Open Access

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

  • Alejandro Tapia-Pizarro1Email author,
  • Paula Figueroa2,
  • Julio Brito2,
  • Juan Carlos Marín3,
  • David J Munroe4 and
  • Horacio B Croxatto5
Reproductive Biology and Endocrinology201412:92

https://doi.org/10.1186/1477-7827-12-92

Received: 30 May 2014

Accepted: 17 September 2014

Published: 23 September 2014

Abstract

Background

Endometrial function is essential for embryo implantation. The aim of this study was to analyze gene expression profiles from individual endometrial samples obtained from women with repeated implantation failure after IVF in oocyte donation programs.

Methods

Seventeen volunteers were recruited: women who had previously participated as recipients in oocyte donation cycles and repeatedly exhibited implantation failure (Group A, study group, n = 5) or had at least one successful cycle (Group B, control group, n = 6) and spontaneously fertile women (Group C, normal fertility group, n = 6). An endometrial cycle was induced with exogenous estradiol (E2) and progesterone (P) and an endometrial sample was collected on the seventh day of P treatment.

Results

Transcriptome analysis showed 82 genes with consistent differential gene expression when comparing A vs. B and A vs. C. One hundred transcripts differentially expressed in group A vs. B have been shown to be regulated by P, suggesting compromised P signaling in the endometrium. The P receptor (PR) mutation PROGINS was not detected in women from group A. Semi-quantitation of immunoreactive PRA/B, PRB and Sp1 (a transcription factor related to P signaling) in paraffin-embedded endometrial sections, did not show statistically significant differences amongst groups. However immunostaining glycodelin was significantly decreased in endometrial samples from group A

Conclusions

We conclude that some cases of repeated implantation failure could be associated with an aberrant gene expression profile. Compromised P signaling might be the underlying mechanism for such endometrial gene expression deregulation in women with repeated implantation failure.

Keywords

Endometrium Gene expression PROGINS Implantation failure

Background

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 [57].

The development of microarray technology has led to many large-scale gene expression profiling studies of human endometrium [810]. 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.

Methods

Subjects

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.

Induction of endometrial cycle

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.

Gene expression profiling

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).

Statistical analyses of microarrays data

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.

Hierarchical clustering

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.

Principal component analysis (PCA)

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.

Functional clustering

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.

DAVID

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.

GATHER

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.

Immunohistochemistry (IHC)

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

DNA isolation and PROGINSdetection

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].

Results

Gene expression profile analysis

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 1A). 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 1B).
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).

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 1C and 1D). When group A was compared to group C, 156 and 884 transcripts were decreased and increased respectively in group A (Figure 1C and 1D). Only 31 and 51 transcripts down- and up-regulated respectively were common when comparing group A with the control groups B and C (Figure 1C and 1D). 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.

Functional associations of transcripts dysregulated in group A vs. control groups

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.

P-regulated genes in women with implantation failure (group A) vs. control (group B)

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 [2432] 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.

[2729, 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.

PROGINS detection

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 2A 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.

IHC analysis

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 3A and 3C), PR-B (Figures 3D and 3F) along with Sp1 (Figure 3G and 3I) and the P-regulated glycoprotein glycodelin (Figures 3 J and 3L) 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 4A). The presence of PR-A/B and PR-B in endometrial tissue was evaluated (Figures 3A-C and 3D-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 4B and 4C 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 4D).
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.

Discussion

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 [4951], as well as associated transcription factors and a variety of upstream signal transduction pathways capable of modifying PR and its coregulators [5256]. 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.

Conclusions

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.

Abbreviations

DAVID: 

Database for annotation visualization and integrated discovery

E2: 

Estradiol

ELS: 

Expression level score

GATHER: 

Gene annotation tool to help explain relationships

IHC: 

Immunohistochemistry

mAdb: 

National cancer institute’s microarrays data base webtool

P: 

Progesterone

PAEP: 

Progestagen-associated endometrial protein

PCA: 

Principal component analysis

Pgr

Progesterone receptor gene

PR: 

Progesterone receptor

PR-A/B: 

Progesterone receptor isoforms A and B

PR-B: 

Progesterone receptor isoform B

PREs: 

Progesterone-response elements

PROGINS: 

Alu insertion in intron G of the progesterone receptor gene

RFLP: 

Restriction fragment length polymorphism

RT-PCR: 

Reverse transcription coupled to polymerase chain reaction

Sp1: 

Specificity Protein 1..

