Kevin G. Osteen

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.

Patterns of matrix metalloproteinase expression in cycling endometrium imply differential functions and regulation by steroid hormones.

Matrix metalloproteinases are a highly regulated family of enzymes, that together can degrade most components of the extracellular matrix. These proteins are active in normal and pathological processes involving tissue remodeling; however, their sites of synthesis and specific roles are poorly understood. Using in situ hybridization, we determined cellular distributions of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, an inhibitor of matrix metalloproteinases, in endometrium during the reproductive cycle. The mRNAs for all the metalloproteinases were detected in menstrual endometrium, but with different tissue distributions. The mRNA for matrilysin was localized to epithelium, while the others were detected in stromal cells. Only the transcripts for the 72-kD gelatinase and tissue inhibitor of metalloproteinases-1 were detected throughout the cycle. Transcripts for stromelysin-2 and the 92-kD gelatinase were only detected in late secretory and menstrual endometrium, while those for matrilysin, the 72-kD gelatinase, and stromelysin-3 were also consistently detected in proliferative endometrium. These data indicate that matrix metalloproteinases are expressed in cell-type, tissue, and reproductive cycle-specific patterns, consistent with regulation by steroid hormones, and with specific roles in the complex tissue growth and remodeling processes occurring in the endometrium during the reproductive cycle.

Suppression of matrix metalloproteinases inhibits establishment of ectopic lesions by human endometrium in nude mice.

Matrix metalloproteinases of the stromelysin family are expressed in the human endometrium as a consequence of cellular events during the menstrual cycle that require extracellular matrix remodeling. We have recently documented the presence of these enzymes in lesions of endometriosis, a benign disease that presents as persistent ectopic sites of endometrial tissue, usually within the peritoneal cavity. Endometriosis can develop after retrograde menstruation of endometrial tissue fragments, and establishment of ectopic sites within the peritoneal cavity requires breakdown of extracellular matrix. To examine whether matrix metalloproteinases might contribute to the steroid-dependent epidemiology and cellular pathophysiology of endometriosis, we have developed an experimental model of endometriosis using athymic nude mice as recipients of human endometrial tissue. Our results demonstrate that estrogen treatment of human endometrial tissue in organ culture maintains secretion of matrix metalloproteinases, and promotes establishment of ectopic peritoneal lesions when injected into recipient animals. In contrast, suppressing metalloproteinase secretion in vitro with progesterone treatment, or blocking enzyme activity with a natural inhibitor of metalloproteinases, inhibits the formation of ectopic lesions in this experimental model.

Cyclic Changes in the Matrix Metalloproteinase System in the Ovary and Uterus

Abstract With each estrous or menstrual cycle, extensive alterations occur in the extracellular matrix and connective tissue of the ovary and uterus. In the ovary, these changes occur during follicular development, breakdown of the follicular wall and extrusion of the oocyte, as well as during the formation and regression of the corpus luteum. In the uterus, the endometrium undergoes dramatic connective tissue turnover associated with tissue breakdown and subsequent regrowth during each menstrual cycle. These changes in the ovarian and uterine extracellular architecture are regulated, in part, by the matrix metalloproteinase (MMP) system. This system is comprised of both a proteolytic component, the MMPs, and associated inhibitors, and it is involved in connective tissue remodeling processes throughout the body. The current review highlights the key features of the MMP system and focuses on the changes in the MMPs and the tissue inhibitors of metalloproteinases during the dynamic remodeling that takes place in the ovary and uterus during the estrous and menstrual cycles.

Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens

Appropriate endometrial maturation is of paramount importance to achieve reproductive success. Practical and ethical considerations require that in vitro methods be available to evaluate regulation of human endometrial function. Additionally, tissue complexity requires separation of individual cell populations. This report describes an improved method for isolation of endometrial epithelial and stromal cells, using biopsy specimens as a tissue source. Separated cells were obtained using selective enzymatic digestion in conjunction with physical separation procedures. Isolated populations exhibited over 95% homogeneity, ascertained immunocytochemically. Using this system, isolated cells from normal endometrium can readily be obtained for in vitro studies. Within the defined conditions of a culture system, important areas of current concern in the endometrium such as ectopic endometrial growth and implantation can be addressed.

Developmental exposure to TCDD reduces fertility and negatively affects pregnancy outcomes across multiple generations

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a ubiquitous environmental contaminant and known endocrine disruptor. Since humans and animals are most sensitive to toxicant exposure during development, we previously developed a mouse model of in utero TCDD exposure in order to examine the impact of this toxicant on adult reproductive function. Our initial in utero toxicant-exposure study revealed a dose-dependent reduction in uterine sensitivity to progesterone; however, we did not previously explore establishment or maintenance of pregnancy. Thus, in the current study, we examined pregnancy outcomes in adult C57BL/6 mice with a history of developmental TCDD exposure. Herein we demonstrate reduced fertility and an increased incidence of premature birth (PTB) in F1 mice exposed in utero to TCDD as well as in three subsequent generations. Finally, our studies revealed that mice with a history of developmental TCDD exposure exhibit an increased sensitivity to inflammation which further negatively impacted gestation length in all generations examined.

