Temporal changes in myeloid cells in the cervix during pregnancy and parturition.

Preterm birth occurs at a rate of 12.7% in the U.S. and is the primary cause of fetal morbidity in the first year of life as well as the cause of later health problems. Elucidation of mechanisms controlling cervical remodeling is critical for development of therapies to reduce the incidence of prematurity. The cervical extracellular matrix must be disorganized during labor to allow birth, followed by a rapid repair postpartum. Leukocytes infiltrate the cervix before and after birth and are proposed to regulate matrix remodeling during cervical ripening via release of proteolytic enzymes. In the current study, flow cytometry and cell sorting were used to determine the role of immune cells in cervical matrix remodeling before, during, and after parturition. Markers of myeloid cell differentiation and activation were assessed to define phenotype and function. Tissue monocytes and eosinophils increased in the cervix before birth in a progesterone-regulated fashion, whereas macrophage numbers were unchanged. Neutrophils increased in the postpartum period. Increased mRNA expression of Csfr1 and markers of alternatively activated M2 macrophages during labor or shortly postpartum suggest a function of M2 macrophages in postpartum tissue repair. Changes in cervical myeloid cell numbers are not reflected in the peripheral blood. These data along with our previous studies suggest that myeloid-derived cells do not orchestrate processes required for initiation of cervical ripening before birth. Additionally, macrophages with diverse phenotypes (M1 and M2) are present in the cervix and are most likely involved in the postpartum repair of tissue.

Cervical remodeling during pregnancy and parturition: molecular characterization of the softening phase in mice.

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.

Dynamic changes in the cervical epithelial tight junction complex and differentiation occur during cervical ripening and parturition.

Cervical epithelia have numerous functions that include proliferation, differentiation, maintenance of fluid balance, protection from environmental hazards, and paracellular transport of solutes via tight junctions (TJs). Epithelial functions must be tightly regulated during pregnancy and parturition as the cervix undergoes extensive growth and remodeling. This study evaluated TJ proteins, as well as markers of epithelial cell differentiation in normal and cervical ripening defective mice to gain insights into how the permeability barrier is regulated during pregnancy and parturition. Although numerous TJ proteins are expressed in the nonpregnant cervix, claudins 1 and 2 are temporally regulated in pregnancy. Claudin 1 mRNA expression is increased, whereas claudin 2 expression declines. The cellular localization of claudin 1 shifts at the end of pregnancy (gestation d 18.75) to the plasma membrane in a lattice pattern, consistent with TJs in the apical cells. The timing of claudin 1-enriched TJs coincides with initiation of terminal differentiation of cervical squamous epithelia as evidenced by the increased expression of genes by differentiated epithelia late on gestation d 18. The cervical ripening defective steroid 5alpha-reductase type 1 deficient mouse, which has an elevated local progesterone concentration, also has aberrant claudin 1 and 2 expressions, fails to form claudin 1-enriched TJs, and lacks normal expression of genes involved in epithelial terminal differentiation. These data suggest that changes in permeability barrier properties during cervical ripening are, in part, negatively regulated by progesterone, and that dynamic changes in barrier properties of the cervix occur during pregnancy and parturition.

Utilization of different aquaporin water channels in the mouse cervix during pregnancy and parturition and in models of preterm and delayed cervical ripening.

Biochemical changes of cervical connective tissue, including progressive disorganization of the collagen network and increased water content, occur during gestation to allow for cervical dilatation during labor, but the mechanisms that regulate cervical fluid balance are not fully understood. We examined whether aquaporins (AQPs), a family of membrane channel proteins that facilitate water transport, help mediate fluid balance in the mouse cervix during parturition. Of the 13 known murine AQPs, AQP0-2, 6, 7, 9, 11, and 12 were absent or at the limits of detection. By Northern blot and real-time PCR, AQP3 expression was low in nongravid and mid-pregnancy cervices with peak expression on d 19 and postpartum d 1 (PP1). AQP4 expression was generally low throughout pregnancy but showed a small upward trend at the time of parturition. AQP5 and AQP8 expression were significantly increased on d 12-15 but fell to nongravid/baseline by d 19 and PP1. By in situ hybridization and immunohistochemistry, AQP3 was preferentially expressed in basal cell layers of the cervical epithelium, whereas AQP4, 5, and 8 were primarily expressed in apical cell layers. Females with LPS-induced preterm labor had similar trends in AQP4, 5, and 8 expression to mice with natural labor at term gestation. Mice with delayed cervical remodeling due to deletion of the steroid 5alpha-reductase type 1 gene showed significant reduction in the levels of AQP3, 4, and 8 on d 19 or PP1. Together, these studies suggest that AQPs 3, 4, 5, and 8 regulate distinct aspects of cervical water balance during pregnancy and parturition.

