The Non-pregnant and Pregnant Human Cervix: a Systematic Proteomic Analysis.

Appropriate timing of cervical remodeling (CR) is key to normal term parturition. To date, mechanisms behind normal and abnormal (premature or delayed) CR remain unclear. Recent studies show regional differences exist in human cervical tissue structure. While the entire cervix contains extracellular matrix (ECM), the internal os is highly cellular containing 50-60% cervical smooth muscle (CSM). The external os contains 10-20% CSM. Previously, we reported ECM rigidity and different ECM proteins influence CSM cell function, highlighting the importance of understanding not only how cervical cells orchestrate cervical ECM remodeling in pregnancy, but also how changes in specific ECM proteins can influence resident cellular function. To understand this dynamic process, we utilized a systematic proteomic approach to understand which soluble ECM and cellular proteins exist in the different regions of the human cervix and how the proteomic profiles change from the non-pregnant (NP) to the pregnant (PG) state. We found the human cervix proteome contains at least 4548 proteins and establish the types and relative abundance of cellular and soluble matrisome proteins found in the NP and PG human cervix. Further, we report the relative abundance of proteins involved with elastic fiber formation and ECM organization/degradation were significantly increased while proteins involved in RNA polymerase I/promoter opening, DNA methylation, senescence, immune system, and compliment activation were decreased in the PG compared to NP cervix. These findings establish an initial platform from which we can further comprehend how changes in the human cervix proteome results in normal and abnormal CR.

Trends, gaps, and future directions of research in cervical remodeling during pregnancy: a bibliometric analysis.

PURPOSE: The cervix undergoes a dynamic remodeling process throughout pregnancy. Appropriate timing of cervical remodeling is essential in maintaining the fetus inside the uterus and ensuring cervical dilatation for safe delivery of the fetus at term. This study aims to determine the characteristics and trends of published articles in the field of cervical remodeling during pregnancy through a bibliometric analysis. MATERIALS AND METHODS: A systematic review of the literature on cervical remodeling during pregnancy was performed on using the Scopus database from inception to 2020. The following information was obtained for each article: authors, year of publication, title, journal, institution, country, title, keywords, citation frequency, and relative citation ratio. The visualization of collaboration networks of countries and keywords related to cervical remodeling during pregnancy was conducted using VOSviewer software. RESULTS: A total of 1979 bibliographic records were obtained from Scopus database. The number of publications increased in the 1980s and peaked in 2010. A total of 80 countries produced research in cervical remodeling during pregnancy. The USA contributed the greatest number of publications (n= 541), total citations (n= 11,971), and number of international collaborations (n= 28 countries). The American Journal of Obstetrics and Gynecology, Obstetrics and Gynecology, and BJOG: An International Journal of Obstetrics and Gynaecology are the top three contributors in this field in terms of number of publications and total citations. The Karolinska Institutet produced the greatest number of publications while UT Southwestern Medical Center was the most cited institution in this field. The topics of the top cited articles were studies regarding the role of collagen degradation in cervical remodeling during pregnancy; dynamics, anatomy, and physiology of cervical remodeling; and the use of misoprostol for cervical ripening and labor induction. CONCLUSIONS: Our bibliometric analysis shows the trends and development, scientific impact, and collaboration in the field of cervical remodeling research. These results show the important discoveries in the past and provided new avenues for scientific and clinical investigations in the field.

Correction to: Activation of an Endogenous Opsin 3 Light Receptor Mediates Photo-Relaxation of Pre-Contracting Late Gestation Human Uterine Smooth Muscle Ex Vivo.

This article was updated to correct Joy Y. Vink's name in the author listing.

Activation of an Endogenous Opsin 3 Light Receptor Mediates Photo-Relaxation of Pre-Contracting Late Gestation Human Uterine Smooth Muscle Ex Vivo.

