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Endocrinology, doi:10.1210/en.2003-0043
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Endocrinology Vol. 144, No. 7 2870-2881
Copyright © 2003 by The Endocrine Society

Expression Profiling of Endometrium from Women with Endometriosis Reveals Candidate Genes for Disease-Based Implantation Failure and Infertility

L. C. Kao, A. Germeyer, S. Tulac, S. Lobo, J. P. Yang, R. N. Taylor, K. Osteen, B. A. Lessey and L. C. Giudice

Department of Obstetrics and Gynecology (L.C.K., A.G., S.T., S.L., J.P.Y., L.C.G.), Stanford University, Stanford, California 94305; Department of Obstetrics, Gynecology, and Reproductive Sciences (R.N.T.), University of California, San Francisco, California 94143; Department of Obstetrics & Gynecology (K.O.), Vanderbilt University, Nashville, Tennessee 37232; and Department of Obstetrics & Gynecology (B.A.L.), University of North Carolina, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Linda C. Giudice, M.D., Ph.D., Center for Research on Women’s Health and Reproductive Medicine, Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Stanford University, Stanford, California 94305-5317. E-mail: giudice{at}stanford.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen {alpha}2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin {alpha}2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein {alpha}. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIOSIS IS AN estrogen-dependent, benign gynecologic disorder affecting 10–15% of women of reproductive age (1, 2). It is characterized by endometrial tissue found outside of the uterus (primarily in the pelvic cavity) and is associated with pelvic pain and infertility. A recent meta-analysis of assisted reproductive outcomes revealed that women with endometriosis and infertility who undergo in vitro fertilization and embryo transfer (IVF-ET) have pregnancy rates that are about 50% of women who undergo IVF-ET for tubal factor infertility (3). Abnormalities in the endometrium resulting in failure of embryonic implantation are believed largely to account for the lower pregnancy rates in women with endometriosis. However, because the pathogenesis of endometriosis per se is uncertain, the basis of implantation failure in women with endometriosis has been difficult to define.

The implantation process involves complex interactions between the embryo and the maternal endometrium, the latter of which is receptive to the embryo only during a restricted period, the window of implantation that spans cycle d 20–24 (or LH + 6–10) (reviewed in Ref. 4). In humans, the implantation process begins with attachment of the embryo to the endometrial epithelium, intrusion through the epithelium and then invasion into the decidualizing stromal compartment, eventually resulting in anchoring of the conceptus and establishment of the fetal placenta and blood supply (5). Molecular definition of the window of implantation in human endometrium is beginning to be understood, and several molecular markers of the window and of uterine receptivity to embryonic implantation have been identified (6). These markers derive from animal models of homologous recombination and gene deletion, analysis of particular genes or proteins in human endometrium during the implantation window and in vitro studies with human endometrial cell cultures. Insight into the molecular basis of implantation failure in women with endometriosis over the past 10 yr derives from select molecules observed to be aberrantly expressed in the implantation window (or at other times of the menstrual cycle) in women with this disorder. These include aromatase, endometrial bleeding-associated factor, hepatocyte growth factor, 17-ß-hydroxysteroid dehydrogenase, HoxA-10, HoxA-11, {alpha}vß3 integrin, leukemia inhibitory factor, matrix metalloproteinases, and progesterone receptors (PRs) (reviewed in Ref. 4). Some of these molecules are important to the implantation process, such as {alpha}vß3 integrin which has a proposed role in embryonic attachment to the endometrial epithelium (7), leukemia inhibitory factor, which is important in embryonic attachment and decidualization of endometrial stromal cells (8), and endometrial bleeding-associated factor, which may predispose to endometrial bleeding if overexpressed during the implantation window (9). Some dysregulated molecules are believed to be important in the pathogenesis of endometriosis [e.g. estrogen synthesis (aromatase) and metabolism (17-ß-hydroxysteroid dehydrogenase) (10, 11) or invasion of endometrial cells into the pelvic peritoneum (matrix metalloproteinases) (12)].