Declarations

Acknowledgements

We acknowledge to all volunteer women participating in the present study for their generous contribution. Also we acknowledge Dr. Fernando Gabler for dating the endometrial samples, Dr. H. Koistinen for providing the glycodelin antibody and Felipe Argandona for excellent technical assistance. This work was supported by FONDECYT 11100443 / 1140614 and FONDAP 15010006.

Authors’ Affiliations

(1)
Instituto de Investigaciones Materno Infantil, Facultad de Medicina, Universidad de Chile,
(2)
Facultad de Medicina, Universidad Mayor,
(3)
Facultad de Ciencias, Universidad del Bio-Bío,
(4)
Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick,
(5)
Center for Integrative Medicine and Innovative Sciences, Universidad Andrés Bello,

References

  1. O’Malley BW, Tsai MJ: Molecular pathways of steroid receptor action. Biol Reprod. 1992, 46: 163-167. 10.1095/biolreprod46.2.163.View ArticlePubMedGoogle Scholar
  2. Gellersen B, Brosens IA, Brosens JJ: Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives. Semin Reprod Med. 2007, 25: 445-453. 10.1055/s-2007-991042.View ArticlePubMedGoogle Scholar
  3. Beato M: Gene regulation by steroid hormones. Cell. 1989, 56: 335-344. 10.1016/0092-8674(89)90237-7.View ArticlePubMedGoogle Scholar
  4. Burney RO, Talbi S, Hamilton AE, Vo KC, Nyegaard M, Nezhat CR, Lessey BA, Giudice LC: Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology. 2007, 148: 3814-3826. 10.1210/en.2006-1692.View ArticlePubMedGoogle Scholar
  5. Hapangama DK, Critchley HO, Henderson TA, Baird DT: Mifepristone-induced vaginal bleeding is associated with increased immunostaining for cyclooxygenase-2 and decrease in prostaglandin dehydrogenase in luteal phase endometrium. J Clin Endocrinol Metab. 2002, 87: 5229-5234. 10.1210/jc.2002-020429.View ArticlePubMedGoogle Scholar
  6. Danielsson KG, Marions L, Bygdeman M: Effects of mifepristone on endometrial receptivity. Steroids. 2003, 68: 1069-1075. 10.1016/S0039-128X(03)00131-4.View ArticlePubMedGoogle Scholar
  7. Catalano RD, Critchley HO, Heikinheimo O, Baird DT, Hapangama D, Sherwin JR, Charnock-Jones DS, Smith SK, Sharkey AM: Mifepristone induced progesterone withdrawal reveals novel regulatory pathways in human endometrium. Mol Hum Reprod. 2007, 13: 641-654. 10.1093/molehr/gam021.View ArticlePubMedGoogle Scholar
  8. Horcajadas JA, Pellicer A, Simon C: Wide genomic analysis of human endometrial receptivity: new times, new opportunities. Hum Reprod Update. 2007, 13: 77-86.View ArticlePubMedGoogle Scholar
  9. Ruiz-Alonso M, Blesa D, Simon C: The genomics of the human endometrium. Biochim Biophys Acta. 1822, 2012: 1931-1942.Google Scholar
  10. Haouzi D, Dechaud H, Assou S, De Vos J, Hamamah S: Insights into human endometrial receptivity from transcriptomic and proteomic data. Reprod Biomed Online. 2012, 24: 23-34. 10.1016/j.rbmo.2011.09.009.View ArticlePubMedGoogle Scholar
  11. Tapia A, Vilos C, Marin JC, Croxatto HB, Devoto L: Bioinformatic detection of E47, E2F1 and SREBP1 transcription factors as potential regulators of genes associated to acquisition of endometrial receptivity. Reprod Biol Endocrinol. 2011, 9: 14-10.1186/1477-7827-9-14.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Tapia A, Gangi LM, Zegers-Hochschild F, Balmaceda J, Pommer R, Trejo L, Pacheco IM, Salvatierra AM, Henriquez S, Quezada M, Vargas M, Ríos M, Munroe DJ, Croxatto HB, Velasquez L: Differences in the endometrial transcript profile during the receptive period between women who were refractory to implantation and those who achieved pregnancy. Hum Reprod. 2008, 23: 340-351.View ArticlePubMedGoogle Scholar
