Nitric oxide synthase activity and localization do not change in uterus and placenta during human parturition

Animal studies have suggested that nitric oxide, a smooth muscle relaxant, is a fundamental mediator in the initiation of parturition. The purpose of this study was to test the hypothesis that the onset of human labour is associated with a reduction in the activity of the enzyme nitric oxide synthase (NOS), within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were collected during Caesarean section from 11 women before and 11 women after the onset of labour at term. Immunocytochemistry was used to localize each of the three isoforms of NOS (endothelial NOS, brain NOS, and inducible NOS) in each of these tissues and the intensity of staining was qualitatively assessed. NOS enzyme activity was determined in homogenates of frozen myometrium, placenta and fetal membranes (with attached decidua), by measuring conversion of radio-labelled L-arginine to L-citrulline. Each of the three isoforms of NOS was localized in each of the tissues. We found no difference in either the expression or enzyme activity of NOS in myometrium, placenta or fetal membranes before and during labour at term. These results suggest that, in contrast to animal studies, a decrease in NOS enzyme activity may not be involved in the onset of parturition at term in the human.     

3D-FEA of osseointegration percentages and patterns on implant-bone interfacial stresses

The aim of this study was to investigate the expression and distribution patterns of the inducible isoform of nitric oxide synthase (iNOS) in rat placenta during gestation and term labour. The expression of iNOS isoform was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with monoclonal antibodies. Two specific bands were detected corresponding to 135 and 124 kDa in all placenta samples. The upper band (135 kDa) was identified as iNOS due to its correspondence with the band obtained with mouse macrophages (positive control). Compared with its concentrations on day 16, iNOS decreased steadily toward the end of gestation to approximately 37% on day 20, 20% on day 22 before labour and 12% during labour (p < 0.01). The lower band (124 kDa) drastically increased (to almost double) from day 16 to day 18 but returned to initial values on day 22, during delivery. Immunohistochemical staining of placentae at day 16 and 22 using rabbit polyclonal anti-iNOS antibody revealed labelling specifically concentrated in the trophospongial cell layer, at the fetal-maternal interface. The most conspicuous iNOS staining was associated with islands of cells referred to as vacuolated 'glycogen cells'. Staining was greatly decreased during labour. The changes in placental iNOS expression suggest a 'paracrine' role for NO in regulating uterine contractility, blood flow and immunosuppression required for pregnancy maintenance. NO withdrawal at term may also be involved in the initiation of labour.     

Expression of nitric oxide synthase isoforms in bone and bone cell cultures

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Nitric oxide synthase regulation during embryonic implantation

It has previously been demonstrated that uterine nitric oxide synthase (NOS) activity increases before embryonic implantation in rats. The aim of the present work was to investigate the regulation and the physiological relevance of the nitric oxide (NO) system in ovoimplantation. The increase in NOS activity in early pregnancy was found to be independent of the presence of embryos in the uterus. Whereas the Ca2+-dependent isoform of NOS increased gradually in the preimplantation days, the Ca2+-independent isoform increased just at the beginning of implantation (Day 5, 1800 hours); then the activity of both isoforms declined. Oestradiol, whose concentration peaks before implantation, might be regulating NOS activity in the uterus, since treatment of rats with tamoxifen, a receptor antagonist, reduces the activity of both isoforms to preimplantation levels. Intraluminal injections of L-NAME (0.5 mg kg[-1]), a competitive inhibitor of NOS, reduced by 50% the number of implanted embryos; this suggests that the NO system plays a role during implantation. The data suggest that oestradiol might be a modulator of NOS activity during nidation and that NO production is necessary to achieve a successful embryo implantation.     

