Molecular Human Reproduction, Vol. 8, No. 9, 871-879,
September 2002
© 2002 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening
1 Department of Biological Sciences, University of Delaware, Newark, DE 19716, 2 UNC Neuroscience Center and 3 Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, NC 27599–0001, USA
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Abstract |
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High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium. Of
12 000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages. Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results. In addition, novel genes were identified. Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes. Several genes were studied further by Northern blot analyses. These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased 4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition. Immunostaining also revealed strong specific staining for claudin-4/CPE, EP1 and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage. Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans. Some of these gene products, e.g. OPN, may play direct roles in embryo–uterine interactions during the implantation process. endometrium/gene expression/implantation/microarray/uterine receptivity
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Introduction |
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Embryo implantation is critically dependent on the arrival of an attachment-competent blastocyst in co-ordination with generation of a receptive uterine state. The acquisition of attachment competence by the embryo follows hatching from the zona pellucida and is accompanied by expression of cell adhesion-promoting molecules on the external trophectodermal surface (Carson et al., 2000
). Uterine receptivity is regulated by the actions of ovarian steroid hormones produced by the corpus luteum (Psychoyos, 1986). In humans, the period of receptivity is believed to occur 7–9 days post-ovulation during the mid-secretory or mid-luteal phase (Lessey and Castelbaum, 1998; Wilcox et al., 1999). In response to ovarian steroids, characteristic morphological changes occur in the endometrium, and these are responses routinely used to stage the human endometrium (Noyes et al., 1950). Such staging is particularly important as inappropriate or delayed development of the endometrium may cause miscarriage or infertility (Wilcox et al., 1999). Inappropriate endometrial development during IVF/embryo transfer protocols may account for the relatively low individual pregnancy success worldwide (Report ASRM/SARTR, 2000). These observations suggest that additional indicators are needed to detect underlying endometrial defects that may make individuals poor candidates for such procedures.
The best markers of a true receptive state would be those molecules directly involved in the initial stages of the implantation process; however, this necessitates functional testing of these marker molecules, an impractical approach in humans. Such testing can be performed in animal models and several markers of the receptive state have been proposed based on correlative studies in humans as well as a combination of correlative and functional studies in animals (Carson et al., 2000; for review). In most cases, identification of these markers was the result of careful examination of the expression of particular proteins or mRNAs based on preconceived ideas or serendipitous observations. The recent advent of high throughput microarray screening for the expression of human genes has allowed an unbiased approach toward identifying changes in gene expression that accompany transition of the human endometrium from a prereceptive (early luteal) to a receptive (mid-luteal) state. Using an existing microarray display comprising of 12 000 genes (25–33% of the human genome) (Clavrie, 2001) we identified a subset of genes that are altered during this transition. A few of these genes correspond to previously suggested markers, while others represent novel potential markers of human receptivity. These studies provide a basis for performing more detailed studies on the expression and function of these novel markers in both human and animal implantation models.
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Materials and methods |
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Human subjects and tissue staging
Endometrial samples were obtained from a larger pool of fertile volunteers. Subjects monitored their urinary LH excretion daily from cycle day 10 using a commercial ovulation predictor kit (Ovuquik One-Step; Quidel, San Diego, CA, USA) and were instructed to contact the study co-ordinator on the day that the mid-cycle LH surge was first detected (luteal day 0). At time of enrolment, subjects were randomized to undergo endometrial sampling on a specific day defined by a randomization schedule (luteal day 1–14). All returned on the appointed day for endometrial biopsy that was performed using an endometrial pipelle. From a pool of tissue specimens we selected three from the early secretory phase (LH day 2–4) and three from the mid-secretory phase (LH day 7–9). Each subject was sampled in a single menstrual cycle, and all selected samples had histological changes consistent with their LH day. Blood samples for measurement of serum progesterone were obtained on the day of endometrial sampling, and in each case ovulation was confirmed on the basis of an elevated serum progesterone. Endometrial tissues were snap-frozen in liquid nitrogen immediately after collection and a portion of each specimen was fixed in 10% (w/v) buffered formalin before paraffin-embedding for histological examination and dating. The use of human subjects and the procedures were approved by the Institutional Review Boards of both the University of North Carolina and the University of Delaware.
