Laparoscopic surgery improves pregnancy outcomes in women with suspected endometriosis with or without pathological confirmation.

PURPOSE OF THE INVESTIGATION: To verify whether histologic confirmation of endometriosis impacts fertility outcomes. MATERIALS AND METHODS: Women with unexplained infertility (UI) underwent laparoscopic excision or ablation with CO2 laser or electrocautery of all suspected endometriotic lesions, followed by clinical treatment between January 2007 and December 2013; pregnancy (> 12 weeks) within 12 months of monitored cycles was the main outcome measured. RESULTS: Women with histological confirmation (n = 74) did not differ from those not confirmed (n = 29) with age, body mass index, gravidity, parity, ovulation induction protocol, and past duration of infertility. Pregnancy outcome was similar in both groups (39/74 vs. 15/29-p = 0.9--Chi-square) and there was no statistical difference in time to conceive/deliver (p = 0.7) between groups. CONCLUSIONS: There is no difference in fertility outcomes in women with UI, whether or not suspected endometriosis is confirmed pathologically.

Altered expression of microRNA-451 in eutopic endometrium of baboons (Papio anubis) with endometriosis.

STUDY QUESTION: Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? SUMMARY ANSWER: Induction of endometriosis causes significant changes in the expression of eight miRs, including miR-451, in the baboon endometrium as early as 3 months following induction of the disease. WHAT IS KNOWN ALREADY: Endometriosis is one of the most common gynecological disorders and causes chronic pelvic pain and infertility in women of reproductive age. Altered expression of miRs has been reported in women and has been suggested to play an important role in the pathophysiology of several gynecological disorders including endometriosis. STUDY DESIGN, SIZE, DURATION: EuE was obtained from the same group of baboons before and 3 months after the induction of endometriosis. The altered expression of miR-451 was validated in the eutopic and ectopic endometrium of additional baboons between 3 and 15 months following disease induction. Timed endometrial biopsies from women with and without endometriosis were also used to validate the expression of miR-451. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was extracted from EuE samples before and after the induction of endometriosis, and miRNA expression was analyzed using a 8 × 15 K miR microarray. Microarray signal data were preprocessed by AgiMiRna software, and an empirical Bayes model was used to estimate the changes. The present study focused on quantitative RT-PCR validation of the microarray data, specifically on miR-451 and its target genes in both baboons (n = 3) and women [control (n = 7) and endometriosis (n = 19)]. Descriptive and correlative analysis of miR-451 and target gene expression was conducted using in situ hybridization and immunohistochemistry, while functional analysis utilized an in vitro 3' untranslated region (UTR) luciferase assay and overexpression of miR-451 in human endometrial and endometriotic cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of endometriosis results in the altered expression of miR-451, -141, -29c, -21, -424, -19b, -200a and -181a in the baboon endometrium. In the baboon, induction of endometriosis significantly decreased the expression of miR-451 at 3 months (P < 0.001), which was also associated with increased expression of its target gene YWHAZ (14.3.3ζ). A similar significant (P < 0.0001) decrease in miR-451 expression was observed in women with endometriosis. The 3' UTR luciferase assay confirmed the regulation of YWHAZ expression by miR-451. Furthermore, overexpression of miR-451 in 12Z cells (immortalized human endometriotic epithelial cell line) led to the decreased expression of its target YWHAZ and this was correlated with decreased cell proliferation. LIMITATIONS, REASONS FOR CAUTION: The study focused only on miR-451 and one of its targets, namely YWHAZ. A single miR could target number of genes and a single gene could also be regulated by number of miRs; hence, it is possible that other miRs and their regulated genes may contribute to the pathophysiology of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.

Expression of the transmembrane mucins, MUC1, MUC4 and MUC16, in normal endometrium and in endometriosis.

