Utility of Optical Density of Picrosirius Red Birefringence for Analysis of Cross-Linked Collagen in Remodeling of the Peripartum Cervix for Parturition.

We report on development of a rapid, quantitative analysis technique of collagen fibers in cross-linked structures to assess remodeling of the cervix during the transition from soft to ripening in preparation for birth. Optical density analysis of picrosirius red stain tissue using circular polarized birefringence light from fixed paraffin-embedded or frozen cervix from pregnant mice during phases of remodeling prior to birth. Data were analyzed using NIH Image J and extended recently to include studies of prepartum cervix in peripartum women. Our results, developed a rapid, consistent, technique to quantify cervical organization. This approach assesses the structure of collagen organization (the principle component of the cervix) and is essential for analysis of experimental outcomes that disrupt cervical morphology in rodent models of preterm birth. The technique, in this report has, for the first time permitted rapid, accurate assessment of the stages that define cervical ripening with large numbers of slides from individual animals. The approach integrates analysis of collagen organization, with distensability and inflammation, processes associated with cervical change before birth. This analysis further holds promise to evaluate other tissues, but also fibrolytic and fibrogenic changes in collagen associated with physiological or pathophysiological conditions.

Progesterone Receptor-Mediated Actions Regulate Remodeling of the Cervix in Preparation for Preterm Parturition.

This study determined whether a progesterone (P) receptor (PR)-mediated mechanism regulates morphological characteristics associated with prepartum cervix remodeling at term and with preterm birth. With focus on the transition from a soft to ripe cervix, the cervix stroma of untreated controls had reduced cell nuclei density/area and less organized extracellular collagen, while the density of macrophages/area, but not neutrophils, increased just 2 days before birth (day 17 vs day 15 or 16.5 postbreeding). Preterm birth was induced within 24 hours of treatment on day 16 postbreeding with PR antagonist or ovariectomy (Ovx). Pure or mixed PR antagonists increased the density of macrophages in the cervix within 8 hours (day 16.5 postbreeding), in advance of preterm birth. However, neither PR antagonists nor P withdrawal after Ovx affected the densities of cell nuclei and neutrophils or extracellular collagen compared to the same day controls-an indication that the cervix was sufficiently remodeled for birth to occur. To block the effect of systemic P withdrawal, Ovx pregnant mice were given a PR agonist, either pure or mixed. These treatments forestalled preterm birth and prevented further morphological remodeling of the cervix. The resulting increase in macrophage density in cervix stroma following Ovx was only blocked by a pure PR agonist. These findings support the hypothesis that inflammatory processes in the prepartum cervix that include residency of macrophages, cellular hypertrophy, and extracellular collagen structure are regulated by genomic actions of PR in a final common mechanism both at term and with induced preterm birth.

Loss of progesterone receptor-mediated actions induce preterm cellular and structural remodeling of the cervix and premature birth.

A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term.

Transection of the pelvic or vagus nerve forestalls ripening of the cervix and delays birth in rats.

Innervation of the cervix is important for normal timing of birth because transection of the pelvic nerve forestalls birth and causes dystocia. To discover whether transection of the parasympathetic innervation of the cervix affects cervical ripening in the process of parturition was the objective of the present study. Rats on Day 16 of pregnancy had the pelvic nerve (PnX) or the vagus nerve (VnX) or both pathways (PnX+VnX) transected, sham-operated (Sham) or nonpregnant rats served as controls. Sections of fixed peripartum cervix were stained for collagen or processed by immunohistochemistry to identify macrophages and nerve fibers. All Sham controls delivered by the morning of Day 22 postbreeding, while births were delayed in more than 75% of neurectomized rats by more than 12 h. Dystocia was evident in more than 25% of the PnX and PnX+VnX rats. Moreover, on prepartum Day 21, serum progesterone was increased severalfold in neurectomized versus Sham rats. Assessments of cell nuclei counts indicated that the cervix of neurectomized rats and Sham controls had become equally hypertrophied compared to the unripe cervix in nonpregnant rats. Collagen content and structure were reduced in the cervix of all pregnant rats, whether neurectomized or Shams, versus that in nonpregnant rats. Stereological analysis of cervix sections found reduced numbers of resident macrophages in prepartum PnX and PnX+VnX rats on Day 21 postbreeding, as well as in VnX rats on Day 22 postbreeding compared to that in Sham controls. Finally, nerve transections blocked the prepartum increase in innervation that occurred in Sham rats on Day 21 postbreeding. These findings indicate that parasympathetic innervation of the cervix mediates local inflammatory processes, withdrawal of progesterone in circulation, and the normal timing of birth. Therefore, pelvic and vagal nerves regulate macrophage immigration and nerve fiber density but may not be involved in final remodeling of the extracellular matrix in the prepartum cervix. These findings support the contention that immigration of immune cells and enhanced innervation are involved in processes that remodel the cervix and time parturition.