Northern blot analysis
Total RNA was extracted from frozen human endometrial tissue samples using Trizol Reagent according to the manufacturer’s instructions (Life Technologies, Gaithersburg, MD, USA). Quantitation and estimation of purity were performed by measuring absorbance of each RNA sample at UV wavelengths of 260 and 280 nm, and integrity was determined by visual inspection of RNA fractionated by agarose gel electrophoresis. Northern blot analysis was performed using the NorthernMax-Gly kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Briefly, 9 µg of glyoxylated total RNA from each sample were separated on 1% (w/v) agarose gels and downward-transferred to BrightStar-Plus positively-charged nylon membrane (Ambion). After transfer, RNA was crosslinked to the membrane by exposure to ultraviolet light. Prehybridization and hybridization were carried out at 42°C in UltraHyb buffer (Ambion). The cDNA probe used for detection of claudin-4 mRNA was a 470 bp RT–PCR product, confirmed by sequencing, made from late luteal phase endometrial RNA. Primers were designed to amplify a segment of claudin-4 (GenBank accession no.: NM_001305.1). Respective forward and reverse primers used for PCR amplification of claudin-4 cDNA were as follows: 5'-CCGCACAGACAAGCCTTACT-3' and 5'-TACCCGGAACAGAGGAGATG-3'. Primers were also designed to amplify a segment of human OPN cDNA (GenBank accession no.: NM_000582) and found to be unique by a BLAST search. Respective forward and reverse primers were 5'-TTGCTTTTGCCTCCTAGGCA-3' and 5'-GTGAAAATTCGGTTGCTGG-3'. Forward and reverse primers for BIGH3 cDNA (GenBank accession no.: M77349) were 5'-TCTCCCGTGACTTTCGTCTT-3' and 5'-CCAGCAGCAATCTTTTAGCC-3' respectively. All primers were designed to amplify unique sequences in each case as determined by a BLAST search. Oligonucleotides were purchased from Sigma Genosys (St Louis, MO, USA). A cDNA probe for human 18S ribosomal RNA (American Type Culture Collection, Manassas, VA, USA) was used as a load control. Probes were non-isotopically labelled using the BrightStar Psoralen-Biotin Nonisotopic Labelling Kit (Ambion). Hybridization signals were detected using the BrightStar BioDetect Kit (Ambion). Blots were exposed to X-ray film, and bands were quantified using the NIH/Scion Image Beta 3b program (available online at http://www.scioncorp.com). Blots were stripped between probings with boiling 0.1% (w/v) sodium dodecyl sulphate in water, then stored at 4°C. Northern blots were performed twice on two separate series of samples with similar results.
Microarray screening
Total RNA was extracted from the frozen human endometrial tissue samples using Trizol reagent (Life Technologies). RNA was then purified by the Rneasy kit (Qiagen, Valencia, CA, USA) and used to probe the Human Genome U95A Array (Affymetrix, Sunnyvale, CA, USA). For microarray screening, 7 µg of total RNA was used to synthesize cDNA. A custom cDNA kit from Life Technologies was used with a T7-(dT)24 primer for this reaction. Biotinylated cRNA was then generated from the cDNA reaction using the BioArray High Yield RNA Transcript Kit. The cRNA was fragmented in fragmentation buffer (5x stock: 200 mmol/l Tris-acetate, pH 8.1, 500 mmol/l KOAc, 150 mmol/l MgOAc) at 94°C for 35 min before chip hybridization. A total of 15 µg of fragmented cRNA was then added to a hybridization cocktail [0.05 µg/µl fragmented cRNA, 50 pmol/l control oligonucleotide B2, BioB, BioC, BioD, and cre hybridization controls, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated bovine serum albumin, 100 mmol/l MES, 1 mol/l Na+, 20 mmol/l EDTA, 0.01% (v/v) Tween 20]. Arrays were hybridized for 16 h in the GeneChip Fluidics Station 400 and then washed and scanned with the Hewlett Packard GeneArray Scanner. Affymetrix GeneChip Microarray Suite 4.0 software was used for washing, scanning and basic analysis. Detailed information on the gene array system is available at www.affymetrix.com. The samples were analysed in duplicate and sample quality was assessed by examination of 3' to 5' intensity ratios of certain genes. GeneSpring software (version 4.0.2; Silicon Genetics) was used to organize and statistically analyse the results.