STUDY QUESTION: Are the transmembrane mucins, MUC1, MUC4 and MUC16, differentially expressed in endometriosis compared with normal endometrium? SUMMARY ANSWER: This study revealed that transmembrane mucin expression does not vary significantly in normal endometrium during the menstrual cycle and is not altered in endometriosis relative to the epithelial marker, cytokeratin-18 (KRT18). WHAT IS KNOWN ALREADY: Increased serum levels of the transmembrane mucin fragments MUC1, MUC4 and MUC16 that normally dominate the apical surface of simple epithelia are found in several pathological conditions, including endometriosis. Altered mucin expression in gynecologic diseases may promote infertility or endometrial pathologies. STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study of samples from 12 endometriosis patients as well as non-endometriosis control samples obtained from 31 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was isolated from endometrial biopsies of ectopic and eutopic endometrium from women with endometriosis and control patients from different stages of the menstrual cycle. Quantitative (q)-RT-PCR analyses were performed for the mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, cytokeratin-18 (KRT18), or ß-actin (ACTB). Frozen sections from endometrial biopsies of proliferative and mid-secretory stage women with endometriosis were immunostained for MUC1, MUC4 and MUC16. MAIN RESULTS AND THE ROLE OF CHANCE: qRT-PCR analyses of MUC1 and MUC16 mRNA revealed that these mucins do not vary significantly during the menstrual cycle nor are they altered in women with endometriosis relative to the epithelial marker, KRT18. MUC4 mRNA is expressed at very low levels relative to MUC1 and MUC16 under all conditions. There was little difference in MUC1 and MUC16 expression between eutopic endometrial and ectopic endometriotic tissues. MUC4 expression also was not significantly higher in the ectopic endometriotic tissues. Immunostaining for all three mucins reveals robust expression of MUC1 and MUC16 at the apical surfaces of endometrial epithelia, but little to no staining for MUC4. LIMITATIONS, REASONS FOR CAUTION: qRT-PCR analysis was the main method used for mucin detection. Additional studies with stage III-IV endometriotic tissue would be useful to determine if changes in MUC1 and MUC16 expression occur, or if MUC4 expression increases, at later stages of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: We report a comprehensive comparative profile of the major transmembrane mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, KRT18, in normal cycling endometrium and in endometriosis, and indicate constitutive expression. Previous studies have profiled the expression of individual mucins relative to ß-actin and indicate accumulation in the luteal phase. Thus, these differences in interpretation appear to reflect the increased epithelial content of endometrium during the luteal phase. STUDY FUNDING: This study was supported by: NIH R01HD29963 to D.D.C.; NIH U54HD007495 to S.M.H.; and NIH R01HD067721 to S.L.Y. and B.A.L. The authors have no competing interests to declare.

MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis.

Endometriosis is a common gynecologic disorder characterized by pain and infertility. In addition to estrogen dependence, progesterone resistance is an emerging feature of this disorder. Specifically, a delayed transition from the proliferative to secretory phase as evidenced by dysregulation of progesterone target genes and maintenance of a proliferative molecular fingerprint in the early secretory endometrium (ESE) has been reported. MicroRNAs (miRNAs) are small noncoding RNAs that collectively represent a novel class of regulators of gene expression. In an effort to investigate further the observed progesterone resistance in the ESE of women with endometriosis, we conducted array-based, global miRNA profiling. We report distinct miRNA expression profiles in the ESE of women with versus without endometriosis in a subset of samples previously used in global gene expression analysis. Specifically, the miR-9 and miR-34 miRNA families evidenced dysregulation. Integration of the miRNA and gene expression profiles provides unique insights into the molecular basis of this enigmatic disorder and, possibly, the regulation of the proliferative phenotype during the early secretory phase of the menstrual cycle in affected women.

Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle.

Integrins are a class of cell adhesion molecules that participate in cell-cell and cell-substratum interactions and are present on essentially all human cells. The distribution of nine different alpha and beta integrin subunits in human endometrial tissue at different stages of the menstrual cycle was determined using immunoperoxidase staining. Glandular epithelial cells expressed primarily alpha 2, alpha 3, and alpha 6 (collagen/laminin receptors), while stromal cells expressed predominantly alpha 5 (fibronectin receptor). The presence of alpha 1 on glandular epithelial cells was cycle specific, found only during the secretory phase. Expression of both subunits of the vitronectin receptor, alpha v beta 3, also underwent cycle specific changes on endometrial epithelial cells. Immunostaining for alpha v increased throughout the menstrual cycle, while the beta 3 subunit appeared abruptly on cycle day 20 on luminal as well as glandular epithelial cells. Discordant luteal phase biopsies (greater than or equal to 3 d "out of phase") from infertility patients exhibited delayed epithelial beta 3 immunostaining. These results demonstrate similarities, as well as specific differences, between endometrium and other epithelial tissues. Certain integrin moieties appear to be regulated within the cycling endometrium and disruption of integrin expression may be associated with decreased uterine receptivity and infertility.

Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.

Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE-->ESE, ESE-->MSE, and MSE-->LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy specimens) from subjects who are as normal as possible in a human study and including those with unknown histology, can be classified by their molecular signatures and correspond to known phases of the menstrual cycle with identical results using two independent analytical methods. Also, the results enable global identification of biological processes and molecular mechanisms that occur dynamically in the endometrium in the changing steroid hormone milieu across the menstrual cycle in normo-ovulatory women. The results underscore the potential of gene expression profiling for developing molecular diagnostics of endometrial normalcy and abnormalities and identifying molecular targets for therapeutic purposes in endometrial disorders.

Extracellular matrix metalloproteinase inducer regulates metalloproteinases in human uterine endometrium.

CONTEXT: Endometrial remodeling occurs during each menstrual cycle in women and also during the establishment of endometriosis. Both processes involve the production of metalloproteinases (MMPs) by uterine endometrial cells. OBJECTIVE: The objective of this study was to determine whether tissue remodeling and endometrial invasion involve activation of MMPs by extracellular matrix metalloproteinase inducer (EMMPRIN). MAIN OUTCOME MEASURES: EMMPRIN expression was examined by immunohistochemistry and real-time PCR in ectopic and eutopic endometria. For functional assays, human uterine fibroblasts were treated in the absence or presence of IL-1beta (10 ng/ml) or purified native EMMPRIN (0.5 or 1 microg/ml) for 24 h. Cellular RNA and conditioned medium were assayed by real-time PCR or immunoblotting. RESULTS: EMMPRIN protein localized to epithelial and fibroblast cells of eutopic and ectopic endometria. The pattern of localization was regulated by ovarian hormones. EMMPRIN mRNA levels varied throughout the menstrual cycle in parallel with the cyclic changes in estradiol. EMMPRIN treatment (0.5 microg/ml) of human uterine fibroblast cells stimulated MMP-1 (5.23-fold) and MMP-2 (8.55-fold), but not MMP-3, mRNA levels over levels in control cells (P < 0.05). EMMPRIN treatment (1 microg/ml) stimulated endogenous EMMPRIN (1.6-fold) mRNA levels (P > 0.05). IL-1beta stimulated MMP-1 (5.6-fold), MMP-2 (2.8-fold), and MMP-3 (75-fold) gene expression, but not EMMPRIN, over levels in control cells (P < 0.05). Both EMMPRIN and IL-1beta treatments stimulated MMP-1, -2, and -3, but not EMMPRIN protein secretion, with 0.5 microg/ml producing the greatest response. CONCLUSIONS: The ability of EMMPRIN to stimulate MMP secretion by endometrial fibroblasts indicates its potential role in uterine remodeling and the pathogenesis of endometriosis.

cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.

Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner.

Diagnostic accuracy of urinary cytokeratin 19 fragment for endometriosis.

Endometriosis affects up to 10% of women of reproductive age and 176 million women worldwide. The prevalence in women with infertility is between 30% and 50% but may be higher in women with pelvic pain, interstitial cystitis, or irritable bowel syndrome. Cytokeratin 19 has been suggested as a potential biomarker in urine for the diagnosis of this condition. The objective of this study was to prospectively determine the accuracy and the performance of a urinary cytokeratin 19 (uCYFRA 21-1) test for diagnosing endometriosis. Ninety-eight consecutive women who underwent laparoscopy had a urinary sample obtained before surgery and were included in the study. Endometriosis was diagnosed by laparoscopy and pathology in 64.3% (63 of 98 women). The estimates and 95% confidence intervals for sensitivity, specificity, positive and negative predictive values, and likelihood ratios were 11.1% (4.5%-21.5%), 94.3% (80.8%-99.3%), 77.7% (39.9-97.1), 37% (27-47.9), 1.94 (0.43-8.86), and 0.94 (0.84-1.06), respectively. Despite the high specificity, the uCYFRA 21-1 test has limited value for clinical practice to discriminate between women with and without endometriosis.

