Early pregnancy prediction of spontaneous preterm birth before 32 completed weeks of pregnancy using plasma RNA: transcriptome discovery and initial validation of an RNA panel of markers.

OBJECTIVE: To compare the second-trimester plasma cell-free (PCF) transcriptome of women who delivered at term with that of women with spontaneous preterm birth (sPTB) at or before 32 weeks of gestation and identify/validate PCF RNA markers present by 16 weeks of gestation. DESIGN: Prospective case-control study. SETTING: Academic tertiary care centre. POPULATION: Pregnant women with known outcomes prospectively sampled. METHODS: PCF RNAs extracted from women at 22-24 weeks of gestation (five sPTB up to 32 weeks and five at term) were hybridised to gene expression arrays. Differentially regulated RNAs for sPTB up to 32 weeks were initially selected based on P value compared with control (P < 0.01) and fold change (≥1.5×). Potential markers were then reordered by narrowness of distribution. Final marker selection was made by searching the Metacore™ database to determine whether the PCF RNAs interacted with a reported set of myometrial Preterm Initiator genes. RNAs were confirmed by quantitative reverse transcription polymerase chain reaction and tested in a second group of 40 women: 20 with sPTB up to 32 weeks (mean gestation 26.5 weeks, standard deviation ±2.6 weeks), 20 with spontaneous term delivery (40.1 ± 0.9 weeks) sampled at 16-19+5  weeks of gestation. MAIN OUTCOME MEASURE: Identification of PCF RNAs predictive of sPTB up to 32 weeks. RESULTS: Two hundred and ninety-seven PCR RNAs were differentially expressed in sPTB up to 32 weeks of gestation. Further selection retained 99 RNAs (86 mRNAs and 13 microRNAs) and five of these interacted in silica with seven Preterm Initiator genes. Four of five RNAs were confirmed and tested on the validation group. The expression of each confirmed PCF RNA was significantly higher in sPTB up to 32 weeks of gestation. In vitro study of the four mRNAs revealed higher expression in placentas of women with sPTB up to 32 weeks and the potential to interfere with myometrial quiescence. CONCLUSIONS: The PCF RNA markers are highly associated with sPTB up to 32 weeks by 16 weeks of gestation. TWEETABLE ABSTRACT: Women destined for spontaneous preterm birth can be identified by 16 weeks of gestation with a panel of maternal plasma RNAs.

Extravillous trophoblast invasion in placenta accreta is associated with differential local expression of angiogenic and growth factors: a cross-sectional study.

OBJECTIVE: Placenta accreta is clinically associated with maternal uterine scar. Our objective was to investigate the biochemical contribution of maternal scarring to hyperinvasive trophoblast. We hypothesised that trophoblast over-invasion in placenta accreta is associated with aberrant invasion-site signalling of growth and angiogenic factors known to be involved in wound healing and promotion of cell invasion through the epithelial to mesenchymal cellular programme. DESIGN: Cross-sectional series. SETTING: Yale-New Haven Hospital. POPULATION: Women with histologically confirmed normal and abnormal placentation. METHODS: Placental invasion site tissue sections were immunostained for endoglin and other angiogenic regulators, and transforming growth factor ß (TGFß) proteins. Maternal serum endoglin, and the vascular endothelial growth factor (VEGF) mediators hypoxia-inducible factor-1α (HIF1α) and endostatin, were assessed using immunoassay. MAIN OUTCOME MEASURES: Differences in median H-score by immunostaining and in mean serum level by immunoassay. RESULTS: By immunostaining, placenta accreta samples demonstrated intervillous endoglin shedding and increased trophoblast expression of its cleavage protein matrix metalloproteinase-14. Absent decidual HIF1α and endostatin were observed in areas of VEGF upregulation. TGFß1 was present in myocytes but not in collagen bundles into which accreta trophoblast invaded. Maternal serum endoglin decreased in praevia and accreta when corrected for gestational age. CONCLUSION: Angiogenic and growth factors at the placental invasion site are altered in accreta, both by decidual absence and within myometrial scar. We postulate this promotes the invasive phenotype of placenta accreta by activating hyperinvasive trophoblast and by dysregulating placental vascular remodelling. FUNDING: Yale Department of Obstetrics, Gynecology and Reproductive Sciences funds. TWEETABLE ABSTRACT: Placenta accreta histology shows dysregulation of angiogenic and growth factors.