Spontaneous preterm birth (sPTB) remains a worldwide healthcare challenge. Preterm labor (PTL) is thought to be the largest reversible cause of sPTB, but current tocolytic therapies are ineffective and associated with systemic side effects from chronic use. Therefore, identifying novel mechanisms that promote human uterine smooth muscle (hUSM) relaxation is essential to improving clinical management of PTL. Here, we aimed to determine if an extraocular opsin receptor (OPN 3,4,5) system is expressed in pregnant hUSM and to characterize how photo-mediated relaxation of pre-contracting hUSM may be facilitated by external application of light. Translational studies were performed with hUSM from healthy late gestation patients (n = 8) and non-pregnant, similarly aged patients undergoing hysterectomy (n = 4). First, RT-PCR screened for mRNA coding for components of the classical extraocular light receptors (OPN 3,4,5). We found a restricted repertoire of opsin receptors (OPN3) expressed in pregnant hUSM tissue. Immunohistochemistry was performed to confirm protein expression. Pre-contracting late gestation hUSM strips were studied in functional organ bath studies to determine if photo-mediated relaxation is intensity or wavelength dependent. Functional organ bath studies revealed acute photo-mediated relaxation occurring in an intensity- and wavelength-dependent manner. Finally, coimmunoprecipitation of OPN3 with Gs following light activation suggests that a component of photo-relaxation occurs via G protein-coupled receptor machinery. This is the first report of light-mediated relaxation of pre-contracted human myometrium. Activation of endogenous light receptors on human myometrium may become a novel, non-invasive tocolytic strategy.

Quantitative Ultrasound Detects Smooth Muscle Activity at the Cervical Internal Os in Vitro.

The cervix has two biomechanical functions: to remain closed while the fetus develops throughout pregnancy, and to open for delivery of the fetus at full term. This dual function is principally attributed to collagen within the extracellular matrix (ECM). However, recent evidence suggests that other ECM, and non-ECM, components play a role as well. One component is smooth muscle cells arranged circumferentially near the internal os. In this study, we investigate correlations between cervical smooth muscle cell force generation and the effective scatterer diameter (ESD), a quantitative ultrasound parameter directly related to the acoustic impedance distribution and, therefore, a potential biomarker of muscle contractility. Using whole cervical slices (N = 5), we determined significant positive correlations (quantified with Pearson's r) between muscle force generation and ESD immediately after administration of oxytocin (median r = 0.90). In summary, the ESD may prove a useful biomarker for studying structure and function of cervical smooth muscle in vivo.

Characterization of the collagen microstructural organization of human cervical tissue.

The cervix shortens and softens as its collagen microstructure remodels in preparation for birth. Altered cervical tissue collagen microstructure can contribute to a mechanically weak cervix and premature cervical dilation and delivery. To investigate the local microstructural changes associated with anatomic location and pregnancy, we used second-harmonic generation microscopy to quantify the orientation and spatial distribution of collagen throughout cervical tissue from 4 pregnant and 14 non-pregnant women. Across patients, the alignment and concentration of collagen within the cervix was more variable near the internal os and less variable near the external os. Across anatomic locations, the spatial distribution of collagen within a radial zone adjacent to the inner canal of the cervix was more homogeneous than that of a region comprising the middle and outer radial zones. Two regions with different collagen distribution characteristics were found. The anterior and posterior sections in the outer radial zone were characterized by greater spatial heterogeneity of collagen than that of the rest of the sections. Our findings suggest that the microstructural alignment and distribution of collagen varies with anatomic location within the human cervix. These observed differences in collagen microstructural alignment may reflect local anatomic differences in cervical mechanical loading and function. Our study deepens the understanding of specific microstructural cervical changes in pregnancy and informs investigations of potential mechanisms for normal and premature cervical remodeling.

Collagen Fiber Orientation and Dispersion in the Upper Cervix of Non-Pregnant and Pregnant Women.

The structural integrity of the cervix in pregnancy is necessary for carrying a pregnancy until term, and the organization of human cervical tissue collagen likely plays an important role in the tissue's structural function. Collagen fibers in the cervical extracellular matrix exhibit preferential directionality, and this collagen network ultrastructure is hypothesized to reorient and remodel during cervical softening and dilation at time of parturition. Within the cervix, the upper half is substantially loaded during pregnancy and is where the premature funneling starts to happen. To characterize the cervical collagen ultrastructure for the upper half of the human cervix, we imaged whole axial tissue slices from non-pregnant and pregnant women undergoing hysterectomy or cesarean hysterectomy respectively using optical coherence tomography (OCT) and implemented a pixel-wise fiber orientation tracking method to measure the distribution of fiber orientation. The collagen fiber orientation maps show that there are two radial zones and the preferential fiber direction is circumferential in a dominant outer radial zone. The OCT data also reveal that there are two anatomic regions with distinct fiber orientation and dispersion properties. These regions are labeled: Region 1-the posterior and anterior quadrants in the outer radial zone and Region 2-the left and right quadrants in the outer radial zone and all quadrants in the inner radial zone. When comparing samples from nulliparous vs multiparous women, no differences in these fiber properties were noted. Pregnant tissue samples exhibit an overall higher fiber dispersion and more heterogeneous fiber properties within the sample than non-pregnant tissue. Collectively, these OCT data suggest that collagen fiber dispersion and directionality may play a role in cervical remodeling during pregnancy, where distinct remodeling properties exist according to anatomical quadrant.