Before the genomic era, the traditional one-by-one approach has been used to investigate candidate molecules for uterine receptivity or to investigate endometrial abnormalities in women with or without endometriosis during the implantation window or at other times of the cycle. Recently, discovery-based genome-wide microarray comparisons have been used to broadly investigate various systems from yeast to cancers (13, 14, 15). In pursuit of a global understanding of human embryonic implantation, we (16) and others (17, 18) have recently applied array-based experiments to investigate differentially expressed genes and generate an endometrial signature for the window of implantation in normally cycling women without endometriosis. This strategy resulted in the identification of gene families and signaling pathways that are candidates for uterine receptivity and generation of hypotheses of molecular mechanisms underlying the process of human implantation (16, 17, 18). In the current study, we have applied paralleled expression profiling using high-density oligonucleotide microarrays to endometrium from women with endometriosis, compared with disease-free women, during the window of implantation. Validation of results was achieved for select up- and down-regulated genes using RT-PCR and Northern/dot-blot analyses. The data provide an expression signature of endometrial genes during the window of implantation that provides insight into the pathogenesis of implantation failure in women with endometriosis, the pathogenesis of establishing endometriosis, and a unique opportunity to design diagnostic tests for endometriosis and targeted drug discovery for endometriosis-based implantation failure and treatment of this enigmatic disorder.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue specimens
Tissues.
Endometrial biopsies were obtained from normally cycling women after informed consent, under a protocol approved by the Stanford University Committee on the Use of Human Subjects in Medical Research and the Human Subjects Committees at the University of North Carolina, Vanderbilt University, and the University of California at San Francisco. All specimens were obtained in accordance with the Declaration of Helsinki. A total of 20 biopsy samples were obtained during the window of implantation (midsecretory phase/peak estradiol and progesterone), which were timed to the LH surge (LH + 8 to LH +10, where LH = 0 is the day of the LH surge) from women surgically demonstrated to be without (n = 12) and with (n = 8) mild/moderate endometriosis. Timing to the LH surge assured sampling during the window of implantation. Of the 20 biopsies, 15 were used for microarray studies and 5 were used for Northern or dot-blot analyses and RT-PCR validation (see below) and 2 were used for both. The subjects were 28–39 yr old, had regular menstrual cycles (26–35 d), were documented not to be pregnant, and were taking no medications. Endometrial biopsies were performed with Pipelle catheters under sterile conditions, from the uterine fundus. A portion of each sample was processed for histologic confirmation, and the remainder was immediately frozen in liquid nitrogen for subsequent RNA isolation. Secretory phase histologies were confirmed independently by three observers: L.C.G., B.AL., and an independent pathologist.

Gene expression profiling
RNA preparation/target preparation/array hybridization and scanning.
Of the fifteen window of implantation samples used for microarray analysis, n = 8 were from patients with surgically confirmed pelvic endometriosis (mild/moderate disease) and n = 7 from women surgically confirmed to be without endometriosis. The latter samples served as the basis for our recent study on gene expression in the window of implantation compared with the late proliferative phase in normally cycling women without endometriosis (16). Each endometrial biopsy sample was processed individually for microarray hybridization following the Affymetrix (Affymetrix, Santa Clara, CA) protocol. Briefly, poly (A)+-RNA was initially isolated from the tissue samples using Oligotex Direct mRNA isolation kits (QIAGEN, Valencia, CA), and a T7-(deoxythymidine)24 oligo-primer was subsequently used for double-stranded cDNA synthesis by the Superscript Choice System (Invitrogen, Carlsbad, CA). In vitro transcription was subsequently carried out with ENZO BioArray High Yield RNA T7 Transcript Labeling Kits (ENZO, Farmingdale, NY) to generate biotinylated cRNAs. After chemical fragmentation with 5x fragmentation buffer (200 mM Tris, pH 8.1; 500 mM KOAc; 150 mM MgOAc), biotinylated cRNAs were mixed with controls and were hybridized to Affymetrix Genechip Hu95A oligonucleotide microarrays on an Affymetrix fluidics station at the Stanford University School of Medicine Protein and Nucleic Acid (PAN) Facility. Fluorescent labeling and laser confocal scanning were conducted in the PAN Facility and generated the data for analysis.

Data analysis.
The data were analyzed using GeneChip Analysis Suite version 4.01 (Affymetrix), GeneSpring version 4.0.4 (Silicon Genetics, Redwood City, CA), and Microsoft Corp. (Redmond, WA) Excel/Mac2001 software, as previously described (16). To assess the expression ratios between the two groups, expression profile data were first prepared using GeneChip Microarray Analysis Suite and subsequently exported to GeneSpring for rank-sum normalization and statistical analysis. We have recently demonstrated in preliminary experiments (16) that chip-to-chip variability when RNA from one endometrial tissue sample was subjected to two independent hybridizations, less than 2.7% of the total genes on the array showed more than 3-fold variation, providing a greater than 95% confidence level, consistent with the manufacturer’s published claims (19, 20). With GeneSpring version 4.0.4 software, within each hybridization panel the 50th percentile of all measurements was used as a positive control for normalization, and each measurement for each gene was divided by this control, using the bottom 10th percentile for background subtraction. Between different hybridization outputs/arrays, each gene was further normalized by synthesizing a positive control for that gene, using the median of the gene’s expression values over all samples of an experimental group, and dividing the measurements for that gene by this positive control, as per the manufacturer’s instructions. Mean values were then calculated among individual experimental groups for each gene probeset, and between-group fold-change ratios were derived [i.e. with endometriosis (n = 8): without disease (n = 7) ratios]. A difference of 2-fold was applied to select up-regulated and down-regulated genes. Nonparametric Mann-Whitney U test was conducted to calculate the P values, applying P < 0.05 to assign statistical significance between the two groups.