Oligomerization of FVFLM peptides and their ability to inhibit beta amyloid peptides aggregation: consideration as a possible model.

Preeclampsia, a pregnancy-specific disorder, shares typical pathophysiological features with protein misfolding disorders including Alzheimer's disease. Characteristic for preeclampsia is the involvement of multiple proteins of which fragments of SERPINA1 and ß-amyloid co-aggregate in urine and placenta of preeclamptic women. To explore the biophysical basis of this interaction, we investigated the multidimensional efficacy of the FVFLM sequence in SERPINA1, as a model inhibitory agent of ß-amyloid aggregation. After studying the oligomerization of FVFLM peptides using all-atom molecular dynamics simulations with the GROMOS43a1 force field and explicit water, we report that FVFLM can aggregate and its aggregation is spontaneous with a remarkably faster rate than that recorded for KLVFF (aggregation "hot-spot" from ß-amyloid). The fast kinetics of FVFLM aggregation was found to be driven primarily by core-like aromatic interactions originating from the anti-parallel orientation of complementarily uncharged strands. The conspicuously stable aggregation mechanism observed for FVFLM peptides is found not to conform to the popular 'dock-lock' scheme. We also found high propensity of FVFLM for KLVFF binding. When present, FVFLM disrupts the ß-amyloid aggregation pathway and we propose that FVFLM-like peptides might be used to prevent the assembly of full-length Aß or other pro-amyloidogenic peptides into amyloid fibrils.

The invasive phenotype of placenta accreta extravillous trophoblasts associates with loss of E-cadherin.

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is a process of molecular and phenotypic epithelial cell alteration promoting invasiveness. Loss of E-cadherin (E-CAD), a transmembrane protein involved in cell adhesion, is a marker of EMT. Proteolysis into N- and C-terminus fragments by ADAM10 and presenilin-1 (PSEN-1) generates soluble (sE-CAD) and transcriptionally active forms. We studied the protein expression patterns of E-CAD in the serum and placenta of women with histologically-confirmed over-invasive placentation. METHODS: The patterns of expression and levels of sE-CAD were analyzed by Western blot, immunoassay, and immunoprecipitation. Tissue immunostaining for E-CAD, cytokeratin-7 (epithelial marker), vimentin (mesenchymal marker), ADAM10, PSEN-1 and ß-catenin expression were investigated in parallel. RESULTS: N-terminus cleaved 80 kDa sE-CAD fragments were present in serum of pregnant women with gestational age regulation of the circulatory levels. Women with advanced trophoblast invasion did not display circulatory levels of sE-CAD different from those of women with normal placentation. Histologically, extravillous trophoblasts (EVT) closer to the placental-myometrial interface demonstrated less E-CAD staining than those found deeper in the myometrium. These cells expressed both vimentin and cytokeratin, an additional feature of EMT. EVT of placentas with advanced invasion displayed intracellular E-CAD C-terminus immunoreactivity predominating over that of the extracellular N-terminus, a pattern consistent with preferential PSEN-1 processing. DISCUSSION: Local processing of E-CAD may be an important molecular mechanism controlling the invasive phenotype of accreta EVT.

Hyperferritinemia and markers of inflammation and oxidative stress in the cord blood of HIV-exposed, uninfected (HEU) infants.