A new paradigm for the role of smooth muscle cells in the human cervix.

BACKGROUND: Premature cervical remodeling resulting in spontaneous preterm birth may begin with premature failure or relaxation at the internal os (termed "funneling"). To date, we do not understand why the internal os fails or why funneling occurs in some cases of premature cervical remodeling. Although the human cervix is thought to be mostly collagen with minimal cellular content, cervical smooth muscle cells are present in the cervix and can cause cervical tissue contractility. OBJECTIVE: To understand why the internal os relaxes or why funneling occurs in some cases of premature cervical remodeling, we sought to evaluate cervical smooth muscle cell content and distribution throughout human cervix and correlate if cervical smooth muscle organization influences regional cervical tissue contractility. STUDY DESIGN: Using institutional review board-approved protocols, nonpregnant women <50 years old undergoing hysterectomy for benign indications were consented. Cervical tissue from the internal and external os were immunostained for smooth muscle cell markers (α-smooth muscle actin, smooth muscle protein 22 calponin) and contraction-associated proteins (connexin 43, cyclooxygenase-2, oxytocin receptor). To evaluate cervical smooth muscle cell morphology throughout the entire cervix, whole cervical slices were obtained from the internal os, midcervix, and external os and immunostained with smooth muscle actin. To correlate tissue structure with function, whole slices from the internal and external os were stimulated to contract with 1 µmol/L of oxytocin in organ baths. In separate samples, we tested if the cervix responds to a common tocolytic, nifedipine. Cervical slices from the internal os were treated with oxytocin alone or oxytocin + increasing doses of nifedipine to generate a dose response and half maximal inhibitory concentration. Student t test was used where appropriate. RESULTS: Cervical tissue was collected from 41 women. Immunohistochemistry showed cervical smooth muscle cells at the internal and external os expressed mature smooth muscle cell markers and contraction-associated proteins. The cervix exhibited a gradient of cervical smooth muscle cells. The area of the internal os contained 50-60% cervical smooth muscle cells that were circumferentially organized in the periphery of the stroma, which may resemble a sphincter-like pattern. The external os contained approximately 10% cervical smooth muscle cells that were randomly scattered in the tissue. In organ bath studies, oxytocin stimulated the internal os to contract with more than double the force of the external os (1341 ± 693 vs 523 ± 536 integrated grams × seconds, respectively, P = .009). Nifedipine significantly decreased cervical tissue muscle force compared to timed vehicle control (oxytocin alone) at doses of 10(-5) mol/L (vehicle 47% ± 15% vs oxytocin + nifedipine 24% ± 16%, P = .007), 10(-4) mol/L (vehicle 46% ± 16% vs oxytocin + nifedipine -4% ± 20%, P = .003), and 10(-3) mol/L (vehicle 42% ± 14% vs oxytocin + nifedipine -15% ± 18%, P = .0006). The half maximal inhibitory concentration for nifedipine was 1.35 × 10(-5) mol/L. CONCLUSION: Our findings suggest a new paradigm for cervical tissue morphology-one that includes the possibility of a specialized sphincter at the internal os. This new paradigm introduces novel avenues to further investigate potential mechanisms of normal and premature cervical remodeling.

Analyzing three-dimensional ultrastructure of human cervical tissue using optical coherence tomography.

During pregnancy, the uterine cervix is the mechanical barrier that prevents delivery of a fetus. The underlying cervical collagen ultrastructure, which influences the overall mechanical properties of the cervix, plays a role in maintaining a successful pregnancy until term. Yet, not much is known about this collagen ultrastructure in pregnant and nonpregnant human tissue. We used optical coherence tomography to investigate the directionality and dispersion of collagen fiber bundles in the human cervix. An image analysis tool has been developed, combining a stitching method with a fiber orientation measurement, to study axially sliced cervix samples. This tool was used to analyze the ultrastructure of ex-vivo pregnant and non-pregnant hysterectomy tissue samples taken at the internal os, which is the region of the cervix adjacent to the uterus. With this tool, directionality maps of collagen fiber bundles and dispersion of collagen fiber orientation were analyzed. It was found that that the overall preferred directionality of the collagen fibers for both the nonpregnant and pregnant samples were circling around the inner cervical canal. Pregnant samples showed greater dispersion than non-pregnant samples. Lastly, we observed regional differences in collagen fiber dispersion. Fibers closer to the inner canal showed more dispersion than the fibers on the radial edges.