Validation of gene expression data
RT-PCR.
Genes of different expression fold changes were randomly selected for validation by RT-PCR and/or Northern or dot-blot analyses. Total RNA from whole endometrial tissue was isolated using Trizol (Invitrogen) protocol, digested with deoxyribonuclease (QIAGEN) and then purified by RNeasy Spin Columns (QIAGEN). Four window of implantation endometrial biopsy samples were used for these experiments: two from female infertility patients with surgically proven endometriosis and two from normal fertile volunteers. Reverse transcription was first performed with Omniscript kit (QIAGEN) for 1 h at 37 C, followed by a 50-µl reaction volume PCR with 40 pmol of specific oligo-primer pairs (see below) and Taq polymerase (QIAGEN), using the Eppendorf Mastercycler Gradient (Brinkmann Instruments, Westbury, NY). The amplification consisted of a hot start at 94 C for 15 min, followed by 25–35 cycles of: denaturation at 94 C, annealing at optimized temperature, and extension at 72 C, each for 45 sec. Specific oligo-primer pairs were derived from public databases and synthesized by the PAN Facility, Stanford University School of Medicine (Table 1Go). All PCR products used for Northern and dot-blot analyses were purified with QIAquick Gel Extraction Kit (QIAGEN) and verified by the Stanford PAN Sequencing Facility.


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  		Table 1. PCR primer pairs sequences and anticipated product length

 
Northern and dot-blot analyses.
Five window of implantation endometrial biopsy samples were used for these experiments, two from patients with endometriosis previously used in RT-PCR validation, and three from normal fertile volunteers, not used before. Total RNA (10–20 µg) was denatured and electrophoresed on 1% formaldehyde agarose gels and transferred for Northern analyses, or directly blotted for dot-blot analysis through the Convertible Filtration Manifold System (Invitrogen), onto Nylon membranes. Specific radioactive probes were generated with Ready-to-Go random primer kit (Pharmacia Biotech, Peapack, NJ), using PCR generated cDNAs, ranging 296–609 bp, and 32{alpha}P-deoxy-CTP (NEN Life Science Products, Boston, MA), followed by MicroSpin S-200 HR Columns (Pharmacia Biotech) cleanup. Membranes were prehybridized at 68 C for 60 min in ExpressHyb buffer (CLONTECH Laboratories, Inc., Palo Alto, CA) containing salmon sperm DNA (Invitrogen), and hybridization carried out for another hour at 68 C using buffer containing 1–2 x 106 cpm/ml of labeled probe. After washing according to the manufacturer’s instructions, membranes were exposed to Kodak MS x-ray films (Eastman Kodak, Rochester, NY), scanned by a GS-710 Imaging Densitometer from Bio-Rad Laboratories, Inc. (Hercules, CA), and analyzed by its accompanied software Quantity One, version 4.0.2. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA intensities were used for normalization before comparison. Mean values of relative expression intensities from different blots were used for final data presentation. Stripping and reprobing were performed using the same membranes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Data analysis
The data were analyzed as detailed in Materials and Methods, and as generally adopted for oligonucleotide microarray profile analysis, a minimal change of 2-fold was applied to select up-regulated and down-regulated genes. Nonparametric statistical testing was subsequently applied with a P value of 0.05 used to designate significance between groups. Applying this strategy, we identified in the window of implantation in endometrium from women with vs. without endometriosis, 91 genes that were significantly up-regulated, of which 28 were expressed sequence tags, and 115 genes that were significantly down-regulated, of which 29 were expressed sequence tags. Tables 2Go and 3GoGo show, in descending order, respectively, the fold-increase and fold-decrease, the P values (P < 0.05), and the GenBank accession numbers for the 63 specifically up-regulated genes and the 86 down-regulated genes in eutopic endometrium of women with endometriosis during the window of implantation, compared with normal fertile women, according to clustering assignments (see below).


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  		Table 2. Genes up-regulated in women with endometriosis

 

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  		Table 3. Genes down-regulated in women with endometriosis

 