OBJECTIVES: The purpose of this study was to evaluate markers of iron status and inflammation/oxidative stress in maternal and cord blood (CB) of HIV-infected and HIV-uninfected women as potential mechanisms for poor outcomes among HIV-exposed, uninfected (HEU) infants. METHODS: Maternal venous blood and CB specimens were obtained from 87 pregnant women (45 HIV-infected and 42 HIV-uninfected) enrolled at Kalafong Hospital, Pretoria, South Africa. Iron status [serum iron, ferritin and transferrin concentrations, transferrin saturation, soluble transferrin receptor (sTfR) concentration and the sTfR/log ferritin (sTfR/F) index], antenatal exposure to inflammation (CB C-reactive protein and interleukin-6 concentrations and haptoglobin switch-on status) and oxidative stress [total radical trapping ability of CB plasma (TRAP) and chronic oxidative stress (soluble receptor of advanced glycation end-products (sRAGE) concentration] were assessed in laboratory studies. RESULTS: There were no differences between the HIV-infected and HIV-uninfected groups in maternal haematological and iron indices, except that HIV-infected mothers had decreased white blood cell counts (P = 0.048) and increased serum ferritin concentrations (P = 0.032). Ferritin levels were significantly higher in CB than in maternal blood (P < 0.001) in both groups and further elevated in the CB of HEU infants (P = 0.044). There was also an inverse relationship between CB sTfR/F index and sRAGE (r = -0.43; P = 0.003) in the HIV-infected but not in the HIV-uninfected group. CONCLUSIONS: Our study showed for the first time that ferritin was significantly elevated in CB of HEU infants. The inverse relationship between sTfR/F index and sRAGE in CB suggests that chronic oxidative stress or RAGE axis activation in HIV-infected mothers may play a role in modulating ferritin levels.

Risk factors associated with preterm delivery after fetoscopic laser ablation for twin-twin transfusion syndrome.

OBJECTIVE: Despite improved perinatal survival following fetoscopic laser ablation (FLA) for twin-twin transfusion syndrome (TTTS), prematurity remains an important contributor to perinatal mortality and morbidity. The objective of the study was to identify risk factors for complicated preterm delivery after FLA. METHODS: Retrospective cohort study of prospectively collected data on maternal/fetal demographics and pre-operative, operative and postoperative variables of 459 patients treated with FLA in three USA fetal centers. Multivariate linear regression was performed to identify significant risk factors associated with preterm delivery, which were cross-validated using the k-fold method. Multivariate logistic regression was performed to identify risk factors for early compared with late preterm delivery based on median gestational age at delivery of 32 weeks. RESULTS: There were significant differences in case selection and outcomes between the centers. After controlling for the center of surgery, multivariate analysis indicated that a lower maternal age at procedure, a history of previous prematurity, shortened cervical length, use of amnioinfusion, a cannula diameter of 12 French (Fr), lack of a collagen plug placement and iatrogenic preterm premature rupture of membranes (iPPROM) were significantly associated with a lower gestational age at delivery. CONCLUSIONS: Specific fetal/maternal and operative variables are associated with preterm delivery after FLA for the treatment of TTTS. Further studies to modify some of these variables may decrease the perinatal morbidity after laser therapy.

The role of urinary soluble endoglin in the diagnosis of pre-eclampsia: comparison with soluble fms-like tyrosine kinase 1 to placental growth factor ratio.