Estrogen-insensitive progesterone receptors in a human breast cancer cell line: characterization of receptors and of a ligand exchange assay.

In modified culture conditions, T47D human breast cancer cells synthesize extraordinary amounts of progesterone receptors (PgR), but, unlike other progesterone target cells, the PgR are entirely independent of estrogen controls. In the present studies we characterize some physicochemical properties of the PgR in T47D cells. We also describe an exchange assay for cytoplasmic and nuclear forms of the receptors which has enabled us to demonstrate that after progesterone treatment, translocation is stoichiometric. Despite the anomalous regulation of PgR levels, these receptors are typical of steroid receptors; they sediment at 7-8S on sucrose density gradients, they bind ligands with high affinity (Kd approximately 4 nM for R5020; Kd approximately 2 nM for progesterone), they bind only progestins specifically, and they are thermolabile (t1/2 at 37 C is approximately 15 min). Receptor levels range from 15-40 pmol/mg DNA, or more than 300,000 sites/cell. The ability of ligands to dissociate from and rebind to the receptors was measured and used in an exchange assay for nuclear PgR. The synthetic progestin R5020 dissociates readily from receptors (t1/2 approximately 3 h at 0 C and 1.5 h at 10 C), and the dissociation of progesterone is even faster (t1/2 approximately 30 min at 0 C). To quantify steroid exchange, receptor levels were measured in mixtures of hormone-filled and unfilled cytosols. These studies assess ligand dissociation and subsequent ligand rebinding. At 0 C for 4-18 H or at 10 C for 4 h, unlabeled progesterone dissociates from receptors, and R5020 rebinds all sites, resulting in 100% exchange. In contrast, despite the use of a variety of incubation times and temperatures, no more than 50% of receptors previously filled with R5020 can exchange for [3H]R5020. The progesterone to [3H]R5020 exchange assay was used to measure salt-extracted nuclear progesterone receptors. In cells treated for 5 min with 0.1 microM progesterone, all depleted cytoplasmic sites were quantitatively recovered from nuclei. These cells provide a new model system to study the molecular biology of human PgR.

Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility.

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 alpha2 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 alpha2, 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.

Global gene profiling in human endometrium during the window of implantation.

Implantation in humans is a complex process that is temporally and spatially restricted. Over the past decade, using a one-by-one approach, several genes and gene products that may participate in this process have been identified in secretory phase endometrium. Herein, we have investigated global gene expression during the window of implantation (peak E2 and progesterone levels) in well characterized human endometrial biopsies timed to the LH surge, compared with the late proliferative phase (peak E2 level) of the menstrual cycle. Tissues were processed for poly(A(+)) RNA and hybridization of chemically fragmented, biotinylated cRNAs on high density oligonucleotide microarrays, screening for 12,686 genes and expressed sequence tags. After data normalization, mean values were obtained for gene readouts and fold ratios were derived comparing genes up- and down-regulated in the window of implantation vs. the late proliferative phase. Nonparametric testing revealed 156 significantly (P < 0.05) up-regulated genes and 377 significantly down-regulated genes in the implantation window. Up-regulated genes included those for cholesterol trafficking and transport [apolipoprotein (Apo)E being the most induced gene, 100-fold], prostaglandin (PG) biosynthesis (PLA2) and action (PGE2 receptor), proteoglycan synthesis (glucuronyltransferase), secretory proteins [glycodelin, mammaglobin, Dickkopf-1 (Dkk-1, a Wnt inhibitor)], IGF binding protein (IGFBP), and TGF-beta superfamilies, signal transduction, extracellular matrix components (osteopontin, laminin), neurotransmitter synthesis (monoamine oxidase) and receptors (gamma aminobutyric acid A receptor pi subunit), numerous immune modulators, detoxification genes (metallothioneins), and genes involved in water and ion transport [e.g. Clostridia Perfringens Enterotoxin (CPE) 1 receptor (CPE1-R) and K(+) ion channel], among others. Down-regulated genes included intestinal trefoil factor (ITF) [the most repressed gene (50-fold)], matrilysin, members of the G protein-coupled receptor signaling pathway, frizzled-related protein (FrpHE, a Wnt antagonist), transcription factors, TGF-beta signaling pathway members, immune modulators (major histocompatibility complex class II subunits), and other cellular functions. Validation of select genes was conducted by Northern analysis and RT-PCR using RNA from endometrial biopsies obtained in the proliferative phase and the implantation window and by RT-PCR using RNA from cultured endometrial epithelial and stromal cells. These approaches confirmed up-regulation of genes corresponding to IGFBP-1, glycodelin, CPE1-R, Dkk-1, mammaglobin, and ApoD and down-regulation for PR membrane component 1, FrpHE, matrilysin, and ITF, as with the microarray data. Cultured endometrial epithelial cells were found to express mRNAs for glycodelin, CPE-1R, Dkk-1, the gamma aminobutyric acid A receptor pi subunit, mammaglobin, matrilysin, ITF and PR membrane component 1. The expression of IGFBP-1, CPE1-R, Dkk-1, and ApoD mRNAs increased upon decidualization of stromal cells in vitro with progesterone after E2 priming, whereas FrpHE decreased, consistent with the microarray results. Overall, the data demonstrate numerous genes and gene families not heretofore recognized in human endometrium or associated with the implantation process. Reassuringly, several gene products, known to be differentially expressed in the implantation window or in secretory endometrium, were verified, and the striking regulation of select secretory proteins, water and ion channels, signaling molecules, and immune modulators underscores the important roles of these systems in endometrial development and endometrial-embryonic interactions. In addition, the current study validates using high density oligonucleotide microarray technology to investigate global changes in gene expression in human endometrium.