Immunostaining
Frozen mid-luteal human endometrial sections (8 µm) were fixed for 10 min at room temperature in methanol and rehydrated in phosphate-buffered saline (PBS) for 10 min with one change of buffer. Sections were then incubated at 37°C with EP1 polyclonal receptor antibody (Cayman, Ann. Arbor, MI, USA) diluted to 20 ng/µl with PBS or leukotriene B4 polyclonal receptor antibody (Cayman) diluted to 4 ng/µl in PBS for 1 h. Non-immune rabbit IgG (Zymed) was used as a negative control for each antibody. Sections were rinsed three times for 5 min in PBS at room temperature and incubated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Amersham, Piscataway, NJ, USA) diluted 1:10 in PBS at 37°C for 40 min. Following three additional 5 min rinses in PBS, samples were mounted in glycerol:PBS containing 0.01% (w/v) p-phenylenediamine to prevent fading and photographed using an epifluorescence microscope. At least two samples from separate patients were stained at each stage.
The anti-claudin-4 antibody (3E2C1; Zymed) is a monoclonal mouse antibody, raised against the synthetic peptide corresponding to a 22 aa sequence at the C-terminus of human claudin-4 protein. Paraffin-embedded sections (5 mm) were deparaffinized in xylene and rehydrated in ethanol with increasing concentrations of water. Endogenous peroxidase activity was quenched with 3% (v/v) hydrogen peroxide for 30 min. Non-specific binding sites were blocked with 2% (v/v) normal goat serum (NGS) for 30 min at room temperature. Primarily antibody was serially diluted in a solution of PBS containing 1% (v/v) NGS and 0.1% (w/v) sodium azide to optimize the appropriate concentrations in order to achieve maximum sensitivity and specificity. Tissue sections were incubated with primary antibody at 4°C overnight at 1:1000 dilution. Negative control sections were treated with non-immune serum diluted in the same manner. After primary antibody incubation, sections were washed twice with PBS followed by treatment with 1% (v/v) NGS for an additional 30 min. Subsequently, sections were washed with PBS and incubated with biotinylated goat anti-mouse secondary antibody (Vectastain Elite ABC Kit; Vector Laboratories, Inc., Burlingame, CA, USA) at a dilution of 1:400 for 30 min at room temperature. After rinsing with PBS, the immunoreactive antigen was visualized using avidin–biotin peroxidase complex (Vectastain Elite ABC kit) and 3,3'-diaminobenzidine as chromagen. Slides were counterstained with Mayer’s haematoxylin blue/Toulidine Blue followed by dehydration in a graded series of ethanols, cleared in xylene, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ, USA). The resulting stain was evaluated on a Nikon microscope by a single blinded observer.
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Results |
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Microarray screening
Total endometrial RNA from six fertile women was extracted and pooled from the early luteal (n = 3) and mid-luteal (n = 3) phases. These RNA preparations were then used to probe the U95 human gene microarray comprising of
12 000 genes. The results of this screening are compiled in the scattergram shown in Figure 1. The vast majority (94.2%) of the genes represented displayed <2-fold changes between early and mid-luteal phases. These included many genes previously shown to display no or modest changes during the menstrual cycle. A list of all genes that were noted to increase or decrease by 
2-fold can be viewed as supplementary data online (www.molehr.oupjournals.org).
