Clinical effects and cervical tissue changes after treatment with 16, 16 dimethyl-trans Δ2 PGE1 methylester in the first trimester

Cervagem pessaries, containing 1 mg of 16, 16-dimethyl-trans Δ 2-PGE₁ methylester, was administered in the posterior vaginal fornix 4 h before vacuum aspiration in the 1st trimester. The effects of cervical dilatation, cervical collagen metabolism and cervical smooth muscle activity were investigated. In treated women the mean cervical dilatation was 7.9 mm as compared with 3.9 mm in controls. The in vitro incorporation of 14C proline in cervical tissue as well as the hydrolytic activity against a synthetic “collagen-like” polypeptide was increased after treatment with cervagem. The cervical smooth muscle sensitivity to prostaglandins, as revealed by inhibition of muscle activity, was higher in treated women than in controls. It is concluded that cervical dilatation, as induced by cervagem, involves an adaptation of both connective tissue and smooth muscle components.     

Has cervical smooth muscle any physiological role in the human?

Strips of human cervical tissue were obtained by needle biopsy and contractile activity was registered isometrically in a tissue chamber perfused by Krebs-Ringer bicarbonate buffer. The most frequently encountered pattern of contractile activity was high frequency-short duration. Prostaglandin (PG)E2, PGI2 and 6-keto-PGF1 alpha had an inhibitory effect on the muscular activity. Cervical muscle from pregnant women was more sensitive to PGE2 than specimens from non-pregnant women. PGF2 alpha had no apparent effect on cervical contractility in non-pregnant and early pregnant patients. In late pregnancy, however, PGF2 alpha inhibited muscle contractions. The present results point to a physiological role of the cervical muscles for the control of cervical competence during pregnancy. The inhibitory effect of PGs on the muscle activity may promote cervical dilatation and retraction.     

Cervical mucus concentration of prostaglandins E2 and F2α after pretreatment with mifepristone in the first trimester of pregnancy

Cervical dilatation and softening after pretreatment with mifepristone are well documented. As this effect is similar to that observed after local application of prostaglandin E₂ (PGE₂) it is tempting to speculate that the effect of mifepristone is mediated via an increase of the endogenous secretion of prostaglandins from the cervical mucosa. Eighteen healthy women in the first trimester of pregnancy were treated with oral mifepristone (200 mg) 48 and 24 hours before legal abortion by vacuum aspiration and 18 women in the same age of gestation without any pretreatment served as controls. Cervical mucus was collected for measurement of prostaglandins by radioimmunoassay before administration of the drug and in connection with vacuum aspiration. The cervical dilatation at the time of surgery was significantly increased in women given mifepristone as compared with untreated women (7.6 versus 5.8 mm). The wet weight of collected cervical mucus was significantly increased in mifepristone treated women. The amount of PGE₂ and prostaglandin F_2α per sample was unchanged in mifepristone-treated women, whereas the concentration was lower as an effect of dilution due to an increased yield in cervical secretion observed after mifepristone treatment. The present observation does not give any support to the hypothesis that mifepristone-induced cervical maturation is mediated via an increase in cervical prostaglandin production.     

Cervical diameter after suction termination of pregnancy.

The diameter of the internal cervical os was measured in several groups of patients in an attempt to assess any damage caused by suction termination of pregnancy. Pregnant women who had had a previous abortion by vacuum aspiration had significantly greater cervical diameters than those who had not, and there was a statistically significant correlation between dilatation of the cervix at operation and cervical diameter at six weeks'' follow-up. Cervical dilatation to 10 mm or less was subsequently associated with a normal cervical diameter, but the diameter was often large when the extent of dilatation was greater than 12 mm or not known. Cervical dilatation at termination of pregnancy should, if possible not exceed 10 mm.     

Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle

We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12-14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists.     

Cervical collagen and biomechanical strength in non-pregnant women with a history of cervical insufficiency

Background  

It has been suggested that cervical insufficiency (CI) is characterized by a "muscular cervix" with low collagen and high smooth muscle concentrations also in the non-pregnant state. Therefore, the aim of this study was to investigate the biomechanical properties, collagen concentration, smooth muscle cell density, and collagen fiber orientation in cervical biopsies from non-pregnant women with a history of CI.     