Timing of neutrophil activation and expression of proinflammatory markers do not support a role for neutrophils in cervical ripening in the mouse.

The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1-/-), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1-/- mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, and after birth. The effect of neutrophil depletion on parturition was tested. Tissue macrophages, myeloperoxidase activity, and expression of proinflammatory molecules are not increased within the cervix until after birth. Neutrophil numbers do not change after birth and neutrophil depletion before term has no effect on timing or success of parturition. These results suggest that cervical ripening does not require neutrophils. Moreover, neutrophil activation and a general inflammatory response are not initiated within the cervix until shortly after parturition. The timing of inflammatory cell migration and activation in pregnant cervix suggest a role for these cells in postpartum remodeling of the cervix rather than in the initiation of cervical ripening at parturition.

Transgene insertion on mouse chromosome 6 impairs function of the uterine cervix and causes failure of parturition.

The molecular mechanisms controlling the initiation of parturition remain largely undefined. We report a new animal model in which parturition does not occur. A line of mice expressing a human apolipoprotein B (APOB) gene fail to deliver their young if the transgene is present in homozygous (Tg/Tg), but not in heterozygous (Tg/Wt), form. Cloning and mapping of the transgene insertion locus indicate that 10 copies of the 80-kilobase APOB genomic fragment inserted into mouse chromosome 6 result in a small, 390-base pair deletion of mouse genomic DNA. Nine other lines expressing the transgene have normal labor, suggesting that transgene insertion in this mutant line disrupted a mouse gene crucial for successful parturition. The pathophysiology of parturition failure in these animals was defined using physiological, endocrinological, and morphological techniques. Results indicate that luteolysis occurs in Tg/Tg mice but is delayed by 1 day. Delivery did not occur in mutant mice at term after spontaneous luteolysis or even after removal of progesterone action by ovariectomy or antiprogestin treatment. Biomechanical and functional studies of the uterus and cervix revealed that the primary cause of failed parturition in Tg/Tg mice was not inadequate uterine contractions of labor but, rather, a rigid, inelastic cervix at term that was abnormally rich in neutrophils and tissue monocytes. Characterization of the transgene insertional mutant, Tg/Tg, indicates that progesterone withdrawal is insufficient to complete parturition in the presence of inadequate cervical ripening at term.

Regulation of hyaluronan expression during cervical ripening.

In preparation for birth, the uterine cervix undergoes a remarkable transformation from a closed, rigid structure to a distensible, remodeled configuration that stretches to allow passage of a fetus. Cervical ripening requires changes in the composition and structure of the extracellular matrix. These include an increase in the glycosaminoglycan hyaluronan (HA) prior to parturition. We show that the increase in cervical HA with advancing gestation correlates with the temporal increase in transcription of hyaluronan synthase 2 (HAS2) in the mouse. On gestation day 18, 1 day prior to birth, HAS2 transcripts are most abundant and begin to decline after birth. The steroid 5alpha-reductase type 1 deficient mouse, which fails to undergo cervical remodeling, has decreased expression of HAS2 mRNA and decreased tissue HA. HAS2 transcripts are expressed by cervical epithelium, and HA is localized to the matrix surrounding the stroma and to a lesser extent around the epithelium. HAS2 expression is suppressed in mice treated with progesterone. The mRNA expression levels of HA metabolizing enzymes hyaluronidase 1 and 2 were unchanged during pregnancy but increased after birth. Thus the net increase in HA content at term correlates with increased transcription of HAS2. Regulation of HA content is conserved in women because HAS2 transcripts are up-regulated in cervices of women in labor as compared to pregnant women not in labor. These results provide insights into the regulation of HA biosynthesis during cervical ripening and underscore the physiological role of HA in this essential process.