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A total of 323 genes displayed significant increases in expression of
2-fold between the early luteal and mid-luteal phase. Cell surface proteins, extracellular matrix components and growth factors/cytokines were the major group of genes of known function altered under these conditions and accounted for 23% of the total (Tables I and II). DNA binding proteins, transcription factors and DNA modifying enzymes accounted for 8% of these genes (Tables I and III), intracellular signalling/cell cycle proteins, metabolic enzymes, genes encoding proteins of miscellaneous other functions and proteins of unknown function or expressed sequence tagges (ESTs) accounted for 12, 17, 7 and 30% of the genes with increased expression respectively (Table I; http://www.med.unc.edu/obgyn/tables/increase.htm).
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A similar number of genes (370) were identified as displaying decreases
50% during the early to mid-luteal phase transition. Again, genes encoding cell surface proteins, extracellular matrix components and growth factors/cytokines were the major group of genes of known function altered and constituted 19% of the total (Tables I and IV
). Transcription factors and DNA modifying enzymes accounted for 13% of this list (Tables I and V). A number of genes associated with cell cycle progression were down-regulated during this transition including p55CDC, cyclin B, CD22 and cdk3. These, along with intracellular signalling proteins, constituted 10% of the decreases while metabolic enzymes, proteins of miscellaneous other functions and proteins of unknown function or ESTs accounted for 12, 11 and 35% of the changes respectively (Table I; www.med.unc.edu/obgyn/tables/decrease.htm).
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From these results, it was evident that genes encoding many different types of proteins were significantly altered in expression during the early to mid-luteal transition. Nonetheless, cell surface, extracellular matrix components and growth factors/cytokines constituted a large proportion of the overall changes (20%). Verification of these observations relied on several criteria: (i) statistical analyses of changes in the relative expression of particular genes in RNA from multiple, fertile women; (ii) detection of similar changes in relative expression of the same gene at independent sites on the microarray chip; and (iii) detection of previously reported changes in gene expression. In the latter case, similar changes in fibroblast growth factor (FGF) pathway components (Sangha et al., 1997
), PP5 (Butzow et al., 1986) decay accelerating factor/CD55 (Kaul et al., 1996), matrix metalloproteinase (MMP)-7/matrilysin (Rodgers et al., 1993) and cyclin B (Shiozawa et al., 1996) have been reported. Nonetheless, there were many examples of genes displaying large changes that had not been reported previously. We performed Northern blots for a select group of genes (of known functions) that displayed large changes to further confirm the results of the microarray analyses.
Northern blot analyses confirms changes detected by microarray analyses
Several genes displaying >4-fold changes during the early to mid-luteal transition were chosen for verification by Northern blot analyses (Figures 2 and 3). Claudin-4/Clostridium perfringens enterotoxin (CPE) receptor (Kaul et al., 1996) showed a large (4-fold) increase in expression during the early to mid-luteal transition (Figure 3), although the increase was not as large as predicted by the microarray results. Osteopontin (OPN) displayed a very large increase (>10-fold) in both the microarray and Northern analyses (Figure 2). More detailed studies have been performed recently revealing similar changes in OPN expression associated with uterine epithelium (Apparao et al., 2001). Betaig-H3 (BIGH3), a transforming growth factor ß-induced extracellular matrix protein with cell adhesion-promoting activity (Kim et al., 2000), decreased by 92% by the microarray analysis and >80% by Northern blot analysis (Figure 3).
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Immunostaining confirms marker protein expression detected by microarray analyses
Antibodies to several proteins encoded by genes displaying
4-fold changes during the early to mid-luteal transition were used to stain mid-luteal stage uterine tissues. Prostaglandin receptor EP1 was strongly expressed in epithelial cells closely associated with cell boundaries (Figure 4A). In contrast, leukotriene B4 receptor was found in both epithelial and stromal cells in a pattern suggesting nuclear association (Figure 4B). Non-immune rabbit IgG did not stain any of these structures (Figure 4C). Antibodies to claudin-4/CPE receptor strongly stained both lumenal and glandular epithelia, but not stroma (Figure 5B). Collectively, these studies confirmed several of the results of mRNA expression by demonstrating protein accumulation in particular uterine cell types during the receptive phase.