Effect of dehydroepiandrosterone sulfate on uterine cervical ripening in late pregnancy

In order to elucidate the effects of dehydroepiandrosterone sulfate (DHAS) on softening and dilatation of the uterine cervix, changes of oestriol, 17 beta-oestradiol and progesterone levels in serum and cervix, Bishop score and collagenase activity in the cervical tissue were assessed in pregnant women before and after treatment with DHAS. 17 beta-oestradiol level in the serum and cervical tissue markedly increased after the administration of DHAS, while oestriol level remained unchanged. Serum progesterone level did not change in the majority of cases, while it decreased within several hours in patients in whom delivery was accomplished within 24 hours after the administration of DHAS. Among the factors connected with the Bishop score, effacement and consistency of the cervix were remarkably improved by DHAS administration. Total collagenase activity in the cervical tissue of patients treated with DHAS was elevated by an average of 152%. These results suggest that DHAS is potent in ripening the uterine cervix through an activation of collagenase activity induced by the enhanced conversion to 17 beta-oestradiol. Thus, DHAS administration in the late stage of pregnancy is valuable in prepartal treatment for induction of labour.     

Collagenase activity in cervical tissue of the non-pregnant and pregnant human cervix

Collagen degradation has been suggested to play an important role in the process of cervical ripening at term. After giving informed consent tissue samples were taken from the posterior lip of 10 non-pregnant women of child-bearing age, 10 women having a termination of pregnancy at 9-12 weeks' gestation and 12 women who had had normal spontaneous deliveries at term. Collagenase activity was measured by a specific technique using native, triple helical collagen. Proteolytic activity was determined with commercially available 14C-methaemoglobin as substrate. For identification of collagen fragments SDS-polyacrylamid-gel-electrophoresis was performed using a modified Laemmli-technique. Collagenase and protease activity were found in all extracts of the different groups. There was a marked increase in enzymatic activities in the postpartum tissue samples suggesting that significant collagen breakdown had taken place. The absence of typical collagen fragments in the SDS-electrophoresis seemed to be due to a very rapid further degradation of the fragments by proteases into small peptides which then readily left the cervix. The present findings confirm the essential role of proteolytic enzymes in cervical dilatation at term.     

Effects of 9-deoxo-16, 16-dimethyl-9-methylene PGE2 on muscle contractile activity and collagen synthesis in the human cervix

The in vitro effects of a stable PGE-analogue (9-deoxo-16, 16-dimethyl-9-methylene PGE₂ (9-methylene PGE₂) on human cervical tissue was investigated. The influence of the analogue on collagen biosynthesis was studied by measuring the incorporation of ³H-proline, while smooth muscle effects were evaluated by isometric recording of contractile activity. The specimens were obtained by needle biopsy from women in early and late pregnancy and from nonpregnant women of fertile age.9-methylene PGE₂ compared with controls increased the incorporation of ³H-proline in the secretory phase and before the 9th week of pregnancy, whereas radiolabelling was decreased in the follicular phase, in the 9th–12th week and at term. With respect to incorporation of 3-H-proline, 9-methylene PGE₂ was equipotent to PGE₂. 9-methylene PGE₂ inhibited spontaneous contractile activity in early as well as in late pregnancy but increased muscular activity in nonpregnant patients. The inhibitory effects of the analogue was similar to that of PGE₂ but the natural compound was considerably more potent in this respect.     

Effects of 9-deoxo-16,16-dimethyl-9-methylene PGE2 on muscle contractile activity and collagen synthesis in the human cervix

The in vitro effects of a stable PGE-analogue (9-deoxo-16,16-dimethyl-9-methylene PGE2 (9-methylene PGE2) on human cervical tissue was investigated. The influence of the analogue on collagen biosynthesis was studied by measuring the incorporation of 3H-proline, while smooth muscle effects were evaluated by isometric recording of contractile activity. The specimens were obtained by needle biopsy from women in early and late pregnancy and from nonpregnant women of fertile age. 9-methylene PGE2 compared with controls increased the incorporation of 3H-proline in the secretory phase and before the 9th week of pregnancy, whereas radiolabelling was decreased in the follicular phase, in the 9th-12th week and at term. With respect to incorporation of 3H-proline,9-methylene PGE2 was equipotent to PGE2. 9-methylene PGE2 inhibited spontaneous contractile activity in early as well as in late pregnancy but increased muscular activity in nonpregnant patients. The inhibitory effects of the analogue was similar to that of PGE2 but the natural compound was considerably more potent in this respect.     