OBJECTIVE: Endoglin, an anti-angiogenic glycoprotein expressed on endothelial cells, has been proposed recently as a biomarker of pre-eclampsia (PE). Given that PE is characterised by an imbalance of angiogenic factors, we sought to determine the clinical utility of urinary soluble endoglin, relative to the soluble fms-like tyrosine kinase 1 to placental growth factor (PlGF) ratio, in the diagnosis of PE during gestation. DESIGN: Prospective observational cohort. SETTING: Tertiary referral university hospital. POPULATION: Two hundred and thirty-four pregnant women were enrolled prospectively in the following groups: healthy controls, n = 63; gestational age (GA), median (interquartile range), 33 weeks (27-39 weeks); chronic hypertension, n = 27; GA, 33 weeks (30-36 weeks); mild PE, n = 38; GA, 37 weeks (34-40 weeks); severe PE, n = 106; GA, 32 weeks (29-37 weeks). METHODS: Free urinary levels of soluble endoglin, soluble fms-like tyrosine kinase 1 and PlGF were measured by sensitive and specific immunoassay. Levels for all urinary analytes were normalised to creatinine. MAIN OUTCOME MEASURES: Urinary soluble endoglin, and the soluble fms-like tyrosine kinase 1 to PlGF ratio. RESULTS: In healthy controls, urinary soluble endoglin levels were increased significantly at term relative to those earlier in gestation. Severe PE was characterised by an increased urinary level of soluble endoglin, soluble fms-like tyrosine kinase 1, protein to creatinine ratio and soluble fms-like tyrosine kinase 1 to PlGF ratio compared with all other groups. There was a direct correlation between urinary soluble endoglin and proteinuria that remained after GA correction (R = 0.382, P < 0.001). Urinary soluble endoglin could not differentiate mild PE from severe preterm PE. Overall, soluble endoglin had the ability to discriminate PE from chronic hypertension and healthy controls only in women who were evaluated at <37 weeks of GA. The sensitivity, specificity and accuracy of urinary soluble endoglin alone in the diagnosis of PE or in the identification of women with PE requiring a mandated delivery before 37 weeks of gestation were 70%, 86% and 76%, respectively. These values were inferior to those of the soluble fms-like tyrosine kinase 1 to PlGF ratio (P < 0.001). The addition of urinary soluble endoglin did not improve the diagnostic accuracy of the soluble fms-like tyrosine kinase 1 to PlGF ratio alone. CONCLUSIONS: We have provided evidence that soluble endoglin is present and elevated in the urine of women who develop preterm PE. Urinary soluble endoglin has only limited ability to determine the severity of PE and to distinguish between PE and chronic hypertension both preterm and at term. Compared with urinary soluble endoglin, the soluble fms-like tyrosine kinase 1 to PlGF ratio remains a better marker of disease presence, severity and outcome.

Fetal inflammatory response in women with proteomic biomarkers characteristic of intra-amniotic inflammation and preterm birth.

OBJECTIVE: To determine the relationship between presence of amniotic fluid (AF) biomarkers characteristic of inflammation (defensins 2 and 1 and calgranulins C and A) and fetal inflammatory status at birth. DESIGN: Prospective observational cohort. SETTING: Tertiary referral University hospital. POPULATION: One hundred and thirty-two consecutive mothers (gestational age, median [interquartile range]: 29.6 [24.1-33.1] weeks) who had a clinically indicated amniocentesis to rule out infection and their newborns. METHODS: Intra-amniotic inflammation was diagnosed by mass spectrometry surface-enhanced-laser-desorption-ionization time of flight (SELDI-TOF). The AF proteomic fingerprint (mass-restricted [MR] score) ranges from 0-4 (none to all biomarkers present). The intensity of intra-amniotic inflammation was graded based on the number of proteomic biomarkers: MR score 0: 'no' inflammation, MR score 1-2: 'minimal' inflammation and MR score 3-4: 'severe' inflammation. At birth, cord blood was obtained for all women. Severity of histological chorioamnionitis and early-onset neonatal sepsis (EONS) was based on established histological and haematological criteria. Interleukin-6 (IL-6) levels were measured by sensitive immunoassays. The cord blood-to-AF IL-6 ratio was used as an indicator of the differential inflammatory response in the fetal versus the AF compartment. MAIN OUTCOME MEASURES: To relate proteomic biomarkers of intra-amniotic infection to cord blood IL-6 and to use the latter as the primary marker of fetal inflammatory response. RESULTS: Women with intra-amniotic inflammation delivered at an earlier gestational age (analysis of variance, P<0.001) and had higher AF IL-6 levels (P<0.001). At birth, neonates of women with severe intra-amniotic inflammation had higher cord blood IL-6 levels (P=0.002) and a higher frequency of EONS (P=0.002). EONS was characterised by significantly elevated cord blood IL-6 levels (P<0.001). Of the 39 neonates delivered by mothers with minimal intra-amniotic inflammation, 15 (39%) neonates had umbilical cord blood IL-6 levels above the mean for the group and 2 neonates had confirmed sepsis. The severity of the neutrophilic infiltrate in the chorionic plate (P<0.001), choriodecidua (P=0.002), umbilical cord (P<0.001) but not in the amnion (P>0.05) was an independent predictor of the cord blood-to-AF IL-6 ratio. Relationships were maintained following correction for gestational age, birthweight, amniocentesis-to-delivery interval, caesarean delivery, status of the membranes, race, MR score and antibiotics and steroid exposure. CONCLUSIONS: We provide evidence that presence of proteomic biomarkers characteristic of inflammation in the AF is associated with an increased inflammatory status of the fetus at birth. Neonates mount an increased inflammatory status and have positive blood cultures even in the context of minimal intra-amniotic inflammation.