Cervical ripening with prostaglandin E2 in a scarred uterus during the third trimester of pregnancy. Report of 82 cases

AIM: To study the feasibility, the results and the complications of cervical ripening using PG2 in scarred uterus in the third trimester of pregnancy. METHOD: A retrospective study of 82 cases of which 10 had ruptured membranes. The administration of 0.5 mgs of PG2 by the intracervical route after Beta-mimetic drugs by the intramuscular route. RESULTS: In 78% of the cases it was possible to improve the condition of the cervix so that labour could be induced or that it would start spontaneously. It was possible to deliver the baby vaginally in 67% of cases. There was no case of ruptured uterus. CONCLUSION: It seems possible that when there is a medical indication to induce labour to ripen the cervix using PG2 in a scarred uterus in the third trimester of pregnancy. The administration of beta-mimetic drugs for ripening and the use of tocometry to monitor labour seemed to be important precautions that have to be taken.     

Changes in cervical fibronectin levels during pregnancy, labor and in the postpartum period

The authors analysed the fibronectin content of the uterine cervix during pregnancy and delivery. The aim of their investigation was furnishing data to the biochemical changes during cervical maturation. The ripening of the uterine cervix during pregnancy is a result of complicated interactions between different macromolecules, where the fibronectin plays a key role. The quantitative determination of the extracellular matrix fibronectin is impossible because its extraction from tissues recently is not solved. Taking this fact in consideration the authors choose a semiquantitative method, being reliable indicator of changes in fibronectin content of uterine cervix. They took small pieces of materials from portio vaginalis uteri of 139 women being in postmenopause and premenopause, in different stages of pregnancy and parturition concerning directly after delivery. The slides were incubated, with rabbit-anti-human fibronectin-FITC. The evaluation of fluorescence happened with an Axiophot (Zeiss) microscope. Authors stated that the fibronectin content in the cervical extracellular matrix and in the cellular membrane of fibroblasts increases during the 1st trimester pregnancy. This increase can be shown in the 3rd trimester as well and it drops significantly during delivery. They could not found any relationship between the leucocyte invasion observed during delivery and the changes of cervical fibronectin content. These observations call our attention to the importance of fibronectin in cervical ripening respectively dilatation and the need of further examinations.     

Endocervical pathogens in women with preterm and term labour

The purpose of this study was to compare the endocervical microflora of women in preterm and term labour and to determine whether the presence of a specific microflora is significantly associated with preterm labor. A prospective study was performed in Lithuania among 212 women in preterm labour (latent phase, n = 110; active phase, n = 102) and among 62 healthy women in term labour. Microbiological assessment included cultures for aerobic bacteria, yeasts, and Trichomonas vaginalis and direct immunofluorescence reaction for Chlamydia trachomatis, Escherichia coli (odds ratio 8.16; 95% confidence interval 1.27-340.23) and Staphylococcus aureus (odds ratio 7.79; 95% confidence interval 1.21-325.40) were significantly more often isolated from women in preterm than from women in term labour. The prevalence of C. trachomatis was the same in the preterm and in the term labour group. The pregnancy outcome during the latent or active phase of preterm labour with or without C. trachomatis infection did not differ. It is concluded that E. coli and S. aureus are significantly more prevalent in endocervical cultures from Lithuanian women in preterm than from those in term labour.     

Ultrasonographic and Biochemical Markers of Preterm Labor

> At present preterm delivery is the leading cause of perinatal morbidity and mortality and its incidence is remained stable during the past 10 years. Conventional methods of identifying patients at risk of preterm delivery such as obstetrics history, demographic factors or evaluation of uterine contractions and cervix by digital examination show disappointintly low sensitivity and positive predictive value. In this review we describe new ultrasonographic and biochemical approaches that have been recently proposed to screen for preterm labor both in patients with intact and with premature rupture of the membranes. The ultrasonographic detection of a short uterine cervix and/or of a dilation of the internal os, expression of weakening of the lower uterine segment or cervical ripening, seems to efficiently predict patients at risk of preterm delivery. The efficiency of this marker may be improved by the association with the assay of fetal fibronectin or pro inflammatory cytokines (interleukin-6 and interleukin-8) in cervical secretions. Further by the concentrations of interleukin-6 and interleukin-8 in cervical secretions seems to be possible to predict among patients in preterm labor those secondary to subclinical endoamniotic infection or chorioamnionitis. The use of these new markers in the future may allow a better identification of patients at risk of preterm labor and a proper selection of the treatment (medical or surgical) required for such patients.     