Prostaglandin release from human cervical tissue in the first trimester of pregnancy after preoperative dilatation with hygroscopic tents

Preoperative dilatation with hygroscopic tents before first trimester abortion by vacuum aspiration is widely accepted and reduces the risk of early and late complications. A softening effect and a reduced compliance to mechanical dilatation occurs in addition to pure mechanical dilatation of the cervix. If this softening is an effect of local prostaglandin release, however, is unknown. Prostaglandin (PG) release in vitro from cervical biopsies following dilatation in vivo by a synthetic hygroscopic tent (Dilapan) for periods of 4 h and 18 h was compared with that of biopsies from untreated women. No difference was observed between the release of PGE₂, PGF_2α, or 6-keto-PGF_1α. No significant difference was found in the tissue water content between treated and untreated women (83.8% versus 83.2%). Prostaglandins were also extracted from an alternative cervical dilator, Lamicel (a polyvinyl sponge impregnated with magnesium sulfate), and compared with the corresponding values from women pretreated with the cyclooxygenase inhibitor indomethacin before application of the tent. Significantly higher concentrations of PGE₂ and PGF_2α but not of 6-keto-PGF_1α were found in women who had not been indomethacin-treated compared with indomethacin-treated women. Slices of the cervix from non-pregnant women operated upon for benign conditions were divided into an outer stromal layer and an inner layer, including the mucosa, and the PG-release in vitro was measured. The inner layer of the cervix showed a significantly higher release of PGE₂ and PGF_2α compared with the outer layer. Lamicel treatment before first trimester abortion results in a significant dilatation of the cervix and a reduced complaince to mechanical dilatation, and this study supports the hypothesis that this effect is mediated via a local PG-release from the cervix. It seems reasonable to believe that Dilapan treatment too has the capacity to induce PG-release from the cervix, but this could not be demonstrated in this study, probably because needle biopsies taken mainly from the outer cervical layers were analyzed.     

Etude clinique préliminaire sur l'emploi de la dihexyvérine en obstétrique

This study attempted to evaluate the effect of dihexyverine HCl (Metaspas), an anticholinergic and direct smooth muscle relaxant (spasmolytic), in shortening the first stage of labour. One hundred and five patients constituted the series: 31 were primiparae, 27 secundiparae and 47 multiparae. The majority of these (95) received one intramuscular injection (10 mg.) of dihexyverine. Alphaprodine (Nisentil), 40 mg., was administered in all but 10 cases, since dihexyverine has no analgesic action. Seventy-five control patients were used: 25 were primiparae, 25 secundiparae and 25 multiparae. Alphaprodine alone was administered to these control patients. The length of time for dilatation from 4 cm. to 10 cm. was the selected criterion. In the control group, the average time for complete cervical dilatation was 4 hours, 10 minutes. In the group treated by dihexyverine, complete dilatation was achieved in 1 hour, 16 minutes. On the average, the first stage of labour was 2 hours and 54 minutes shorter with dihexyverine; the clinical effect begins 10-30 minutes after administration. The only side effect encountered was a skin rash in one patient.     

Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.     

Phasic Contractions of the Mouse Vagina and Cervix at Different Phases of the Estrus Cycle and during Late Pregnancy

Background/Aims

The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca²⁺ release from sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) -dependent intracellular Ca²⁺ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking.

Methodology/Principal Findings

Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K⁺ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca²⁺ stores, but were inhibited in low Ca²⁺ solution or in the presence of nifedipine, an inhibitor of L-type Ca²⁺channels. ICs were found in small numbers in the mouse cervix but not in the vagina.

Conclusions/Significance

Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca²⁺ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges.     