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  		Table 3A. Continued

 
Clustering
Stringent data filtering permits identification of significantly and consistently changed genes. Clustering further allows grouping of genes of biological relevance in eutopic endometrium during the window of implantation of women with endometriosis. We performed unsupervised cluster analysis, based on NCBI (National Center for Biotechnology Information)/Entrez/OMIM (Online Mendelian Inheritance in Man) database searches, which segregated genes of interest into various categories (Tables 2Go and 3GoGo). The most highly up-regulated genes, reaching the upper limit of the program algorithm of 100-fold, include those involved in: apoptosis [cysteine protease (ICErel-III)], protein or RNA processing (putative mono-ADP-ribosyltransferase) (RNA-binding protein CUG-binding protein), transporter protein [bile salt export pump (BSEP)], zinc metalloenzyme (carbonic anhydrase I), DNA repair [postmeiotic segregation increased 7 mRNA (yeast mismatch repair gene postmeiotic segregation increased 1 homolog), DNA primase], immune function ({alpha}-interferon, TGFß-3), secretory protein (secretogranin II), signal transduction (serine/threonine kinase, ether-a-go-go-like potassium channel receptor tyrosine kinase ligand, MAPK kinase 1), cell surface protein (mucin, major histocompatibility complex-related antigen) and transcription factors (Brain 4, G13). Other genes of unspecified biological pathways such as retinoic acid-inducible endogenous retroviral DNA, heat shock protein 40 homolog, kallikrein, testis- specific protein and alkaline phosphatase also were up- regulated to the algorithm maximum of 100-fold. Other up-regulated genes include members of cytokine receptor families, secretory proteins, signal transduction, cell surface receptors, membrane-associated proteins and extracellular matrix/cell-cell contact, potassium channel, cytoskeleton/cell structure, neurokinin receptor, and others.

The most highly down-regulated genes include those involved in: calcium-binding (S100E calcium binding protein), regulator of vesicular transport (Rab9 expressed pseudogene), RNA polymerase (p300/CREB-binding protein-associated factor-associated factor 65{alpha}), serine protease/inhibitor (hepatocyte growth factor activator-like protein, cytoplasmic antiproteinase 2), signal transduction (GTPase-activating protein, ras interactor, protein kinase c-Jun NH2-terminal kinase 1, protein tyrosine phosphatase), transcription factor (double homeodomain protein 1), immune function [activating natural killer (NK)-receptor (NK-p46), monocyte chemotactic protein-2], cell surface proteins (T cell receptor, leptin receptor splice variant, integrin {alpha}-2 subunit), all reaching 100-fold difference, as did endonuclease G-like protein-2 and DNA glycosylase. Down-regulated genes also included signal transduction, immune function, and cytokine/receptor genes, cell surface glycoproteins/receptors, retinol binding protein, ion transporters, secretory proteins including inhibin ß-B, B61, Dickkopf-1, and glycodelin, N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST/GlcNAC), and others.

Validation of gene expression
Expression of select up-regulated and down-regulated genes was validated by RT-PCR and/or Northern or dot-blot analysis, using RNA isolated from endometrial biopsy samples in the window of implantation, from women with and without endometriosis. The results are shown in Figs. 1–3GoGoGo. Although real-time quantitative PCR was not performed, the RT-PCR data in Figs. 1Go and 2Go demonstrate clearly in the women with endometriosis, compared with normal fertile women, up-regulation of semaphorin E, collagen {alpha}2 type I, ankyrin G, and down-regulation of integrin {alpha}2, platelet- derived endothelial cell growth factor, GlcNAc6ST/GlcNAC, B61/ephrin, and glycodelin. These data are consistent with the observations from the microarray data (Tables 2Go and 3GoGo).



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  		Figure 1. Equal cycle RT-PCR of selected genes up-regulated in eutopic human endometrium during the window of implantation, from women without (N) and with (D) endometriosis. Specific primer pairs used are shown in Table 1Go. Appropriate size bands are depicted for the housekeeping gene GAPDH (lane 1), as well as for the up-regulated genes: semaphorin E (lane 2), collagen {alpha}-2 type I (lane 3), and ankyrin G (lane 4). Two samples from women without and two from women with endometriosis were used for this study; representative results are shown.

 


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  		Figure 2. Equal cycle RT-PCR of selected genes down-regulated in eutopic human endometrium during the window of implantation, from women without (N) and with (D) endometriosis. Specific primer pairs used are shown in Table 1Go. Appropriate size bands are depicted for: integrin {alpha}2 subunit (lanes 1), platelet-derived endothelial cell growth factor (PD-EGF) (lanes 2), GlcNAC (lanes 3), B61/Ephrin (lanes 4), and glycodelin (lanes 5). Two samples from women without and two from women with endometriosis were used for this study; representative results are shown.

 


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  		Figure 3. Northern blot analyses demonstrating: (A) up-regulation of collagen {alpha}-2 type I, (B) down-regulation of GlcNAC, glycodelin, integrin 2 {alpha}-subunit and B61, in eutopic human endometrium during the window of implantation, from women without (a) or with (b) endometriosis. C, Dot-blot analysis for up-regulation of BSEP. Three samples from women without and two from women with endometriosis were used and representative results are shown. GAPDH hybridization densities of corresponding lanes are also shown for comparison (see also Table 4Go).

