OBJECTIVE-To validate a rat model of endometriosis using cDNA microarrays by identifying common gene expression patterns beween experimental and natural disease. DESIGN-Autotransplantation rat model. SETTING-Medical school department. ANIMALS-Female Sprague-Dawley rats.INTERVENTIONS-Endometriosis was surgically-induced by suturing uterine horn implants next to the small intestine's mesentery. Control rats received sutures with no implants. After 60 days, endometriotic implants and uterine horn were obtained. MAIN OUTCOME MEASURES-Gene expression levels determined by cDNA microarrays and QRT-PCR.METHODS-Cy5-labeled cDNA was synthesized from total RNA obtained from endometriotic implants. Cy3-labeled cDNA was synthesized using uterine RNA from a control rat. Gene expression levels were analyzed after hybridizing experimental and control labeled cDNA to PIQOR ™ Toxicology Rat Microarrays (Miltenyi Biotec) containing 1,252 known genes. Cy5/Cy3 ratios were determined and genes with >2-fold higher or <0.5-fold lower expression levels were selected. Microarray results were validated by QRT-PCR.RESULTS-We observed differential expression of genes previously shown to be upregulated in patients, including growth factors, inflammatory cytokines/receptors, tumor invasion/metastasis factors, adhesion molecules, and anti-apoptotic factors.CONCLUSIONS-This study presents evidence in support of using this rat model to study the natural history of endometriosis and test novel therapeutics for this incurable disease.

Endometriosis is associated with activation of local and systemic inflammatory mechanisms, including increased levels of chemokines and other proinflammatory cytokines. We have previously reported increased gene expression of chemokine receptor 4 (CXCR4), the receptor for CXCL12, in lesions of the rat model of endometriosis. The CXCR4-CXCL12 axis has been shown to have both immune (HIV infection, lymphocyte chemotaxis) and nonimmune functions, including roles in tissue repair, angiogenesis, invasion, and migration. There is evidence indicating that these mechanisms are also at play in endometriosis; therefore, we hypothesized that activation of the CXCR4-CXCL12 axis could be responsible, at least in part, for the survival and establishment of endometrial cells ectopically. Immunohistochemistry (IHC) showed that CXCR4 protein levels were significantly higher in endometriotic lesions compared to the endometrium of controls. Next, we determined basal gene and protein expression of CXCR4 and CXCL12 and regulation by estradiol (E2) and/or progesterone (P4) in endometrial cell lines using quantitative polymerase chain reaction (qPCR), and Western blots. Basal CXCR4 gene expression levels were higher in epithelial versus stromal cells; conversely, CXCL12 was expressed at higher levels in stromal vs epithelial cells. CXCR4 gene expression was significantly downregulated by ovarian steroid hormones in endometrial epithelial. These data suggest that steroid modulation of CXCR4 is defective in endometriosis, although the specific mechanism involved remains to be elucidated. These findings have implications for future therapeutic strategies specifically targeting the inflammatory component in endometriosis.

This study documents that expression of LOX is differentially regulated in endometriotic lesions and endometrium. A role for LOX in mediating proliferation, migration, and invasion of endometrial and endometriotic cells was observed, which may be implicated in the establishment and progression of endometriotic lesions.

High levels of inflammatory factors including chemokines have been reported in peritoneal fluid and blood of women with endometriosis. CXCL12 mediates its action by interaction with its specific receptor, CXCR4, reported to be elevated in human endometriosis lesions and in the rat model of endometriosis. Activation of the CXCR4-CXCL12 axis increases cell proliferation, migration, and invasion of cancer cells. To obtain insights into the CXCR4 expression profile in lesions and endometrium, as well as functionality of the CXCR4-CXCL12 axis in endometriosis, we analyzed the expression of CXCR4 in tissues on a human tissue array and studied CXCL12-mediated activation of proliferation, invasion, and migration in vitro. We observed differences in levels of nuclear CXCR4 expression among lesion types, being higher in ovarian lesions. Endometriotic cell lines (12Z) showed higher levels of CXCR4, proliferative and migratory potential, and AKT phosphorylation/kinase activity compared to untreated control cells (endometrial epithelial cells). CXCL12 and endometriotic stromal cell-enriched media increased proliferation of non-endometriotic epithelial cells. CXCL12 caused a significant increase in 12Z cell invasion but had no effect on migration; AMD3100, a CXCR4-specific inhibitor, significantly increased invasion of 12Z cells but decreased their migration. However, treatment with CXCL12 plus AMD3100 significantly decreased invasion and migration of 12Z cells. In conclusion, the CXCR4-CXCL12 axis is functional in endometriosis cells, but the expression of CXCR4 varies among lesions. CXCL12 promoted proliferation, migration, and invasion of endometriotic cells, while inducing AKT phosphorylation and activity, but pharmacologically blocking this axis in the absence of the ligand induced their invasiveness.

This study was conducted to assess genetic associations to endometriosis in a Puerto Rican population. Significant differences in allelic frequencies and genotype distribution of genetic variants in LOXL4 and C3 were documented in patients with endometriosis-associated infertility vs controls, and in patients with endometriosis vs controls, respectively, and in women having the risk genotype at both SNPs, the estimated risk for endometriosis nearly doubled.