Calcium-activated chloride channels anoctamin 1 and 2 promote murine uterine smooth muscle contractility.

OBJECTIVE: To determine the presence of calcium activated chloride channels anoctamin 1 (ANO1) and 2 (ANO2) in human and murine uterine smooth muscle (MUSM) and evaluate the physiologic role for these ion channels in murine myometrial contractility. STUDY DESIGN: We performed reverse transcription polymerase chain reaction to determine whether ANO1 and 2 are expressed in human and murine uterine tissue to validate the study of this protein in mouse models. Immunohistochemical staining of ANO1 and 2 was then performed to determine protein expression in murine myometrial tissue. The function of ANO1 and 2 in murine uterine tissue was evaluated using electrophysiologic studies, organ bath, and calcium flux experiments. RESULTS: ANO1 and 2 are expressed in human and MUSM cells. Functional studies show that selective antagonism of these channels promotes relaxation of spontaneous MUSM contractions. Blockade of ANO1 and 2 inhibits both agonist-induced and spontaneous transient inward currents and abolishes G-protein coupled receptor (oxytocin) mediated elevations in intracellular calcium. CONCLUSION: The calcium activated chloride channels ANO1 and 2 are present in human and murine myometrial tissue and may provide novel potential therapeutic targets to achieve effective tocolysis.

Anthrax toxin receptor 2 functions in ECM homeostasis of the murine reproductive tract and promotes MMP activity.

Anthrax Toxin Receptor proteins function as receptors for anthrax toxin, however physiological activity remains unclear. To evaluate the biological role of Antxr2, we generated Antxr2-/- mice. Antxr2-/- mice were viable, however Antxr2 is required for parturition in young females and for preserving fertility in older female mice. Histological analysis of the uterus and cervix revealed aberrant deposition of extracellular matrix proteins such as type I collagen, type VI collagen and fibronectin. A marked disruption of both the circular and longitudinal myometrial cell layers was evident in Antxr2-/- mice. These changes progressed as the mice aged, resulting in a thickened, collagen dense, acellular stroma and the disappearance of normal uterine architecture. To investigate the molecular mechanism underlying the uterine fibrosis we performed immunoblotting for MMP2 using uterine lysates and zymography using conditioned medium from Antxr2-/- mouse embryonic fibroblasts and found reduced levels of activated MMP2 in both. This prompted us to investigate MT1-MMP status, as MMP2 processing is regulated by MT1-MMP. We found MT1-MMP activity, as measured by MMP2 processing and activation, was enhanced by expression of either ANTXR1 or ANTXR2. We identified an ANTXR2/MT1-MMP complex and demonstrated that MT1-MMP activity is dependent on ANTXR2 expression levels in cells. Thus, we have discovered that ANTXR1 and ANTXR2 function as positive regulators of MT1-MMP activity.

Amniotic fluid index and birth weight: is there a relationship in diabetics with poor glycemic control?

OBJECTIVE: This study was undertaken to evaluate if the previously demonstrated relationship between macrosomia (> 4000 g) and polyhydramnios (> 25 cm) is linear across birth weights (BW) in diabetic patients with poor glycemic control. STUDY DESIGN: Using a prospectively collected database of patients undergoing amniocentesis for fetal lung maturity for various indications with amniotic fluid index (AFI) obtained < or = 7 days before delivery and BWs available (n = 69), we computed gestational age (GA) specific AFI and BW centiles using standard tables. BW and AFI centiles were analyzed in diabetic patients with poor glycemic control using linear regression and ANOVA, with P < .05 significant. RESULTS: In the poorly controlled diabetic population, a linear relationship existed between AFI and BW centiles, with the largest BW centiles having the highest AFI centiles (P < .0001). CONCLUSION: The previously noted relationship between elevated AFI and BW centiles in the general patient population is linear in diabetic patients with poor glycemic control.