  13. Damario MA, Lesnick TG, Lessey BA, Kowalik A, Mandelin E, Seppala M, Rosenwaks Z: Endometrial markers of uterine receptivity utilizing the donor oocyte model. Hum Reprod. 2001, 16: 1893-1899. 10.1093/humrep/16.9.1893.View ArticlePubMedGoogle Scholar
  14. Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy. Am J Obstet Gynecol. 1975, 122: 262-263.PubMedGoogle Scholar
  15. Benjamini Y, Hochberg Y: Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J Roy Statist Soc Ser B (Method). 1995, 57: 289-300.Google Scholar
  16. Joliffe IT, Morgan BJ: Principal component analysis and exploratory factor analysis. Stat Methods Med Res. 1992, 1: 69-95. 10.1177/096228029200100105.View ArticlePubMedGoogle Scholar
  17. Dennis G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA: DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003, 4: P3-10.1186/gb-2003-4-5-p3.View ArticlePubMedGoogle Scholar
  18. Chang JT, Nevins JR: GATHER: a systems approach to interpreting genomic signatures. Bioinformatics. 2006, 22: 2926-2933. 10.1093/bioinformatics/btl483.View ArticlePubMedGoogle Scholar
  19. Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M: From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 2006, 34: D354-357. 10.1093/nar/gkj102.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. Gene Ontology Consortium Nat Genet. 2000, 25: 25-29.PubMedGoogle Scholar
  21. Gonzalez-Ramos R, Rocco J, Rojas C, Sovino H, Poch A, Kohen P, Alvarado-Diaz C, Devoto L: Physiologic activation of nuclear factor kappa-B in the endometrium during the menstrual cycle is altered in endometriosis patients. Fertil Steril. 2012, 97: 645-651. 10.1016/j.fertnstert.2011.12.006.View ArticlePubMedGoogle Scholar
  22. Koistinen H, Koistinen R, Dell A, Morris HR, Easton RL, Patankar MS, Oehninger S, Clark GF, Seppala M: Glycodelin from seminal plasma is a differentially glycosylated form of contraceptive glycodelin-A. Mol Hum Reprod. 1996, 2: 759-765. 10.1093/molehr/2.10.759.View ArticlePubMedGoogle Scholar
  23. Pisarska MD, Carson SA, Casson PR, Tong X, Buster JE, Kieback DG: A mutated progesterone receptor allele is more prevalent in unexplained infertility. Fertil Steril. 2003, 80: 651-653. 10.1016/S0015-0282(03)00755-6.View ArticlePubMedGoogle Scholar
  24. Kao LC, Tulac S, Lobo S, Imani B, Yang JP, Germeyer A, Osteen K, Taylor RN, Lessey BA, Giudice LC: Global gene profiling in human endometrium during the window of implantation. Endocrinology. 2002, 143: 2119-2138. 10.1210/endo.143.6.8885.View ArticlePubMedGoogle Scholar
  25. Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillips SC, Smith SK: Determination of the transcript profile of human endometrium. Mol Hum Reprod. 2003, 9: 19-33. 10.1093/molehr/gag004.View ArticlePubMedGoogle Scholar
  26. Carson DD, Lagow E, Thathiah A, Al-Shami R, Farach-Carson MC, Vernon M, Yuan L, Fritz MA, Lessey B: Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod. 2002, 8: 871-879. 10.1093/molehr/8.9.871.View ArticlePubMedGoogle Scholar
  27. Riesewijk A, Martin J, van Os R, Horcajadas JA, Polman J, Pellicer A, Mosselman S, Simon C: Gene expression profiling of human endometrial receptivity on days LH + 2 versus LH + 7 by microarray technology. Mol Hum Reprod. 2003, 9: 253-264. 10.1093/molehr/gag037.View ArticlePubMedGoogle Scholar
  28. Mirkin S, Arslan M, Churikov D, Corica A, Diaz JI, Williams S, Bocca S, Oehninger S: In search of candidate genes critically expressed in the human endometrium during the window of implantation. Hum Reprod. 2005, 20: 2104-2117. 10.1093/humrep/dei051.View ArticlePubMedGoogle Scholar
  29. Talbi S, Hamilton AE, Vo KC, Tulac S, Overgaard MT, Dosiou C, Le Shay N, Nezhat CN, Kempson R, Lessey BA, Nayak NR, Giudice LC: Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology. 2006, 147: 1097-1121. 10.1210/en.2005-1076.View ArticlePubMedGoogle Scholar