Effect of longitudinal gravitational loads on the structure of the wall of the vertebral artery]

A single exposure to the greatest endurable gravitation stress causes such changes in the wall of the vertebral artery as division into layers, oedema, vacuolization of smooth muscle cells. Repeated exposure according to a training schedule resulted in reconstruction of the vascular wall (division of the medial sheath of the artery into several muscular layers). Concentration of smooth muscle cells in "layers" increased when training was after a "cumulative" schedule. Under conditions of increasing cumulative effect the collagenization of the media and dystrophy of the muscular and elastic tissue began. The greatest changes in the arterial wall took place in the upper parts of the vertebral artery at the level of the 2nd and 3d cervical vertebra.     

Vaginally administered PGF2alpha for cervical dilatation in nulliparas prior to suction curettage

Because of the need for an atraumatic method to dilate the cervix when performing artificial abortion by suction curettage, cervical dilatation following vaginally administered PGF2alpha was studied. A 50 mg PGF2alpha vaginal suppository was administered to 40 (treated group) first trimester nulliparas 3 hours prior to progressive cervical dilatation from a 19 (circumference in mm) Pratt dilator to a 35 Pratt dialator. The smallest-sized dilator that met resistance was interpreted as being the amount of clinically significant cervical dilatation. The results were compared to 20 (control group) first trimester nulliparas who received no PGF2alpha studied in an identical manner. Independent of gestational age, treated patients were dilated significantly more than the control patients. When subjects of similar gestational age were compared, PGF2alpha treated subjects were more often dilated sufficiently to perform abortion (55%) by suction curettage than control group subjects (5%). Those PGF2alpha subjects needing further dilatation to accept an appropriate sized cannula for their gestational age needed less dilatation than did those subjects of similar gestational age in the control group. No serious complications of PGF2alpha per se were observed and the most frequent side effects, vomiting and diarrhea, did not appear severe enough to limit the clinical practicability of the method.     

Inhibitor-1 phosphatase activity in vascular smooth muscle

The histone-H1 and polylysine stimulated "latent" phosphorylase phosphatase, characterized by a molecular weight of 260,000 in gel filtration and 130,000 in sucrose density gradient centrifugation has been identified as a major inhibitor-1 phosphatase in vascular smooth muscle. Its substrate: protein phosphatase inhibitor-1, was also shown to be present in the same tissue. Following treatment with beta-mercaptoethanol the enzyme dissociates into a lower molecular weight (38,000) form with a higher specific activity.     

Subcellular localization of phospholipase C isoforms in vascular smooth muscle

The phospholipase C (PLC) isoform most important during agonist-activated IP(3) production in vascular smooth muscle is still unknown. When PLC activity in rat tail artery homogenate was determined, this activity was shown to be inhibited by an antibody directed against PLCbeta2. Antibodies directed against the gamma1, beta1, beta3 and delta1 isoforms of PLC failed to inhibit PLC activity in this tissue. Both PLCbeta2 and PLCgamma1 were isolated from rat tail artery by DEAE column chromatography and PLCbeta2 activity was shown to be 3-fold greater than PLCgamma1 activity. When rat tail artery was treated with norepinephrine (10 mM), PLCbeta2 was shown to translocate from cytosol to membranes. When subcellular fractions of rat tail artery were isolated by sucrose density gradient centrifugation, including nuclei, plasma membrane, and cytosol, PLCbeta2 was detected in the plasma membrane and the cytosol but not in the nuclei. PLCdelta1 and PLCgamma1 were found only in cytosol. This evidence is consistent with the model wherein an agonist such as norepinephrine can activate smooth muscle contraction via interaction with a plasma membrane receptor which can easily interact with a plasma membrane-associated isoform of PLC, such as PLCbeta2.     

Forces required for dilatation of human cervix in first trimester of pregnancy

Cervical resistance to dilatation was measured in 76 patients undergoing first trimester legal abortion; a specially designed force-sensing instrument was used. No correlation between cervical resistance and patient age or gestational age was found. Increasing parity and earlier legal abortions were significantly correlated with a lowering of the cervical resistance. In patients dilated to 11 mm a lowering of resistance was noted suggesting a tear in cervical tissue.     

Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

Background

The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases.

Methods

Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance.

Results

We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction.

Conclusion

Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.