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Discussion |
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Recent technological advances have aided the practice of reproductive medicine in the last 15 years (Wilcox et al., 1999
). Much of this success can be attributed to improvements in embryo selection and culture procedures. Nonetheless, overall embryo implantation success rates remain low (<33%) in many areas, and a significant proportion of women receiving embryo transfers either require multiple procedures or never achieve a successful pregnancy (Report ASRM/SARTR, 2000). Various pathologies are associated with female infertility including endometriosis (Lu and Ory, 1995) and tubal disease (Hurst et al., 2001). Studies have suggested that in some women with endometriosis or hydrosalpinges, uterine receptivity may be diminished (Lessey et al., 1994; Meyer et al., 1997). An underlying problem is the fundamental lack of useful predictors of the uterine state of receptivity for embryo implantation. It is generally agreed that the initial phase of human embryo implantation occurs during the mid-luteal phase with low success occurring during the early luteal phase (Lessey et al., 1999). Similar observations have been made under well-controlled conditions in laboratory animals (Noyes et al., 1963). Information already exists implicating various genes in the implantation process (Salamonsen et al., 2001). Furthermore, recent studies using microarray-based approaches have revealed changes in gene expression during human stromal cell differentiation (decidualization) in vitro (Popovici et al., 2000). While useful information can be gained by comparing other uterine stages, e.g. proliferative or late luteal/menstrual phases, large changes in the hormonal milieu and gross, histologically evident changes in endometrial architecture indicate that many changes in gene expression occur and have little bearing on implantation-related processes (Parr and Parr, 1989). Thus, we chose to examine changes in gene expression that occur during the early to mid-luteal transition as a means of focusing on a set of changes in gene expression more closely tied to generation of a receptive uterine state. As a result, we report consistent changes in <6% of the genes surveyed. Those genes surveyed represent 25–33% of the human transcriptome (Clavrie, 2001).
Confirmation of the observed changes relied not only upon repeated observations using the microarray approach, but also on results of previous studies identifying similar changes in expression of particular genes or gene products in the human endometrium. These included FGF pathway components (Sangha et al., 1997), PP5 (Butzow et al., 1986), decay accelerating factor/CD55 (Kaul et al., 1996), MMP-7/matrilysin (Rodgers et al., 1993) and cyclin B (Shiozawa et al., 1996). Many other gene products in which changes were observed have been detected in human endometrium, although cycle-related changes have not been reported.
Complex changes in Wnt pathway regulators were observed during the early to mid-luteal transition. A large increase (10-fold) in Dickkopk1/DKK1, a Wnt pathway inhibitor (Semenov et al., 2001), and a smaller (50%) decrease in LRP6, a Wnt co-receptor with frizzled (FrpHE) (Semenov et al., 2001), occurred. This suggests that decreased Wnt-dependent signalling may occur. Paradoxically, these changes were paralleled by a large increase (>45-fold) in Wnt10B expression and large decrease (90%) in the soluble form of FrpHE, another Wnt pathway inhibitor (Salic et al., 1997). Wnt-10b is reported to promote glandular development and hyperplasia in mammary tissue (Lane and Leder, 1997). Therefore, Wnt-10b also may promote endometrial glandular development that occurs during the luteal phase (Parr and Parr, 1989). FrpHE was shown to decrease during the luteal phase in an earlier report (Abu-Jawdeh et al., 1999). In mice, estrogens suppress production of secreted frizzled related protein-2 (Das et al., 2000). Therefore, nidatory estrogen might also be expected to suppress expression during the receptive phase in this species, although this has not been specifically determined. Whole endometrium, rather than isolated cell types, was used in these studies. The complexity of the changes in Wnt pathway-related components suggests that compartmentalization of these changes may occur within the endometrium to balance responses within particular cell types. More detailed studies localizing the cellular source of these changes will be required to determine if this is the case.