 
Northern or dot-blot analyses were also conducted to validate changes in gene expression in the samples from women with endometriosis vs. normal controls. Representative Northern blots and dot-blots are demonstrated in Fig. 3Go. Densitometric analyses of band intensities and dot intensities were conducted, and GAPDH was used as a control to determine relative mRNA expression in each sample. Normalized relative expressions of select mRNAs in endometrium during the implantation window in women with vs. without endometriosis were then calculated, and the data are shown in Table 4Go. Northern and dot-blot analyses parallel results obtained from the microarray expression profiling analysis, although exact fold-change differences are not the same as in the microarray analysis (Tables 2Go and 3GoGo). As we previously speculated (16), one would not expect the fold changes to be the same among various methodologies due to several factors such as tissue heterogeneity, subject-to-subject biologic variation, and the lower abundance of specific mRNAs relative to the highly abundant GAPDH mRNA.


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  		Table 4. Relative abundance of mRNAs in endometrium from women with endometriosis (disease) vs. those without (normal) determined by Northern and dot-blot analyses

 
Target identification
Comparisons were made between differentially expressed genes in the implantation window in endometrium of women with vs. without endometriosis and genes previously identified to be differentially up- or down-regulated in normal human endometrium in the implantation window compared with the late proliferative phase (16). Twelve target genes in three distinct patterns are identified. In group 1, eight genes normally up-regulated in the window of implantation were significantly down-regulated in endometrium of women with endometriosis. In group 2, genes normally down-regulated during the window of implantation, three were up-regulated in endometriosis. In group 3, one gene already down-regulated in the normal window of implantation was further down-regulated with endometriosis. Group 1 consists of IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, GlcNAc6ST, G0S2 protein and purine nucleoside phosphorylase. Group 2 consists of semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA, and Sam68-like phosphotyrosine protein {alpha} (SALP), and group 3 is represented by a single gene, neuronal pentraxin II.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is widely accepted that endometriosis originates from retrograde menstruation of endometrial tissue through patent fallopian tubes into the peritoneal cavity, leading to its ectopic location. Establishment of the disease has been attributed to endometrial fragments attaching to the epithelium of the peritoneum, invading into it, establishing a blood supply, and triggering a suboptimal immune response that does not adequately clear the implants, resulting in continued survival and growth. These phenomena occur against the background of unique genetic, environmental, and/or immunologic factors. Endometrium of women with endometriosis is believed to be abnormal, predisposing to the establishment of ectopic disease. This view is compelling, especially because most women have some degree of retrograde menstruation, but only 15% have endometriosis. This observation is consistent with the hypothesis that endometrium of women with endometriosis also may be abnormal with regard to embryonic receptivity, especially because there is a high correlation of implantation failure in women with endometriosis (21). Herein, we have identified genes that likely have relevance to implantation failure and pathogenesis of the disease, including genes for apoptosis, ion transporters, immune functions, secretory proteins, signal transduction, membrane proteins, transcription factors, and others (Tables 2Go and 3GoGo). Furthermore, we found three unique groups of target genes. One group was up-regulated during the implantation window in women without endometriosis but significantly decreased with endometriosis. Group 2 genes were down-regulated during the normal window of implantation but significantly increased in endometrium in women with endometriosis, and group 3 was comprised of one gene that was down-regulated during the normal window of implantation and further decreased in endometriosis patients. Many genes found herein have not been documented previously to be dysregulated in the endometrium of women with endometriosis and evoke plausible new hypotheses for either the infertility phenotype or the pathogenesis of the disease. Select genes and gene families (see Table 5Go) are discussed below in the context of implantation failure and pathogenesis of endometriosis.


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  		Table 5. Proposed functional categories of select genes in the window of implantation in women with vs. without endometriosis

 
Candidate genes for implantation failure and infertility in endometriosis
When embryos arrive in the endometrial cavity after transport through the fallopian tube, a mechanism of tethering and rolling is thought to mediate this initial process of recognition before implantation. Surface glycosylation-dependent cell adhesion molecules such as mucins have been suggested as candidate mediators of rolling, by analogy with selectins in lymphocyte attachment to the endothelium (22, 23). Endometrial epithelial mucin-1 expression was previously found to be abundant during the secretory phase of the menstrual cycle (22). Most recently, the presence of the selectin adhesion system at the human maternal-fetal interface has been documented during the window of implantation (24), providing further support for this mechanism. GlcNAc6ST, up-regulated during the window of implantation in women without endometriosis (16), is one of the three major enzymes participating in the glycosylation process of these surface molecules. One family member of the GlcNAc6ST had also been previously demonstrated with a specific role in generating L-selectin ligands (25). Down- regulation of this critical enzyme in endometrium of women with endometriosis, found herein, may reduce proper glycosylation of these surface proteins, hindering the proper recognition process, and leading to implantation failure.

Olfactomedin was originally identified as a glycosylated extracellular matrix protein surrounding chemosensory dendrites of olfactory neurons (26). Olfactomedin-related proteins are secretory glycoproteins subsequently discovered in many animals from Xenopus to the human and are proposed to possess varied functions promoting neurogenesis and pattern signaling (27). Overexpression of this protein may interfere with embryo tethering and rolling and contribute to failure of implantation. Alternatively, olfactomedin and its related proteins may exert yet-undiscovered signals for endometrial survival after retrograde shedding and contribute to the establishment of peritoneal endometriotic lesions.