Uterine relaxation responses to calcitonin gene-related peptide and calcitonin gene-related peptide receptors decreased during labor in rats

OBJECTIVES: Our purpose was to investigate (1) whether uterine relaxation responses to calcitonin gene-related peptide are differentially regulated during pregnancy and labor, (2) the involvement of nitric oxide in smooth muscle relaxant action of calcitonin gene-related peptide in the rat uterus, (3) whether receptors for calcitonin gene-related peptide are expressed in rat uterus, and if so (4) whether the concentrations of these receptors are differently regulated during pregnancy and labor. STUDY DESIGN: Rats were killed on day 18 of gestation, at the time of spontaneous labor, or postpartum day 2. The uteri were removed for in vitro contractility measurements, nitric oxide production, and calcitonin gene-related peptide receptor binding assay. RESULTS: (1) Calcitonin gene-related peptide induced a dose-dependent relaxation in spontaneously contracting uterine strips from pregnant rats on day 18 of gestation; (2) the relaxation effects of calcitonin gene-related peptide on the uterus were decreased during spontaneous delivery at term and post partum compared with that during pregnancy; (3) calcitonin gene-related peptide-induced relaxation was inhibited by pretreatment of the uterine tissue with a calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide(8-37); (4) nitric oxide synthesis inhibitor (N(G)-nitro-L-arginine methyl ester) and soluble guanylate cyclase inhibitor (LY83583) significantly decreased calcitonin gene-related peptide-induced relaxation of the rat uterus during pregnancy; (5) calcitonin gene-related peptide increased the uterine nitric oxide production in pregnant rats, and this increase was obliterated in the presence of calcitonin gene-related peptide(8-37); and (6) calcitonin gene-related peptide receptors are present in rat uterus, and the concentration of these receptors dramatically increases during pregnancy and decreases during labor at term. CONCLUSIONS: Calcitonin gene-related peptide inhibits uterine spontaneous contractions in rats during pregnancy but not during labor and post partum. The inhibitory effects of calcitonin gene-related peptide on uterine contractility appear to be modulated, at least in part, by the activation of nitric oxide generation in the rat uterus. Changes in calcitonin gene-related peptide receptors could contribute to the changes in calcitonin gene-related peptide-mediated uterine relaxation during pregnancy and labor.     

Effects of retinoic acid and estrogens on oxytocin gene expression in the rat uterus: in vitro and in vivo studies

We and others have previously identified functional estrogen (E) and retinoic acid (RA) response elements in the human and rat oxytocin (OT) gene promoters. Whereas there is no direct evidence for a significant role of E or RA in the regulation of rat hypothalamic OT gene expression, we have recently demonstrated that in vivo administration of E strongly stimulates uterine OT gene expression. Here, we show that in vivo administration of RA similarly induces a significant increase in uterine OT gene expression. Moreover, we report that the E and RA effects are reproducible in vitro. Using short-term uterine organ explant cultures derived from 18-day pregnant rats, we found that E (50 nM) and RA (0.4 nM) increased OT mRNA levels 5.2- and 3-fold, respectively, suggesting a direct action of these agents on uterine OT gene expression. Finally, we analyzed uterine E and RA receptor gene expression during pregnancy. Using semi-quantitative Northern blot analysis, we found that mRNAs encoding the E receptor, the RA receptor alpha and RA receptor beta are present in rat uterus and that their levels rise by 3.7-, 3.6- and 5.8-fold, respectively, between day 14 of gestation and term. Taken together, the data suggest that, at term, the rat uterus has an increased capacity to respond to E and RA, and that both agents may be involved in mediating the dramatic increase of OT mRNA accumulation observed in the uterus at term.