Paracrine Regulation of Matrix Metalloproteinase Expression in the Normal Human Endometrium

Endometrial expression of matrix metalloproteinase (MMP)-3, MMP-7 and MMP-11 occurs during menstrual breakdown and subsequent estrogen-mediated growth, but not during the secretory phase. These enzymes are suppressed by progesterone treatment. Paracrine factors, including transforming growth factor-β (TGF-β) and retinoic acid, are also critical for MMP regulation in the endometrium. In contrast, inflammatory cytokines such as interleukin-1α may block or interfere with steroid-mediated MMP regulation at ectopic sites of growth. Using in vitro models, our laboratory has investigated the complex interactions between progesterone and locally produced cytokines that may affect MMP expression during the development of endometriosis. Our results indicate that targeting the regulation of MMPs may represent an appropriate therapeutic strategy for the treatment of endometriosis.

Progesterone and transforming growth factor-β coordinately regulate suppression of endometrial matrix metalloproteinases in a model of experimental endometriosis

Endometriosis is a benign, though aggressive, disease of the female reproductive tract that consists of endometrial stromal and epithelial cells growing at an extrauterine site. Although it is widely accepted that the majority of cases of endometriosis result from the ectopic implantation of refluxed menstrual tissue, the precise mechanisms by which this disease becomes established are not well understood. Matrix metalloproteinases (MMPs), enzymes which are important for extracellular matrix turnover, have recently been implicated in the development of endometriosis. MMPs appear to be overexpressed in endometriotic lesions, but expression levels decrease following successful medical therapy. Intriguingly, although transforming growth factor-beta (TGF-beta) mediates progesterone suppression of specific endometrial MMPs, this growth factor is overexpressed in women with endometriosis. In the current study, we used an established experimental model of endometriosis to explore MMP regulation by TGF-beta. Our findings indicate that blocking the action of TGF-beta opposes progesterone-mediated suppression of MMPs and blocks the ability of this steroid to prevent experimental endometriosis. However, we also show that the action of TGF-beta does not lead to a sustained suppression of MMPs as observed following progesterone treatment. Taken together, our data suggest that in the absence of a normal progesterone response, common in ectopic lesions of endometriosis, sensitivity to TGF-beta may be altered, resulting in a failure to regulate MMPs.

Immune interactions in endometriosis

Endometriosis is a common, complex gynecologic disorder characterized by the presence of endometrial glands and stroma at extrauterine (ectopic) sites. In women who develop this disease, alterations in specific biological processes involving both the endocrine and immune systems have been observed, which may explain the survival and growth of displaced endometrial tissue in affected women. In the past decade, a considerable amount of research has implicated a role for alterations in progesterone action at both eutopic and ectopic sites of endometrial growth which may contribute to the excessive inflammation associated with progression of endometriosis; however, it remains unclear whether these anomalies induce the condition or are simply a consequence of the disease process. In this article, we summarize current knowledge of alterations within the immune system of endometriosis patients and discuss how endometrial cells from women with this disease not only have the capacity to escape immunosurveillance, but also use inflammatory mechanisms to promote their growth within the peritoneal cavity. Finally, we discuss evidence that exposure to an environmental endocrine disruptor, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, can mediate the development of an endometrial phenotype that exhibits both reduced progesterone responsiveness and hypersensitivity to proinflammatory stimuli mimicking the endometriosis phenotype. Future studies in women with endometriosis should consider whether a heightened inflammatory response within the peritoneal microenvironment contributes to the development and persistence of this disease.

Simvastatin protects against the development of endometriosis in a nude mouse model.

CONTEXT Endometriosis is a common condition associated with infertility and pelvic pain in women. Recent in vitro studies have shown that statins decrease proliferation of endometrial stroma (ES) and inhibit angiogenesis. OBJECTIVE The aim was to evaluate effects of simvastatin on development of endometriosis in a nude mouse model. METHODS Proliferative phase human endometrial biopsies were obtained from healthy donors and established as organ cultures or used to isolate ES cells. To establish endometriosis in the nude mouse, endometrial tissues were maintained in 1 nm estradiol (E) for 24 h and subsequently injected into ovariectomized nude mice. Mice (n = 37) were treated with E (8 mg, SILASTIC capsule implants; made in author laboratory) alone or with E plus simvastatin (5 or 25 mg/kg x d) for 10 d beginning 1 d after tissue injection (from three donors). Mice were killed and examined for disease. Effects of simvastatin on matrix metalloproteinase-3 (MMP-3) were evaluated in cultures of ES cells. PRIMARY OUTCOME The number and size of endometriotic implants were measured. RESULTS Simvastatin induced a dose-dependent decrease of the number and size of endometrial implants in mice. At the highest dose of simvastatin, the number of endometrial implants decreased by 87%, and the volume by 98%. Simvastatin also induced a concentration-dependent decrease in MMP-3 in the absence and presence of inflammatory challenge (using IL-1alpha). CONCLUSIONS Simvastatin exerted a potent inhibitory effect on the development of endometriosis in the nude mouse. Mechanisms of action of simvastatin may include inhibition of MMP-3. The present findings may lead to the development of novel treatments of endometriosis involving statins.