Proline-rich protein is also known as complement 4 binding protein (C4BP), which is a regulator of the complement system. Multiple components of the complement system have been documented in the uterine fluids in various animal models (28), as well as in humans (29), and reported previously to be dysregulated in endometriosis (30). The presence of the complement system has been postulated to constitute a local antimicrobial defense system in the uterine cavity, particularly during the period of embryonic implantation. Decreased expression of the key regulator of this local defense system, found herein, could allow decreased clearing of bacterial flora, potentially leading to an inhospitable environment for an implanting conceptus in women with endometriosis. Recently, C4BP has been demonstrated to interact with low-density lipoprotein receptor-related protein, LRP, via its heparin-binding sites (31). LRP, traditionally considered as a receptor involved in hepatic clearance of numerous proteins via endocytosis, has recently been recognized as an important component of cell-cell signaling via the Wnt pathway (32). Members of the Wnt family are regulated during the menstrual cycle in normal endometrium (16, 17, 33) and likely play a significant, but as yet undefined, role during the implantation process. Dysregulation of C4BP may interfere with endometrial decidualization and subsequently implantation through disruption of this paracrine/juxtacrine loop.

We have previously found that IL-15 is up-regulated in endometrium during the implantation window, compared with the late proliferative phase. Carson et al. (17) similarly found up-regulation of this cytokine in the implantation window, compared with prereceptive endometrium. Others have demonstrated up-regulation of IL-15 (and its receptor) in secretory compared with proliferative phase endometrium (34, 35). IL-15 is a potent stimulator of endometrial NK cell proliferation. Uterine NK cells play a role in endometrial stromal decidualization and endometrial breakdown at menstruation as they produce cytokines, including granulocyte macrophage-colony stimulating factor, IL-10, IL-13, and interferon-{gamma} (36). IL-15 is essential for type 2 cytokine production by these cells. In the current study, our finding of underexpression of this mediator during the window of implantation may reflect interruption of its physiological role in decidualization and signaling NK cell proliferation, which could contribute to the implantation failure phenotype observed in patients with endometriosis.

Dickkopf-1 inhibits Wnt signaling by binding to LRP5/6, a coreceptor for Wnt ligands (37). It had previously been demonstrated by our microarray analysis to be up-regulated during the window of implantation (16). Wnt signaling, via paracrine/ juxtacrine mechanisms in the endometrium, has been postulated to play a role during decidualization (16). Down-regulation of this coregulator of the Wnt signaling system, possibly involving C4BP, may contribute further to the implantation failure phenotype in women with endometriosis.

Purine nucleoside phosphorylase is an enzyme involved in the purine salvage pathway and is expressed in maternal decidual cells in animal models (38, 39). The high level of expression of this enzyme is postulated to reflect extensive programmed cell death during the implantation process. Additionally, pharmacological inhibition of purine nucleoside phosphorylase had been found to be embryolethal or teratogenic (38). Endometrial underexpression of this enzyme in women with endometriosis, as demonstrated in the current study, may potentially impair local purine salvage, similar to a pharmacological block, leading to implantation failure.

The group 3 target gene, neuronal pentraxin II, was down-regulated during the normal window of implantation and further decreased in endometriosis. Pentraxins are a family of proteins capable of forming pentameric complexes and bind a variety of ligands. These proteins have been suggested to mediate nonspecific uptake of bacteria and cell debris that may be associated with inflammation and immune responses. While pentraxin 3 has been shown to bind apoptotic cell remnants, possibly contributing to prevent autoimmune reactions (40), pentraxin II is observed in mice with highest tissue expression in the testis (41). A significantly reduced level of this protein may impede removal of apoptotic cell debris in the endometrium and contribute to an inhospitable endometrial environment, leading to implantation failure.

Previous clinical observations have documented resistance of endometriosis to treatment with progestins. Comparison studies of eutopic and ectopic endometrium revealed differences in progestin-induced changes (42), leading to subsequent findings of different expression levels of PR variants in the endometrium of women with endometriosis (43, 44). Our finding of dysregulated expression of glycodelin, which is under the regulation of progesterone (45), in women with endometriosis is consistent with this progesterone resistance in the setting of endometriosis. Glycodelin, also known as progesterone-dependent endometrial protein, placental protein 14 or placental {alpha}2- macroglobulin (46, 47), herein has been demonstrated to be underexpressed in the endometrium of women with endometriosis. Although glycodelin has been suggested to interfere with fertilization, several studies support its immunosuppressive properties, including inhibition of NK cell activity (48) within the endometrium during the window of implantation. Underexpression of this protein in endometrium of women with endometriosis may contribute to the low implantation rates and infertility observed.