Tumor-associated glycoprotein (TAG-72) in endometriotic implants.

Tumor-associated glycoprotein (TAG-72) is an antigen detected by the monoclonal antibody B72.3 that is expressed by a wide variety of human malignancies and in normal secretory endometrium. Serum TAG-72 may be elevated in women with endometriosis, but the distribution of TAG-72 in this disorder has not been well studied. Accordingly, TAG-72 was assayed by immunohistochemical staining in 26 endometriotic implants collected at various times of the menstrual cycle and compared to that in 56 samples of normal endometrium, in 8 cases from the same patient. TAG-72 expression was markedly discordant between normal and ectopic endometrium. Unlike proliferative phase endometrial samples, which were uniformly negative, endometriotic implants were TAG-72 positive in 50% of the cases. Like normal endometrium, all except very early secretory phase endometriosis was positive. Based on our current understanding of TAG-72 expression, this unique estrogen-repressed glycoprotein may be a useful probe to study the basis for phenotypic alterations noted in endometriosis, some of which probably contribute to the infertility associated with this disease. Moreover, the presence of B72.3 in nonmalignant tissues puts in question it usefulness in screening for malignancies.

Epidermal growth factor and sex steroids dynamically regulate a marker of endometrial receptivity in Ishikawa cells.

The factors regulating human endometrial receptivity remain poorly understood. The alpha v beta 3 integrin cell adhesion molecule appears to be regulated in the human endometrium, appearing on postovulatory days 5-6, corresponding to the time of initial embryo attachment. This integrin has been extensively studied as a potential marker of endometrial receptivity and is aberrantly expressed in the endometrial epithelium of some infertile women. Ishikawa cells are a well differentiated endometrial adenocarcinoma cell line that maintain functional estrogen and progesterone receptors and are a useful model to study steroid-mediated events in human endometrial epithelium. This cell line expresses most of the normal endometrial epithelial integrins, including the alpha v beta 3 vitronectin receptor. The regulation of this integrin was studied with fluorescence immunocytochemistry, flow cytometry, and Northern blot analysis. Estrogen with or without progesterone treatment down-regulates alpha v beta 3 in this cell line. Several growth factors, including epidermal growth factor and the closely related transforming growth factor-alpha significantly increase the expression of this integrin. We conclude that endometrial differentiation is influenced by both steroid hormones and growth factors. The alpha v beta 3 integrin appears to be an excellent marker to study the molecular events leading to the establishment of uterine receptivity and successful implantation.

