Clinically, vaginal progesterone (VP) and 17 alpha-hydroxyprogesterone caproate (17P) have been shown to prevent preterm birth (PTB) in high-risk populations. We hypothesize that treatment with these agents may prevent PTB by altering molecular pathways involved in uterine contractility or cervical remodeling.
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.
Withdrawal of progestational support for pregnancy is part of the final common pathways for parturition, but the role of nuclear progesterone receptor (PGR) isoforms in this process is not known. To determine if the PGR-B isoform participates in cervical remodeling at term, cervices were obtained from mice lacking PGR-B (PGR-BKO) and from wild-type (WT) controls before or after birth. PGR-BKO mice gave birth to viable pups at the same time as WT controls during the early morning of Day 19 postbreeding. Morphological analyses indicated that by the day before birth, cervices from PGR-BKO and WT mice had increased in size, with fewer cell nuclei/area as well as diminished collagen content and structure, as evidenced by optical density of picrosirius red-stained sections, compared to cervices from nonpregnant mice. Moreover, increased numbers of resident macrophages, but not neutrophils, were found in the prepartum cervix of PGR-BKO compared to nonpregnant mice, parallel to findings in WT mice. These results suggest that PGR-B does not contribute to the growth or degradation of the extracellular matrix or proinflammatory processes associated with recruitment of macrophages in the cervix leading up to birth. Rather, other receptors may contribute to the progesterone-dependent mechanism that promotes remodeling of the cervix during pregnancy and in the proinflammatory process associated with ripening before parturition.
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.
Using a rodent model for neuropathology induced by human amyloid precursor protein, the present study tested the hypothesis that 24 h rest/activity rhythms deteriorate with age. A lifespan of rest/activity patterns was studied in transgenic Tg2576 mice and wild-type controls. Classic indices of circadian timekeeping, including onsets, offsets, and the duration of nighttime activity, were stable throughout the 96-week study. Analyses of ultradian bout activity revealed significant genotype and age-related changes in the duration and intensity of activity bouts, as well as amplitude of the 24 h rhythm. Tg2576 mice had more total activity counts, fewer bouts/24 h, more counts/bout, and longer bout time than wild-type controls. Amyloid deposits and plaques were solely found in specific cortex regions in aged postmortem Tg2576 mice, but were not evident in the hypothalamus or suprachiasmatic nucleus; this neuropathology was absent from brains of wild-type controls. These findings suggest that amyloidosis of the Tg2576 mouse exerts little influence on timing of locomotor activity in the circadian domain but significantly alters the temporal structure of ultradian activity.
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.
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.
This study was designed to test the hypothesis that fetal adrenal nitric oxide synthase (NOS) is elevated in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for approximately the last 100 days of gestation. Between days 138 and 141 of gestation, adrenal glands were collected from LTH fetuses and age-matched normoxic controls. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western analysis were used to quantify NOS expression, and NOS distribution was examined by immunohistochemistry and double-staining immunofluorescence for endothelial NOS (eNOS) and 17alpha-hydroxylase (CYP17). Neuronal NOS (nNOS) was expressed at very low levels and with no differences between groups. Expression of eNOS was significantly greater in the LTH group compared with control. Neuronal NOS was distributed throughout the cortex while the greatest density of eNOS was observed in the zona fasciculata/reticularis area and eNOS co-localized with CYP17. We conclude that LTH enhances eNOS expression in the inner adrenal cortex which may play a role in regulation of cortisol biosynthesis in the LTH fetus.
To determine whether a progestational agent can modify inflammation-induced preterm cervical ripening, mice on day 15 of gestation were given an intrauterine injection of (1) saline, (2) lipopolysaccharide, (3) an intramuscular injection of medroxyprogesterone acetate alone prior to lipopolysaccharide, or (4) medroxyprogesterone acetate alone. Cervices were obtained 6 hours later, then fixed, sectioned, and processed to stain collagen structure or to identify immune cells or nerve fibers. Cervical remodeling was induced by lipopolysaccharide treatment compared with that in saline controls, an effect blocked by medroxyprogesterone acetate pretreatment. Moreover, lipopolysaccharide reduced macrophages and enhanced neutrophils in the cervix, effects also forestalled by medroxyprogesterone acetate pretreatment. Although the density of nerve fibers was not altered by lipopolysaccharide, medroxyprogesterone acetate reduced innervation in the cervix. Thus, progestational treatment forestalls the inflammation-induced reduction in collagen structure and immune cell traffic through a mechanism that is independent of nerve fiber density. These findings raise the possibility that progestational treatment may regulate ripening of the cervix early in the process leading to preterm birth.
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.
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.
Remodeling of the cervix is a critical early component of parturition and resembles an inflammatory process. Infiltration and activation of myeloid immune cells along with production of proinflammatory mediators and proteolytic enzymes are hypothesized to regulate cervical remodeling as pregnancy nears term. The present study standardized an approach to assess resident populations of immune cells and phenotypic markers of functional activities related to the mechanism of extracellular matrix degradation in the cervix in preparation for birth. Analysis of cells from the dispersed cervix of mice that were nonpregnant or pregnant (Days 15 and 18 postbreeding) by multicolor flow cytometry indicated increased total cell numbers with pregnancy as well as increased numbers of macrophages, the predominant myeloid cell, by Day 18, the day before birth. The number of activated macrophages involved in matrix metalloproteinase induction (CD147) and signaling for matrix adhesion (CD169) significantly increased by the day before birth. Expression of the adhesion markers CD54 and CD11b by macrophages decreased in the cervix by Day 18 versus that on Day 15 or in nonpregnant mice. The census of cells that expressed the migration marker CD62L was unaffected by pregnancy. The data suggest that remodeling of the cervix at term in mice is associated with recruitment and selective activation of macrophages that promote extracellular matrix degradation. Indices of immigration and activities by macrophages may thus serve as markers for local immune cell activity that is critical for ripening of the cervix in the final common mechanism for parturition at term.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.