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.

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 ␣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 Lselectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin ␣2, E NDOMETRIOSIS 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 ϩ 6 -10) (reviewed in Ref. 4). In humans, the implantation process begins with attachment of the emAbbreviations: BSEP, Bile salt export pump; C4BP, c...

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...

Members of the Wnt family of signaling molecules are important in cell specification and epithelial-mesenchymal interactions, and targeted gene deletion of Wnt-7a in mice results in complete absence of uterine glands and infertility. To assess potential roles of the Wnt family in human endometrium, an endocrine-responsive tissue, we investigated in the proliferative and secretory phases of the menstrual cycle, endometrial expression of several Wnt ligands (Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, and Wnt-8b), receptors [Frizzled (Fz)-6 and low-density lipoprotein receptor-related protein (LRP)-6], inhibitors [FrpHE and Dickkopf (Dkk)-1], and downstream effectors (Dishevelled-1, glycogen synthase kinase-3beta, and beta-catenin) by RT-PCR, real-time PCR and in situ hybridization. No significant menstrual cycle dependence of the Wnt ligands (except Wnt-3), receptors, or downstream effectors, was observed. Wnt-3 increased 4.7-fold in proliferative compared with secretory endometrium (P < 0.05). However, both inhibitors showed dramatic changes during the cycle, with 22.2-fold down-regulation (P < 0.05) of FrpHE and 234.3-fold up-regulation (P < 0.001) of Dkk-1 in the secretory, compared with the proliferative phase. In situ hybridization revealed cell-specific expression of different Wnt family genes in human endometrium. Wnt-7a was exclusively expressed in the luminal epithelium, and Fz-6 and beta-catenin were expressed in both epithelium and stroma, without any apparent change during the cycle. Both FrpHE and Dkk-1 expression were restricted to the stroma, during the proliferative and secretory phase, respectively. These unique expression patterns of Wnt family genes in different cell types of endometrium and the differential regulation of the inhibitors during the proliferative and secretory phase of the menstrual cycle strongly suggest functions for a Wnt signaling dialog between epithelial and stromal components in human endometrium. Also, they underscore the likely importance of this family during endometrial development, differentiation and implantation.

Although there is significant evidence for progesterone's role as an immunomodulator, nuclear progesterone receptors have not been consistently identified in immune cells. Recently, three new putative membrane progesterone receptors (mPRs), mPRa, mPRb, and mPRg have been described. The objective of this study was to examine whether mPRs are expressed in peripheral blood leukocytes (

These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.

Decidualization of endometrial stromal cells is a prerequisite for human implantation and occurs in vivo in response to progesterone and involves activation of the protein kinase A (PKA) pathway. The objective of this study was to determine the molecular signatures and patterns of gene expression during stimulation of this pathway with an analog of cAMP. Endometrial stromal cells from two subjects were treated with or without 8-Br-cAMP (1 mM) for 0, 2, 12, 24, 36, and 48 h and were processed for microarray analysis, screening for 12,686 genes and ESTs. Most abundantly upregulated genes included neuropeptides, immune genes, IGF family members, cell cycle regulators, extracellular matrix proteases, cholesterol trafficking, cell growth and differentiation, hormone signaling, and signal transduction. Most abundantly downregulated genes included activator of NF-kappaB, actin/tropomyosin/calmodulin binding protein, cyclin B, IGFBP-5, alpha1 type XVI collagen, lipocortin III, l-kynurenine hydrolase, frizzle-related protein, and cyclin E2. RT-PCR validated upregulation of IGFBP-1, preprosomatostatin, and IL-11, and Northern analysis validated their kinetic upregulation. RT-PCR confirmed downregulation of IGFBP-5, cyclin B, and TIL-4. K-means analysis revealed four major patterns of up- and downregulated genes, and genes within each ontological group were categorized into these four kinetic patterns. Within each ontological group different patterns of temporal gene expression were observed, indicating that even genes within one functional category are regulated differently during activation of the PKA pathway in human endometrial stromal cells. Overall, the data demonstrate kinetic reprogramming of genes within specific functional groups and changes in genes associated with nucleic acid binding, cell proliferation, decreased G protein signaling, increased STAT pathway signaling, structural proteins, cellular differentiation, and secretory processes. These changes are consistent with cAMP modulating early events (0-6 h) primarily involving cell cycle regulation, subsequent events (12-24 h) involving cellular differentiation (including changes in morphology and secretory phenotype), and late events (24-48 h) mediating more specialized function, including immune modulators, in the human endometrial stromal cell.

Molecular mechanisms underlying fetal growth restriction due to placental insufficiency and in utero hypoxia are not well understood. In the current study, time-dependent (3 h-11 days) changes in fetal tissue gene expression in a rat model of in utero hypoxia compared with normoxic controls were investigated as an initial approach to understand molecular events underlying fetal development in response to hypoxia. Under hypoxic conditions, litter size was reduced and IGFBP-1 was up-regulated in maternal serum and in fetal liver and heart. Tissue-specific, distinct regulatory patterns of gene expression were observed under acute vs. chronic hypoxic conditions. Induction of glycolytic enzymes was an early event in response to hypoxia during organ development; consistently, tissue-specific induction of calcium homeostasis-related genes and suppression of growth-related genes were observed, suggesting mechanisms underlying hypoxia-related fetal growth restriction. Furthermore, induction of inflammation-related genes in placentas exposed to long-term hypoxia (11 days) suggests a mechanism for placental dysfunction and impaired pregnancy outcome accompanying in utero hypoxia.