Another example of a highly down-regulated gene in endometriosis is S100E. It belongs to a group of tightly clustered genes encoding calcium-binding proteins with variable tissue distributions. Targeted disruption of a single member of this gene family resulted in mouse embryonic resorption, which was not prevented by other family members (49). Though not yet investigated in human endometrium, our finding of 100-fold down-regulation of another member of this family of genes, S100E, supports a potentially important role for regulation of calcium homeostasis in endometrium and provides another hypothesis for the implantation failure phenotype in women with endometriosis.

One of the most highly up-regulated genes in endometrium of women with endometriosis, compared with disease-free women, was human BSEP. BSEP is the major bile salt export pump in mammalian liver, and BSEP null mice have intrahepatic cholestasis (50). Several years ago, cholic acid was isolated by HPLC from human uterine fluid and was proposed to be the embryo-toxic substance found in the uterine fluid during the nonreceptive phase (51). Bile acids are known to induce cellular apoptosis (52) and increase fetal resorption in animals (53). It is possible that up-regulation of BSEP expression in endometrium of women with endometriosis contributes to intraluminal bile acid accumulation, leading to embryo-toxicity and decreased survival rates of embryos in women with endometriosis. This may be particularly relevant to lower implantation rates reported among women with endometriosis treated with IVF-ET (21).

Candidate genes for the pathogenesis of endometriosis
Integrins are likely important in the process of endometrial tissue attachment to the peritoneum. During the process of data analysis, the gene for integrin {alpha}-2 subunit was found to be both up- and down-regulated (Tables 2Go and 3GoGo). Interrogation of this transcript was performed with two probesets on the microarray, one targeting the distal 3' end of the transcript which demonstrated significant up-regulation (Table 2Go), and the other targeting the midregion of the transcript, showing significant down-regulation (Table 3GoGo). After careful inspection of the probeset target sequences on the microarray, we generated different specific PCR primer pairs within these target regions to reconcile, verify and validate the findings. With the support of equal cycle RT-PCR and Northern hybridization, we confirmed the down-regulation of this transcript within the disease state of endometriosis. The aberrant finding of up-regulation might have resulted from initial 3' degeneration of transcripts, corresponding to the down stream location of these target sequences.

Due to the nature of hormonal dependence of this disease, local production of estrogen by endometriotic tissues has been investigated, with a major focus on the expression of cytochrome P450 aromatase, the key enzyme in converting C19 steroids to estrogens. While increased expression of CYP19 aromatase in ectopically located endometriotic lesions, especially ovarian endometriomas, has been demonstrated, its expression in eutopic endometrium has been most consistently shown in women with severe pelvic endometriosis and not in mild disease (10). CYP19 aromatase expression has also been detected in the eutopic endometrium of women with other uterine diseases such as leiomyoma (86%) and adenomyosis (86%) (54). Within the oligonucleotide microarray used for this study, two sequences were designed to interrogate the expression of the CYP19 gene, one targeting exon 10, the other exon 9. Neither probeset detected differential expression in women with endometriosis. Upon careful examination of these sequences, the exon 10 probesets were found to be down stream from the polyadenylation sites, and this absence of differential expression likely reflects the absence of signals from both experimental groups, because the designed probeset is beyond the initial mRNA/oligo-deoxythymidine priming site during target generation (see Materials and Methods, Gene Expression Profiling, Target Preparation). We hypothesize that the inability to detect differential expression by exon 9 targeted probesets most likely reflects differences in disease stages, as mostly mild disease was present in subjects participating in this study.

B61, a protein originally discovered as a secreted ligand for the epithelial cell receptor protein tyrosine kinases, is also known as ephrin A1 (55). Ephrins are expressed in differentiated epithelial elements of skin, kidney, and intestine. In developing neural and cardiovascular systems, ephrin activates a receptor tyrosine kinase and subsequently modulates axonal pathfinding, neural cell migration (56), and development of the cardiovascular system (57). Besides being implicated in embryonic vascular development, ephrins have been shown to regulate tumor neovascularization in in vivo models of angiogenesis (58). Ephrin A1 also inhibits cell proliferation in several epithelial cell lines, exerting antimitogenic functions (59). In a study investigating an intestinal epithelial cell line, ephrin enhanced epithelial barrier integrity (60). These proposed functions for B61 protein resonate important physiological properties of the endometrial epithelium during the window of implantation. Dysregulation of this protein may promote tissue dissemination and potentiate neovascularization, both characteristic of the pathogenesis for endometriosis.

G0S2 protein is a small basic nuclear phosphoprotein that is involved in the G0/G1 switch of the cell cycle (61). The protein contains potential sites for phosphorylation by protein kinase C and the cell cycle control kinase cdc2. Dysregulation of this protein may reflect the propensity of eutopic endometrium of women with endometriosis to survive or remain mitotically competent after retrograde menstruation into the peritoneal cavity, leading to the establishment of the disease.

