Steroid Hormone Action In Female Reproduction

Child Health And Human Development

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Abstract

This project focuses on leiomyomata (fibroids) and endometriosis and examines factors related to their pathogenesis, diagnosis and/or potential treatment. We have hypothesized that gene expression is different in fibroids and myometrium, and these differences may suggest pathophysiologic etiologies. We have evaluated samples obtained from women undergoing routine hysterectomy for fibroids using the 33,000 gene Affymetrix array and subsequent validation studies evaluating messenger RNA and protein. Contrary to expectation, genes involved in estrogen action were not differentially expressed between leiomyoma and normal myometrium. Genes encoding extracellular-matrix proteins were prominently featured, suggesting their involvement in formation of a myofibroblast phenotype. Analysis of the extracellular matrix in the leiomyomas revealed a disordered collagen fibril orientation. Expression of the collagen-binding protein dermatopontin was found to be consistently decreased in leiomyoma by both reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR (mean underexpression = 9.41-fold) regardless of leiomyoma size, leiomyoma location, patient race, and patient age. This expression pattern was observed in 11 subjects and a total of 23 leiomyoma:myometrium pairs. Decreased expression of dermatopontin was also associated with keloid formation, a fibrotic disease that shares epidemiologic similarities with leiomyoma. Immunohistochemical studies of leiomyomas and keloids demonstrated reduced levels of dermatopontin in both tissues. In addition, ultrastructural analysis revealed that the orientation of the collagen fibrils in the keloid tissues strongly resembled that in the leiomyomas. Reduction in dermatopontin was associated with an increase in transforming growth factor-beta3 mRNA levels in leiomyomas, whereas other genes involved in dermatopontin signaling were not differentially expressed. These findings suggest that leiomyoma development involves a myofibroblast cell phenotype characterized by dysregulation of genes encoding extracellular-matrix proteins. In particular, decreased expression of dermatopontin represents a molecular link between the leiomyoma and keloid phenotypes. Ongoing work seeks to evaluate the utility of the progesterone response modulator CDB2914 as a treatment of fibroids. An ongoing randomized, double-blind, placebo-controlled phase II study evaluates whether surgical excision of endometriosis followed by daily administration of raloxifene for six months reduces pain for a longer time than surgery alone. In women participating in this study, we evaluated the utility of histologic diagnosis of endometriosis by adjunctive use of CD10 immunohistochemistry in conjunction with H&E-stained specimens. CD10 was consistently present in endometrial stroma. Of the 70 specimens judged negative initially by H&E staining, CD10 staining led to the diagnosis of endometriosis in 11. The addition of CD10 immunohistochemistry detected more positive endometriosis lesions than H&E staining alone (45% vs. 35%). In three women with minimal endometriosis at surgery but initially negative histopathology, CD10 immunohistochemistry changed the histologic diagnosis to endometriosis. Thus, the adjunctive use of CD10 immunohistochemistry improved diagnostic sensitivity for endometriosis, especially for women with minimal disease. We also evaluated the utility of urine VEGF for the diagnosis of endometriosis. Urine VEGF levels corrected for creatinine excretion were similar in women with (83.6 +/- 11.3 pg/mg Cr) and without (88.5 +/- 10.4 pg/mg Cr) endometriosis (P =.77). No significant difference was noted in urine VEGF levels when comparing endometriosis stages or in those with endometriomas compared to controls. Urine VEGF did not vary significantly over the menstrual cycle or between groups by cycle phase. No cutoff point discriminated individuals with and without the condition. Thus, it is unlikely that urine VEGF-A(121, 165), as measured in this study, will be a useful non-invasive marker for endometriosis.