  30. Ace CI, Okulicz WC: Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase. Reprod Biol Endocrinol. 2004, 2: 54-10.1186/1477-7827-2-54.PubMed CentralView ArticlePubMedGoogle Scholar
  31. Haouzi D, Mahmoud K, Fourar M, Bendhaou K, Dechaud H, De Vos J, Reme T, Dewailly D, Hamamah S: Identification of new biomarkers of human endometrial receptivity in the natural cycle. Hum Reprod. 2009, 24: 198-205.View ArticlePubMedGoogle Scholar
  32. Diaz-Gimeno P, Horcajadas JA, Martinez-Conejero JA, Esteban FJ, Alama P, Pellicer A, Simon C: A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature. Fertil Steril. 2011, 95: 50-60. 10.1016/j.fertnstert.2010.04.063. 60 e51-15View ArticlePubMedGoogle Scholar
  33. Bulun SE, Cheng YH, Yin P, Imir G, Utsunomiya H, Attar E, Innes J, Julie Kim J: Progesterone resistance in endometriosis: link to failure to metabolize estradiol. Mol Cell Endocrinol. 2006, 248: 94-103. 10.1016/j.mce.2005.11.041.View ArticlePubMedGoogle Scholar
  34. Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE: Progesterone receptor isoform A but not B is expressed in endometriosis. J Clin Endocrinol Metab. 2000, 85: 2897-2902.PubMedGoogle Scholar
  35. Coulam CB, Jeyendran RS, Roussev R: Association of progesterone receptor polymorphisms with recurrent implantation failure after in vitro fertilization and embryo transfer. J Assist Reprod Genet. 2008, 25: 119-122. 10.1007/s10815-008-9210-9.PubMed CentralView ArticlePubMedGoogle Scholar
  36. Seppala M, Bohn H, Tatarinov Y: Glycodelins. Tumour Biol. 1998, 19: 213-220. 10.1159/000030009.View ArticlePubMedGoogle Scholar
  37. Seppala M, Taylor RN, Koistinen H, Koistinen R, Milgrom E: Glycodelin: a major lipocalin protein of the reproductive axis with diverse actions in cell recognition and differentiation. Endocr Rev. 2002, 23: 401-430. 10.1210/er.2001-0026.View ArticlePubMedGoogle Scholar
  38. Bell SC, Hales MW, Patel S, Kirwan PH, Drife JO: Protein synthesis and secretion by the human endometrium and decidua during early pregnancy. Br J Obstet Gynaecol. 1985, 92: 793-803. 10.1111/j.1471-0528.1985.tb03048.x.View ArticlePubMedGoogle Scholar
  39. Kao LC, Germeyer A, Tulac S, Lobo S, Yang JP, Taylor RN, Osteen K, Lessey BA, Giudice LC: Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility. Endocrinology. 2003, 144: 2870-2881. 10.1210/en.2003-0043.View ArticlePubMedGoogle Scholar
  40. Mackenna A, Li TC, Dalton C, Bolton A, Cooke I: Placental protein 14 levels in uterine flushing and plasma of women with unexplained infertility. Fertil Steril. 1993, 59: 577-582.PubMedGoogle Scholar
  41. Seppala M, Koistinen H, Koistinen R, Chiu PC, Yeung WS: Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations. Hum Reprod Update. 2007, 13: 275-287. 10.1093/humupd/dmm004.View ArticlePubMedGoogle Scholar
  42. Taylor RN, Vigne JL, Zhang P, Hoang P, Lebovic DI, Mueller MD: Effects of progestins and relaxin on glycodelin gene expression in human endometrial cells. Am J Obstet Gynecol. 2000, 182: 841-847. 10.1016/S0002-9378(00)70333-4. discussion 847–849View ArticlePubMedGoogle Scholar
  43. Taylor RN, Savouret JF, Vaisse C, Vigne JL, Ryan I, Hornung D, Seppala M, Milgrom E: Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells. J Clin Endocrinol Metab. 1998, 83: 4006-4012.PubMedGoogle Scholar
  44. Vaisse C, Atger M, Potier B, Milgrom E: Human placental protein 14 gene: sequence and characterization of a short duplication. DNA Cell Biol. 1990, 9: 401-413. 10.1089/dna.1990.9.401.View ArticlePubMedGoogle Scholar