Tissue kallikrein belongs to a family of serine proteases involved in the generation of bioactive peptide kinins in many organs. Its expression had previously been documented in normal human endometrium and is believed to be regulated by steroid hormones (62). Members of this protein family possess diverse biological functions including tumor suppressors, modulators of cytokines, and inhibitors of angiogenesis. In addition, kallekrein enzymes may participate in proteolysis of extracellular matrix, important in establishing the disease in the peritoneal cavity. Prominent kallikreins, normal epithelial cell-specific 1 gene and prostate-specific antigen, have been advocated as disease markers (63, 64, 65). Our finding of 100-fold up-regulation of kallikrein in women with endometriosis may represent a unique pathophysiological link and a new marker for this disease.

Semaphorin E, or semaphorin 3C, is a secretory protein that has been postulated to be a signaling molecule for axonal guidance in the developing nervous system (66). Animals deficient in semaphorin 3C exhibit abnormal cardiac outflow tract development, implicating the involvement of this signaling molecule in neural crest cell migration (67). Along with other signaling molecules such as ephrin A1 (see above), semaphorins are believed to exert coordinated attractive or repulsive actions in the developing nervous system. Semaphorin receptors, neuropilins, have also been demonstrated to bind vascular endothelial growth factor isoforms (68), implicating a potential signaling role for semaphorin in angiogenesis. Down-regulation of this molecule in human endometrium during the window of implantation may reflect the removal of directional signaling and arresting the state of endometrial glandular branching, and/or maximal vascular development and arrest of further vasculogenesis. Overexpression of this molecule may contribute to the pathogenesis of endometriosis where neovascularization is evident in peritoneal lesions.

Sam68 is a nuclear RNA-binding protein and a substrate for Src tyrosine kinase in mitotic cells (69). Cells undergoing mitosis show a dramatic reduction in the level of Sam68, implicating its function in tumorigenesis. When the expression of Sam68 protein was genetically manipulated to less than 25% of the wild-type, murine fibroblasts demonstrated anchorage-independent growth, defective contact inhibition, and the ability to form metastatic tumors in nude mice (70). In chicken embryo fibroblasts, ectopic expression of SALP (Sam 68-like phosphotyrosine protein {alpha}), down-regulates the expression of Sam68 and paradoxically arrests the growth of cells, suggesting that SALP functions as a negative regulator of cell growth (71). Higher expression of this protein in endometrium during the window of implantation can potentially maintain the tissue in a resting state, contributing to the survival of endometrial tissue, rendering a metastatic phenotype and eventually leading to the establishment of peritoneal lesions.

Model of endometrial abnormalities in gene expression leading to endometriosis and implantation failure associated with this disorder
The data presented herein offer new perspectives into molecular processes potentially involved in the pathogenesis of endometriosis and molecular mechanisms underlying implantation failure in women with endometriosis (Table 5Go). The dysregulated expression of candidate genes such as G0S2 and SALP, may alter mitotic activity, as suggested in deficits of tumor suppressor gene products (72). Other genes, e.g. B61 and semaphorin E, may participate in the neovascularization and survival of endometrium, leading to the establishment of endometriosis. These products may also affect or be affected by the peritoneum which may have aberrant gene expression and responses in women with vs. without endometriosis. Dysregulation of other candidate genes such as BSEP, C4BP, Dickkopf-1, glycodelin, IL-15, pentraxin II, and purine nucleoside phosphorylase may promote an inhospitable endometrial milieu for embryonic implantation, due to embryo toxicity, immune dysfunction, inflammatory or apoptotic responses. Concurrently, dysregulated GlcNAc6ST and olfactomedin-related protein may hinder the tethering and attachment mechanisms of human embryo implantation, resulting in implantation failure. The candidate genes identified herein may be used as a biochemical screen for endometriosis and for future investigation into molecular mechanisms involved in human implantation and/or the pathogenesis of endometriosis. Furthermore, they may serve for targeted drug discovery to treat endometrial dysfunction relevant to the pathogenesis of endometriosis and implantation-based failure in women with this disorder.


   Acknowledgments
 
We thank the Endometriosis Association for its assistance in recruiting subjects for this study.


   Footnotes
 
This work was supported by the NIH Specialized Cooperative Centers Program in Reproductive Research (NICHD HD-31398) and the NIH Office of Women’s Health Research (to L.C.G., B.A.L., R.N.T.), the NIH Women’s Reproductive Health Research Career Development Program (L.C.K.), and the German Research Foundation [Deutsche Forschungsgemeinschaft GE 1173/1-1 (AG)].

Abbreviations: BSEP, Bile salt export pump; C4BP, complement 4 binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GlcNAC/GlcNAc6ST, N-acetylglucosamine-6-O-sulfotransferase; IVF-ET, in vitro fertilization and embryo transfer; LRP, lipoprotein receptor-related protein; NK, natural killer; PR, progesterone receptor; SALP, Sam68-like phosphotyrosine protein {alpha}.

Received January 9, 2003.

Accepted for publication March 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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