers a sequence of highly coordinated responses beginning with the detention of the estrogen-induced epithelial cell proliferation and followed by the transformation to a secretory phenotype of the gland, recruitment of leukocytes and angiogenesis [ 2 ]. The P action is mediated primarily through binding to and activation of its cognate receptors; the full length B- and N-terminally truncated A isoforms of the P receptor (PR), classically defined as ligand-activated transcription factors [ 3 ]. Upon exposure to P, the ligand-activated receptor can directly interact with specific P-response elements (PREs) in the promoter regions of target genes. It is accepted that P acts on an estrogen primed endometrium to initiate a pattern of gene expression important for achievement of receptivity and an altered PR signaling has been associated with human endometrial dysfunction [ 4 ]. The antiprogestin mifepristone binds to the PR with high affinity blocking the biological effects of P. In women, the administration of a single dose of oral mifepristone (200 mg) during the secretory phase of the cycle rapidly renders the endometrium unreceptive and modifies gene expression in the uterus within 6 h of administration [ 5 – 7 ]. The development of microarray technology has led to many large-scale gene expression profiling studies of human endometrium [ 8 – 10 ]. Although there seems to be very few consensus genes that have been identified across similar studies [ 11 ], collectively they demonstrate that a multitude of genes are associated with the endometrial transcriptome, whose regulation for the acquisition of the receptive phenotype is ultimately driven by P. The approach our group has used to identify the endometrial receptivity transcript profile in a previous report from our laboratory [ 12 ] was to analyze endometrial tissue obtained from women during a mock hormonal treatment cycle for oocyte donation as a recipient. The endometrial samples are collected during the time interval corresponding to the window of implantation [ 13 ] comparing gene expression profiles from women who were refractory to implantation and those who achieved pregnancy in previous oocyte donation cycles [ 12 ]. Although this previous study provided interesting insights to endometrial gene expression associated with implantation failure, the microarrays analysis was performed with only 3(from a total of 5) samples from women with repeated embryo implantation failure that were pooled and using a microarrays platform that examined only one-third of the human genome; providing only a partial view of the whole picture. The aim of the present report was to examine the individual gene expression profiles in the endometrium from women with implantation failure and compare them with those obtained from fertile women in order to identify compromised transcripts and pathways in the infertile group. We used a microarrays platform for complete coverage of the human genome and bioinformatics tools for data interpretation. Here we report that several transcripts, whose expression level is aberrant in the infertile group, have been described as regulated by P and are related to immune function.

Uterine receptivity is defined as a restricted time-related period when the uterus is receptive to blastocyst attachment and implantation. The establishment of this endometrial transition, which supports embryo implantation, is primarily coordinated by ovarian hormones, E2 and P that modulate uterine events in a spatiotemporal manner. Endometrial factors, at the molecular level, have been suggested to explain some cases of infertility, recurrent miscarriages and implantation failure after IVF. In the present study we assessed the endometrial gene expression profile during the receptive period in mock oocyte donation cycles of women with repeated embryo implantation failure (Group A). Their profiles were compared with those obtained from women who achieved embryo implantation and pregnancy in oocyte donation cycles (Group B), or which got pregnant in natural spontaneous cycles (Group C). The data suggest a strong association between an aberrant endometrial gene expression and implantation failure. The stimulation protocol with steroid hormones performed before the endometrial sample collection was the same for all participating women in this study. Hence, the differential transcript profile in Group A suggests a long-term dysregulation of endometrial gene regulation rendering it not suitable for embryo implantation. The functional annotation analysis of dysregulated transcripts showed an enrichment of decreased genes involved in immune response and complement activation in women with repeated implantation failure. Integration and cross-validation of endometrial transcripts regulated by P could increase the confidence in expression results for many more genes than is tractable with classical one-by-one validation of differentially expressed genes and should provide the up- and down-regulated genes that together orchestrate the acquisition of the receptive phenotype of the endometrium for embryo implantation. Such