Characterization of integrin expression in a well differentiated endometrial adenocarcinoma cell line (Ishikawa).

The pattern of constitutive and cycle-dependent integrins in normal endometrium has recently been established, suggesting a role for cell adhesion molecules in endometrial receptivity and implantation. Currently few, if any, models exist for the study of human endometrial integrins and their role in establishment of the receptive endometrial phenotype. The Ishikawa cell line maintains functional estrogen receptors and progesterone receptors. The progesterone receptors in these cells are inducible by priming with estradiol and down-regulated by treatment with progesterone. In the present study the pattern of integrin expression in this well differentiated endometrial cell line is compared to that in normal endometrial epithelium using immunohistochemistry and flow cytometry and is confirmed by immunoprecipitation, Western immunoblot, and PCR. Like normal endometrial epithelium, Ishikawa cells maintain constitutive expression of alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 4. PCR demonstrates the expected size fragments of each, although evidence for alternatively spliced forms of the alpha 2-subunit was noted. Progesterone treatment of estradiol-primed cells resulted in increased expression of the alpha 1 beta 1 collagen-laminin receptor and suppression of the alpha v beta 3 vitronectin receptor, two of the cycle-dependent integrins expressed by normal endometrial epithelium. These data support the use of Ishikawa cells as an excellent model to study the regulation endometrial integrins and advance our understanding of hormonally mediated events surrounding implantation.

Modulation of integrin expression in endometrial stromal cells in vitro.

Integrins, a class of cell adhesion molecules found on virtually all cells, display dynamic temporal and spatial patterns of expression in the endometrium during the menstrual cycle and in early pregnancy. To study integrin regulation, we measured the expression of eight different integrin subunits on cultured human endometrial stromal cells obtained from proliferative phase endometrium, using immunofluorescence and flow cytometry. Treatment with estrogen and progesterone induced hormonal changes of decidualization but did not alter the expression of any of the integrins. It is presently unknown whether steroid hormones other than estrogen or progesterone affect integrin expression. In contrast, treatment with several growth factors and cytokines resulted in specific alterations of integrin levels. Epidermal growth factor, transforming growth factor-alpha, and transforming growth factor-beta 1 induced expression of the alpha 1 beta 1 collagen/laminin receptor. There also was a trend towards decreased expression of the alpha 6 subunit in response to interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor-alpha. The expression of alpha 1 beta 1 was accompanied by increased adhesion to collagen but there was no change in the binding to fibronectin and vitronectin. Our findings suggest that some aspects of decidualization may be regulated by steroid hormones, whereas others, such as integrin expression, are regulated by cytokines or growth factors, possibly of trophoblast origin. Integrins are likely to play a role in the interaction between trophoblast and endometrium.

Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle.

Estrogen receptors (ER) and progesterone receptors (PgR) were studied immunohistochemically using specific antireceptor monoclonal antibodies in uterine tissue samples from 33 women in various stages of the menstrual cycle. Immunohistochemical localization was quantified as to intensity of staining and tissue distribution in glandular epithelium, stroma, and myometrium, and the results were compared with those of standard ligand binding assays. In all samples ER and PgR localized within the nuclei of target cells. The maximal concentrations of ER and PgR occurred in the mid- to late proliferative phase of the menstrual cycle. ER content declined throughout the secretory phase. In contrast, PgR content underwent unexpectedly complex and dyssynchronous fluctuations during the secretory phase of the menstrual cycle. Specifically, the glandular epithelium had diminished PgR content, while the stroma and myometrium maintained a significant PgR content. PgR and perhaps ER are not concordant in different cell types within the uterus. Segregation of function through alteration of receptor content may be an important mechanism in steroid-dependent growth and differentiation of target tissues.

A prospective, randomized study of endometrial telomerase during the menstrual cycle.

