The cervix serves as a protective barrier from invading microorganisms and as a structural barrier to delivery of the fetus. Among all biological processes, the phenomenal connective tissue remodeling that occurs in the cervix during and after parturition is unparalleled in scope and magnitude. The process of connective tissue remodeling in the cervix during pregnancy occurs in four stages: softening, ripening, dilation, and repair. Although overlapping in time, each stage is uniquely regulated. Results from studies using serial measurements of cervical length indicate that cervical ripening precedes myometrial contractions of labor by several weeks, suggesting that parturition in women is a process of long duration and that uterine contractions of labor are late events in the parturition process. A clear understanding of the biologic mechanisms that regulate cervical remodeling during pregnancy is needed to influence the preterm birth rate and to develop strategies to prevent preterm dilation of the cervix.

AECs secrete soluble factors that inhibit cells in both the innate and adaptive immune systems.

Cervical remodeling during pregnancy and parturition is a single progressive process that can be loosely divided into four overlapping phases termed softening, ripening, dilation/labor, and post partum repair. Elucidating the molecular mechanisms that facilitate all phases of cervical remodeling is critical for an understanding of parturition and for identifying processes that are misregulated in preterm labor, a significant cause of perinatal morbidity. In the present study, biomechanical measurements indicate that softening was initiated between gestation days 10 and 12 of mouse pregnancy, and in contrast to cervical ripening on day 18, the softened cervix maintains tissue strength. Although preceded by increased collagen solubility, cervical softening is not characterized by significant increases in cell proliferation, tissue hydration or changes in the distribution of inflammatory cells. Gene expression studies reveal a potentially important role of cervical epithelia during softening and ripening in maintenance of an immunomucosal barrier that protects the stromal compartment during matrix remodeling. Expression of two genes involved in repair and protection of the epithelial permeability barrier in the gut (trefoil factor 1) and skin (serine protease inhibitor Kazal type 5) were increased during softening and/or ripening. Another gene whose function remains to be elucidated, purkinje cell protein 4, declines in expression as remodeling progressed. Collectively, these results indicate that cervical softening during pregnancy is a unique phase of the tissue remodeling process characterized by increased collagen solubility, maintenance of tissue strength, and upregulation of genes involved in mucosal protection.

Aberrant up-regulation of aromatase in eutopic endometrium and implants from women with endometriosis has been reported. Aromatase induction may be mediated by increased cyclooxygenase-2 (COX-2). Recently, we demonstrated that progesterone receptor (PR)-A and PR-B serve an antiinflammatory role in the uterus by antagonizing nuclear factor kappaB activation and COX-2 expression. PR-C, which antagonizes PR-B, is up-regulated by inflammation. Although estrogen receptor alpha (ERalpha) is implicated in endometriosis, an antiinflammatory role of ERbeta has been suggested. We examined stage-specific expression of aromatase, COX-2, ER, and PR isoform expression in eutopic endometrium, implants, peritoneum, and endometrioma samples from endometriosis patients. Endometrial and peritoneal biopsies were obtained from unaffected women and those with fibroids. Aromatase expression in eutopic endometrium from endometriosis patients was significantly increased compared with controls. Aromatase expression in endometriosis implants was markedly increased compared with eutopic endometrium. Aromatase mRNA levels were increased significantly in red implants relative to black implants and endometrioma cyst capsule. Moreover, COX-2 expression was increased in implants and in eutopic endometrium of women with endometriosis as compared with control endometrium. As observed for aromatase mRNA, the highest levels of COX-2 mRNA were found in red implants. The ratio of ERbeta/ERalpha mRNA was significantly elevated in endometriomas compared with endometriosis implants and eutopic endometrium. Expression of PR-C mRNA relative to PR-A and PR-B mRNA was significantly increased in endometriomas compared with eutopic and control endometrium. PR-A protein was barely detectable in endometriomas. Thus, whereas PR-C may enhance disease progression, up-regulation of ERbeta may play an antiinflammatory and opposing role.

Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, fibulin-5 inhibited the β 1 integrin-dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5 RGE/RGE ) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to fibulin-5 knockouts (Fbln5 -/-), Fbln5 RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with β-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5 -/-mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre-and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women. IntroductionPelvic organ prolapse (POP) is characterized by abnormal protrusion of female pelvic organs involving the uterus, bladder, and vagina (reviewed in refs. 1-3). Epidemiologic studies indicate that (a) vaginal birth, aging, and increased body mass index are major risk factors for the development of POP and (b) more than 1 pathology may be involved to exhibit full anatomical loss of support. Although approximately 11% of women have surgery for POP or urinary incontinence in their lifetimes (4), to date, effective therapies to prevent progression of POP have not been established, thereby imposing profound social and financial burden to affected individuals (3).Despite a difference in anatomical position of pelvic organs relative to the body axis and pelvic floor, rodent models of POP have provided important tools to study underlying mechanisms of prolapse. In contrast to an observation that rectal prolapse is frequently associated with the presence of chronic inflammatory bowel disease (5), POP has been found in animals with defective ECM proteins, including fibulin-3, fibulin-5, and lysyl oxidaselike-1 (LOXL-1) (an enzyme that predominantly catalyzes crosslinking of elastin) (6-8). Interestingly, these proteins are abundantly expressed in the vaginal wall and involved in synthesis and assembly of elastic fibers. It is also known that uterosacral ligaments support the vaginal wall and that Hoxa11 is essential for formation of uterosacral ligaments in mice (9). Hoxa11-deficient mice exhibit increased mobility of the uterus; however,

Several strategies have been described for the primary culture of human myometrial cells. However, primary cultures of myometrial cells have a limited life span, making continual tissue acquisition and cell isolation necessary. Recent studies have demonstrated that cell culture life span is related to chromosomal telomere length, and cellular senescence results from progressive telomere shortening and the lack of telomerase expression. Transfection of cells with expression vectors containing the human telomerase reverse transcriptase (hTERT) maintains telomere length and effectively gives normal cells an unlimited life span in culture. In addition, hTERT extends the life span of cultured cells far beyond normal senescence without causing neoplastic transformation. In the present study, we developed a cell line from hTERT-infected myometrial cells (hTERT-HM). Cells were isolated from myometrial tissue obtained from women undergoing hysterectomy, and retroviral infection was used to express the catalytic subunit of telomerase in myometrial cells. Cells expressing hTERT have been in continuous culture for >10 mo, whereas the control culture senesced after approximately 2 mo. Telomerase activity was monitored in cells with a polymerase chain reaction-based telomerase activity assay. Telomerase-expressing cells contained mRNA for alpha smooth muscle actin, smoothelin, oxytocin receptor, and estrogen receptor alpha, but the estrogen receptor beta receptor was lost. Immunoblotting analysis identified the expression of calponin, caldesmon, alpha smooth muscle actin, and oxytocin receptor. Although estrogen receptor expression was below the level of detection with immunoblotting, transfection experiments performed with reporter constructs driven by estrogen response elements demonstrated estrogen responsiveness in the hTERT-HM. In addition, treatment of hTERT-HM with oxytocin caused a concentration-dependent increase in intracellular calcium levels, confirming the presence of functional oxytocin receptors. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in primary cultures of smooth muscle cells. The expression of various smooth muscle/myometrium cell markers suggests that these cells may be an appropriate model system to study certain aspects of human myometrial function.

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Identification of temporal and spatial changes in myometrial gene expression during parturition may further the understanding of the coordinated regulation of myometrial contractions during parturition. The objective of this study was to compare the gene expression profiles of human fundal myometrium from pregnant women before and after the onset of labor using a functional genomics approach, and to further characterize the spatial and temporal expression patterns of three genes believed to be important in parturition. Fundal myometrial mRNA was isolated from five women in labor and five women not in labor, and analyzed using human UniGEM-V microarrays with 9182 cDNA elements. Real-time polymerase chain reaction using myometrial RNA from pregnant women in labor or not in labor was used to examine mRNA levels for three of the genes; namely, prostaglandin-endoperoxide synthase 2 (PTGS2), calgranulin B (S100A9), and oxytocin receptor (OXTR). The spatial expression pattern of these genes throughout the pregnant uterus before and after labor was also determined. Immunolocalization of cyclooxygenase-2 (also known as PTGS2) and S100A9 within the uterine cervix and myometrium were analyzed by immunohistochemistry. Few genes were differentially expressed in fundal myometrial tissues at term with the onset of labor. However, there appears to be a subset of genes important in the parturition cascade. The cellular properties of S100A9, its spatial localization, and dramatic increase in cervix and myometrium of women in labor suggest that this protein may be very important in the initiation or propagation of human labor.