exploration and integration could help to get a comprehensive view of existing data needed to better prioritize experimental efforts. We identified a subset of P-regulated transcripts with differential expression in the endometrium of women from group A compared to the control group B revealing compromised P-signalling in the endometrium. Pisarska et al. [ 23 ] reported that 42% of women with unexplained infertility carry the allele for the PROGINS mutation compared with 14% of control fertile women (with at least 1 term pregnancy). We analyzed the presence of the PROGINS allele in women from groups A, B and C and found no correlation between the PROGINS carrier women and altered transcript levels of P-regulated genes in the endometrium. This result is in line with a study from Coulam et al. [ 35 ] that did not find an association between PR polymorphisms with recurrent implantation failure in women after in vitro fertilization and embryo transfer. Glycodelin, encoded by the gene PAEP [ 36 , 37 ] is the main P-regulated glycoprotein secreted by the endometrial epithelium during the secretory phase and early pregnancy [ 38 ]. The transcript levels for PAEP have also been consistently identified to be one of the most abundant in the endometrium by several gene expression profiling studies [ 24 , 25 , 27 , 31 ], and it has been shown to be decreased in women with endometriosis [ 39 ]. In the present study we found that the transcript levels for PAEP were decreased in the endometrium of women from group A compared to both control groups (Table  5 ). In addition, immunoreactive glycodelin evaluation in endometrial sections showed the protein to be significantly decreased in group A which is in line with the microarrays data. These results are consistent with the reduced concentrations of glycodelin in uterine flushing reported for patients with unexplained infertility [ 40 ]. In normal ovulatory cycles, P secretion is followed by endometrial glycodelin synthesis in epithelial glands from 4 to 5 postovulatory days onwards [ 37 , 41 ]. Endometrial epithelial cells stimulated in vitro with progestins showed an increase in glycodelin transcription, synthesis, and secretion [ 42 ] however a PR-antagonist failed to prevent the induction of glycodelin [ 43 ]. In silico analysis of PAEP gene promoter sequence identified a potential P response element [ 44 ], however functional studies found that the transcription factor Sp1 mediates the effect of P and PR on human glycodelin expression in endometrial cells [ 45 ]. We semiquantified the immunoreactive levels of PR-A/B, PR-B and Sp1 in endometrial sections of women from groups A, B and C and found no significant differences between the groups. The endometrial response to the increased circulating levels of P during the luteal phase has shown to be remarkably different in women with endometriosis compared to healthy controls. Such response has been evidenced by dysregulated specific gene networks of P-dependent genes in patients with endometriosis compared to non-diseased patients in eutopic secretory endometrium [ 4 , 39 , 46 , 47 ]. This transcriptional behavior has led to the concept of ‘P resistance’ which may explain the association between pelvic endometriosis and infertility. We have found that the endometrial transcript profile from women with repeated implantation failure (group A) presents altered gene expression profile including several transcripts reported to be P-regulated, suggesting a women from group A have a compromised P signalling in the endometrium. The cause of this endometrial defect is unknown, although the apparent intrinsic dysregulation in P signalling that renders the endometrium unreceptive in women with repeated embryo implantation failure seems to be beyond perturbations in PR expression such as chaperone proteins involved in receptor recycling and ligand binding [ 48 ], coregulators [ 49 – 51 ], as well as associated transcription factors and a variety of upstream signal transduction pathways capable of modifying PR and its coregulators [ 52 – 56 ]. In addition, the action of the P is not limited to the cell type in which is PR expressed since steroid hormone regulation can be mediated also through epithelial-stromal cross talk in the endometrium [ 57 ]. Also P can elicit a variety of rapid signalling events, independently of a direct transcriptional regulation or even in the absence of its cognate nuclear receptors [ 58 ] which may modulate gene expression. The molecular mechanism behind the defect in P-regulated gene networks in the endometrium of women with repeated embryo implantation failure is yet to be determined.

We conclude that some cases of repeated implantation failure could be associated with an aberrant gene expression profile, particularly of transcripts related to the immune function and complement activation. Compromised P signaling might be the underlying mechanism for such endometrial gene expression deregulation in women with repeated implantation failure. Future research should focus on determining the causes of incomplete P signalling in the endometrium from these women.