The purpose of this study was to characterize telomerase activity during the menstrual cycle, focusing on the luteal phase. A total of 84 endometrial biopsy samples were obtained from 72 participants. Daily urinary LH testing (OvuQuick, Quidel) was used to establish the day of the LH rise, and participants were randomized to return during the secretory phase. Twelve women returned on the identical day during the luteal phase of a subsequent cycle to allow intercycle comparisons of telomerase activity. Telomerase activity was evaluated using a modified TRAP-eze (Intergen) detection protocol. At the time of each endometrial biopsy, serum estrogen and progesterone were measured. Proliferative phase endometrium showed high telomerase activity. At the onset of the luteal phase telomerase activity was high, but it decreased during the early luteal phase, disappeared by the midluteal phase (6 d after LH surge detected), and then rose to moderate levels in the late luteal phase beginning on luteal d 10. Serum progesterone levels were inversely related to telomerase activity. In conclusion, endometrial telomerase activity is dynamic: high during the proliferative phase but inhibited during the midsecretory phase of the menstrual cycle. The timing of expression coincides with the rise and fall of progesterone levels and the time period of maximal uterine receptivity for embryo implantation. This supports a relationship between sex steroid levels and telomerase regulation.

Osteopontin and its receptor alphavbeta(3) integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially.

Osteopontin is an arginine-glycine-aspartic acid-containing acidic glycoprotein component of the extracellular matrix that is postulated to bind to integrin receptors at the cell surface to mediate cellular adhesion and migration during embryo implantation. The primary aim of this study was to examine the uterine expression of osteopontin throughout the menstrual cycle in normal fertile controls sampled prospectively based on urinary LH surge detection. Expression of osteopontin was documented using Northern blot analysis, in situ hybridization, and immunohistochemistry. Furthermore, the temporal pattern of osteopontin expression was compared with that of its receptor, the alphavbeta3 integrin. Using Ishikawa cells, a well differentiated endometrial adenocarcinoma cell line, the in vitro regulation of osteopontin and its receptor alphavbeta3 integrin was studied. By Northern blot analysis, osteopontin mRNA appears during the early secretory phase, with maximal expression occurring in mid to late secretory-phase endometrium. The in situ hybridization analyses showed that osteopontin mRNA specifically localized in epithelial cells within the endometrium. Immunostaining of osteopontin was detected in the glandular secretions and on the apical portions of surface (luminal) epithelium. The patterns of expression of osteopontin by Northern blotting, in situ hybridization, and immunohistochemistry are remarkably similar to the pattern for the alphavbeta3 integrin. Despite these similarities in distribution, in vitro studies demonstrate that osteopontin and beta3 integrin subunit expression are differentially regulated. The expression of osteopontin was primarily induced in response to progesterone, whereas the beta3 integrin subunit was up-regulated by epidermal growth factor or heparin-binding epidermal growth factor. The differential regulation of these two endometrial proteins suggests the existence of two separate pathways regulating epithelial gene expression in human endometrium during the window of implantation. In adhesion assays using Ishikawa cells, alphavbeta3 but not alphavbeta5 or beta1 integrins appear to be the primary receptors for osteopontin. These findings may better define the factors that favor the development of a receptive endometrium.

Aberrant integrin expression in the endometrium of women with endometriosis.

Integrins are ubiquitous cell adhesion molecules that undergo dynamic alterations during the normal menstrual cycle in the human endometrium. The alpha v beta 3 vitronectin receptor integrin is expressed in endometrium at the time of implantation, but its presence is delayed in endometrium that is assessed to be out of phase using classical histological features. To investigate the expression of this integrin in women with endometriosis, we assessed the presence of the beta 3-subunit throughout the menstrual cycle in 268 "in-phase" endometrial biopsies, using immunohistochemistry. The beta 3-subunit was expressed on endometrial epithelium after days 19-20 of the menstrual cycle. In 241 women whose biopsies were obtained after day 19, a lack of beta 3 expression was found to be closely related to the diagnosis of endometriosis (by Wilcoxon test, P = 0.02). This defect in integrin expression was associated with nulliparity, inversely related to the stage of disease, and occurred despite the presence of in-phase histological features. In a prospective double blind assessment of this integrin, we found endometrial beta 3 analysis to have a high specificity and positive predictive value as a nonsurgical diagnostic test for minimal and mild endometriosis. In conclusion, aberrant integrin expression in the native endometrium is associated with the finding of endometriosis and may identify some women with decreased cycle fecundity due to defects in uterine receptivity.