A possible discrepancy with previous studies was the observed decrease in stromelysin-3/MMP-11. A careful study (Rodgers et al., 1994) demonstrated stromelysin-3/MMP-11 transcript expression in proliferative and late luteal/early menstrual endometrium. The results of the present studies indicate that stromelysin-3/MMP-11 mRNA is increased during the early luteal phase, perhaps to support uterine extracellular matrix remodelling that occurs during the luteal phase (Parr and Parr, 1989; Lessey, 1998). Finally, many genes and ESTs displayed consistent changes that have not yet been studied in human endometrium and represent other avenues for more focused research.
Some genes expected to change in expression during the early to mid-luteal transition were not found to change in the current screen. In some cases, e.g. HOXA10 (Taylor et al., 1998), these genes were not represented on the array. However, in other cases, e.g. progesterone receptor (Lessey et al., 1996), the genes were represented. Gene products expressed by more than one cell type may undergo cell type-specific changes that are not of sufficient amplitude to be detected in the entire tissue. This is a potential shortcoming of the microarray approach, but could be overcome by the use of ancillary techniques such as laser capture or cell isolation prior to mRNA isolation. Alternatively, cell lines that mimic normal tissue components may also be useful to study specific cell types or to investigate regulation of select genes.
We chose several genes that consistently displayed large (>4-fold) changes in expression during the early to mid-luteal transition for further study. Northern blot analyses confirmed the large decrease in BIGH3 mRNA as well as increases in OPN and claudin-4/CPE receptor mRNA expression. Detailed examination of OPN mRNA and protein expression and distribution throughout the cycle has been examined recently and further confirms these observations (Apparao et al., 2001). The increase in OPN expression complements increased expression of the OPN receptor complex, integrin 
Vß3 in lumenal epithelium during the mid-luteal phase in humans (Lessey, 1998). Thus, an OPN:Vß3 complex may occur at the apical surface of human uterine lumenal epithelium to promote embryo attachment, as suggested in other species (Johnson et al., 1999). Claudin-4/CPE receptor is an epithelial protein and, presumably, is associated with uterine lumenal and/or glandular epithelium. Immunostaining revealed strong epithelial staining for claudin-4/CPE receptor at the mid-luteal phase confirming both the microarray and Northern blotting results and identifying the cellular site of protein accumulation. Clear demonstration of the function of this protein has not been performed; however, increases in the expression of this component of cell:cell junctional complexes appears to be part of a general increase in expression of other intercellular adhesion components, e.g. cadherins and associated proteins (Fujimoto et al., 1996), that occurs during the transition from the prereceptive early luteal to the receptive mid-luteal phase. The prostaglandin EP1 receptor also accumulated in epithelia during the mid-luteal phase indicating the site of accumulation of this gene product also identified by the microarray analysis. In contrast to the epithelial specific expression of these markers as well as of OPN (Apparao et al., 2001), leukotriene B4 receptor was expressed by both epithelial and stromal cells. Thus, while some markers display cell type-specific expression, others are more broadly expressed.
These studies provide the first transcriptome-based comparison of endometrial gene expression during the transition to the receptive uterine state in humans. From these results, it should be possible to construct much more limited gene arrays that can be used to determine the feasibility of using changes in gene expression for prediction of uterine receptivity as well as for the identification of underlying fertility defects.
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We appreciate the outstanding technical assistance of Palestrina Truong and the excellent typing and graphics support of Ms Sharron Kingston, Mrs Ginger Moore and Mrs Margie Barrett. We are grateful to Dr S.Tsukita (Kyoto University) for his generous gift of the claudin-4 antibody. We thank the members of the Carson, Farach-Carson and Lessey laboratories for their critical reading of this manuscript and many helpful suggestions. This research was supported by NICHD/NIH as part of the Specialized Cooperative Centers Program in Reproduction Research U54 HD34824 (B.L.) and the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (HD35041, B.L. and HD29963, D.D.C.).
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4 To whom correspondence should be addressed. E-mail: dcarson{at}udel.edu
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Submitted on September 20, 2001; resubmitted on February 14, 2002; accepted on May 17, 2002.
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