Pregnancy-related changes in connections from the cervix to forebrain and hypothalamus in mice.

The transneuronal tracer pseudorabies virus was used to test the hypothesis that connections from the cervix to the forebrain and hypothalamus are maintained with pregnancy. The virus was injected into the cervix of nonpregnant or pregnant mice, and, after 5 days, virus-labeled cells and fibers were found in specific forebrain regions and, most prominently, in portions of the hypothalamic paraventricular nucleus. With pregnancy, fewer neurons and fibers were evident in most brain regions compared to that in nonpregnant mice. In particular, little or no virus was found in the medial and ventral parvocellular subdivisions, anteroventral periventricular nucleus, or motor cortex in pregnant mice. By contrast, labeling of virus was sustained in the dorsal hypothalamus and suprachiasmatic nucleus in all groups. Based upon image analysis of digitized photomicrographs, the area with label in the rostral and medial parvocellular paraventricular nucleus and magnocellular subdivisions was significantly reduced in mice whose cervix was injected with virus during pregnancy than in nonpregnant mice. The findings indicate that connections from the cervix to brain regions that are involved in sensory input and integrative autonomic functions are reduced during pregnancy. The findings raise the possibility that remaining pathways from the cervix to the forebrain and hypothalamus may be important for control of pituitary neuroendocrine secretion, as well as for effector functions in the cervix as pregnancy nears term.

Retrograde tracing of spinal cord connections to the cervix with pregnancy in mice.

In contrast to the uterus, the cervix is well innervated during pregnancy and the density of nerve fibers increases before birth. To assess neural connections between the cervix and the spinal cord, the cervix of pregnant mice was injected with the trans-synaptic retrograde neural tract tracer pseudorabies virus (PRV). After 5 days, the virus was present in nerve cells and fibers in specific areas of the sensory, autonomic, and motor subdivisions of the thoracolumbar spinal cord. In nonpregnant controls, the virus was predominantly distributed in laminae I-III in the dorsal gray sensory areas with the heaviest label in the substantia gelatinosa compared with the autonomic or motor areas. Labeled cells and processes were sparse in other regions, except for a prominent cluster in the intermediolateral column (lamina VII). Photomicrographs of spinal cord sections were digitized, and the total area with the virus was estimated. Compared with nonpregnant controls, the area with PRV was significantly decreased in all the spinal cord subdivisions in pregnant mice except in the intermediolateral column. However, areas with the virus were equivalent in mice injected with PRV at 4 days or 1 day before birth. These findings suggest that the predominant innervation of the murine cervix is from the sensory regions of the thoracolumbar spinal cord, and that these connections diminish with pregnancy. The results raise the possibility that the remaining connections from sensory and autonomic subdivisions, particularly the intermediolateral column, of the thoracolumbar spinal cord may be important for increased density of nerve fibers in the cervix as pregnancy nears term.

Progesterone withdrawal promotes remodeling processes in the nonpregnant mouse cervix.