  45. Gao J, Mazella J, Seppala M, Tseng L: Ligand activated hPR modulates the glycodelin promoter activity through the Sp1 sites in human endometrial adenocarcinoma cells. Mol Cell Endocrinol. 2001, 176: 97-102. 10.1016/S0303-7207(01)00450-6.View ArticlePubMedGoogle Scholar
  46. Aghajanova L, Velarde MC, Giudice LC: Altered gene expression profiling in endometrium: evidence for progesterone resistance. Semin Reprod Med. 2010, 28: 51-58. 10.1055/s-0029-1242994.View ArticlePubMedGoogle Scholar
  47. Taylor HS, Bagot C, Kardana A, Olive D, Arici A: HOX gene expression is altered in the endometrium of women with endometriosis. Hum Reprod. 1999, 14: 1328-1331. 10.1093/humrep/14.5.1328.View ArticlePubMedGoogle Scholar
  48. Hirota Y, Tranguch S, Daikoku T, Hasegawa A, Osuga Y, Taketani Y, Dey SK: Deficiency of immunophilin FKBP52 promotes endometriosis. Am J Pathol. 2008, 173: 1747-1757. 10.2353/ajpath.2008.080527.PubMed CentralView ArticlePubMedGoogle Scholar
  49. Aghajanova L, Velarde MC, Giudice LC: The progesterone receptor coactivator Hic-5 is involved in the pathophysiology of endometriosis. Endocrinology. 2009, 150: 3863-3870. 10.1210/en.2009-0008.PubMed CentralView ArticlePubMedGoogle Scholar
  50. Caballero V, Ruiz R, Sainz JA, Cruz M, Lopez-Nevot MA, Galan JJ, Real LM, de Castro F, Lopez-Villaverde V, Ruiz A: Preliminary molecular genetic analysis of the Receptor Interacting Protein 140 (RIP140) in women affected by endometriosis. J Exp Clin Assist Reprod. 2005, 2: 11-10.1186/1743-1050-2-11.PubMed CentralView ArticlePubMedGoogle Scholar
  51. Suzuki A, Horiuchi A, Oka K, Miyamoto T, Kashima H, Shiozawa T: Immunohistochemical detection of steroid receptor cofactors in ovarian endometriosis: involvement of down-regulated SRC-1 expression in the limited growth activity of the endometriotic epithelium. Virchows Arch. 2010, 456: 433-441. 10.1007/s00428-010-0884-x.View ArticlePubMedGoogle Scholar
  52. Kim JJ, Taylor HS, Lu Z, Ladhani O, Hastings JM, Jackson KS, Wu Y, Guo SW, Fazleabas AT: Altered expression of HOXA10 in endometriosis: potential role in decidualization. Mol Hum Reprod. 2007, 13: 323-332. 10.1093/molehr/gam005.View ArticlePubMedGoogle Scholar
  53. Lee B, Du H, Taylor HS: Experimental murine endometriosis induces DNA methylation and altered gene expression in eutopic endometrium. Biol Reprod. 2009, 80: 79-85. 10.1095/biolreprod.108.070391.PubMed CentralView ArticlePubMedGoogle Scholar
  54. Shazand K, Baban S, Prive C, Malette B, Croteau P, Lagace M, Racine JB, Hugo P: FOXO1 and c-jun transcription factors mRNA are modulated in endometriosis. Mol Hum Reprod. 2004, 10: 871-877. 10.1093/molehr/gah119.View ArticlePubMedGoogle Scholar
  55. Wu Y, Halverson G, Basir Z, Strawn E, Yan P, Guo SW: Aberrant methylation at HOXA10 may be responsible for its aberrant expression in the endometrium of patients with endometriosis. Am J Obstet Gynecol. 2005, 193: 371-380. 10.1016/j.ajog.2005.01.034.View ArticlePubMedGoogle Scholar
  56. Yang S, Fang Z, Suzuki T, Sasano H, Zhou J, Gurates B, Tamura M, Ferrer K, Bulun S: Regulation of aromatase P450 expression in endometriotic and endometrial stromal cells by CCAAT/enhancer binding proteins (C/EBPs): decreased C/EBPbeta in endometriosis is associated with overexpression of aromatase. J Clin Endocrinol Metab. 2002, 87: 2336-2345.PubMedGoogle Scholar
  57. Cunha GR, Cooke PS, Kurita T: Role of stromal-epithelial interactions in hormonal responses. Arch Histol Cytol. 2004, 67: 417-434. 10.1679/aohc.67.417.View ArticlePubMedGoogle Scholar
  58. Gellersen B, Fernandes MS, Brosens JJ: Non-genomic progesterone actions in female reproduction. Hum Reprod Update. 2009, 15: 119-138.View ArticlePubMedGoogle Scholar

Copyright

© Tapia-Pizarro et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Advertisement