Prepartum cervical ripening is associated with remodeling of collagen structure and with inflammation. Progesterone withdrawal is critical for parturition, but the effects of progesterone decline on cervical morphology are unknown. The present study tested the hypothesis that progesterone withdrawal promotes processes associated with remodeling of the cervix. Adult, virgin, female C57BL/6 mice received silastic capsules with oil vehicle or estradiol plus progesterone to parallel concentrations in circulation during pregnancy. After 17 days of estradiol and progesterone treatment, the progesterone implant was removed from one group. Mice in each group were killed 15, 18, or 19 days after placement of capsules. Sections of cervix were stained for collagen, and the densities of macrophages, neutrophils, and area with nerve fibers were assessed. Treatment with gonadal steroids promoted hypertrophy of the cervix, as well as reduced collagen and increased area with nerve fibers compared with vehicle-treated controls. Removal of the progesterone capsule did not affect hypertrophy or innervation, but it did reduce collagen. By contrast, significantly more macrophages and neutrophils were present in the cervix on Days 18 and 19 (i.e., by 24 and 48 h after withdrawal of the progesterone capsule); the immune cell census was equivalent to that in vehicle controls. Findings indicate that gonadal steroids, comparable to those during pregnancy, promote hypertrophy and suppress immigration of immune cells in the cervix. Therefore, in a nonpregnant murine model for parturition, progesterone withdrawal is suggested to recruit immune cells and processes that remodel the cervix.

Cervix remodeling and parturition in the rat: lack of a role for hypogastric innervation.

The hypogastric nerve is a major pathway innervating the uterine cervix, yet its contribution to the processes of cervical ripening and parturition is not known. The main objective of this study was to determine the effect of hypogastric nerve transection on remodeling of the cervix and timing of birth. As an initial goal, processes associated with remodeling of the peripartum cervix were studied. The cervix was obtained from time-dated pregnant rats on days 15, 19, 21, and 21.5 of pregnancy, and post partum on the day of birth (day 22). The cervix was excised, post-fixed overnight, and sections stained to evaluate collagen content and structure or processed by immunohistochemistry to identify macrophages or nerve fibers. The census of macrophages and density of nerve fibers in the cervix peaked on day 21, the day before birth, and then declined post partum. These results replicate in time course and magnitude previous studies in mice. To address the main objective, the hypogastric nerve was bilaterally transected on day 15 post-breeding; sham-operated rats served as controls. Pups were born in both groups at normal term. Transection of the hypogastric nerves did not affect remodeling of collagen or the census of macrophages or the density of nerve fibers in the cervix. These findings support the contention that enhanced innervation and immigration of immune cells are associated with remodeling of the cervix and parturition, but that a neural pathway other than the hypogastric nerve may participate in the process of cervical ripening.

Increased innervation and ripening of the prepartum murine cervix.

OBJECTIVE: Ripening of the cervix before birth is coincident with reduced collagen content and leukocyte immigration, characteristics that are analogous to a neurogenic inflammatory-like process. We sought to assess the morphologic relationship between innervation and remodeling of the peripartum cervix. METHODS: Cervix was obtained from C3H/HeN mice on days 15 and 18 of pregnancy, 1 day postpartum, and from non-pregnant controls. Tissues were immersion-fixed, paraffin-embedded, and some sections stained with Picrosirius red to assess collagen content and complexity of organization. By image analysis of optical density, collagen content and structure were significantly decreased by the day before birth. Other sections were processed to visualize nerve fibers by immunohistochemistry with antibodies against neuron-specific epitopes, PGP9.5, peripherin, as well as brain nitric oxide synthase (bNOS), calcitonin gene-related peptide (CGRP), and other neuropeptides. Fiber density was assessed stereologically and normalized to cell density in non-pregnant cervix to correct for tissue hypertrophy due to reproductive status. RESULTS: In groups of non-pregnant, day 15 pregnant, and postpartum mice, cervix contained nerve fibers that were immunoreactive for the pan-neural markers PGP9.5 and peripherin. Punctate and beaded varicosities were sparsely distributed in stroma, subepithelium, and in proximity to vascular structures. By day 18 of pregnancy, 1 day before birth, fiber density was increased fourfold or more compared to other groups. bNOS fibers and, to a lesser extent, CGRP accounted for most of the increased innervation of the murine cervix by the day before birth, a period when macrophage numbers are enhanced. CONCLUSIONS: The findings suggest that increased bNOS and CGRP innervation contribute to early inflammatory-like processes that ripen the cervix before birth.