Proteomics of the amniotic fluid in assessment of the placenta. Relevance for preterm birth.

Proteomics is the study of expressed proteins and has emerged as a complement to genomic research. The major advantage of proteomics over DNA-RNA based technologies is that it more closely relates to phenotypes and not the source code. Proteomics thus holds the promise of providing a direct insight into the true mechanisms of human diseases. Historically, examination of the placenta has been the first modality to subclassify pathogenetic entities responsible for preterm birth. Because placenta is a key pathophysiological participant in several major obstetrical syndromes (preterm birth, pre-eclampsia, intrauterine growth restriction) identification of relevant biomarkers of placental function can profoundly impact on the prediction of fetal outcome and treatment efficacy. Since proteomics is a young science and studies that associate proteomic patterns with long-term outcome require follow-up of children up to school age, using placental pathological footprints of cellular injury as intermediate outcomes can be useful in the interim. Furthermore, knowledge on the identity of the dysregulated proteins may provide the needed breakthrough insight into novel pathophysiological pathways and unravel possible targets for therapeutical intervention that could not have been envisioned through hypothesis-driven approaches.

Placental growth factor in the cerebrospinal fluid of women with preeclampsia.

OBJECTIVE: Recent data suggest that excess circulating soluble fms-like tyrosine kinase-1 (sFlt-1) may causally relate to preeclampsia. This study investigates the levels of sFlt-1, VEGF, and PlGF in cerebrospinal fluid (CSF) of patients with preeclampsia and normotensive controls. METHODS: CSF was collected from preeclamptic patients (n=15) and controls (n=7) at the time of spinal anesthesia and assayed for PlGF, sFlt-1, and VEGF (total and free) by specific immunoassays. RESULTS: All sought angiogenic factors were measurable. Levels of free PlGF but not sFlt-1 or VEGF (total or free) were increased in CSF of preeclamptic women. There was no significant difference in the ratios of angiogenic factors in the CSF of women with preeclampsia. There was no correlation between levels of angiogenic factors and CSF cell counts or severity of symptoms. CONCLUSION: Elevated levels of PlGF in CSF preeclamptic women may promote vascular permeability and contribute to the hypertensive encephalopathy seen in such patients.

Pregnancy and estradiol decrease GTPase activity in the guinea pig uterine artery.

The mechanisms by which pregnancy redistributes cardiac output in an organ-specific manner are poorly understood. We propose that it is consequential to estrogen-mediated alterations in G protein-mediated signal transduction. Aortas and uterine (UAs) and mesenteric arteries (MAs) were obtained from late-pregnant, nonpregnant, or ovariectomized guinea pigs chronically treated with 17beta-estradiol. High-affinity GTPase activity was assayed enzymatically. The cGMP generated in response to the endothelium-dependent agonist ACh was measured in UAs incubated with or without cholera toxin (CTX, which inhibits G(s)alpha). Pregnancy significantly decreased UA but not aorta or MA GTPase activity. 17beta-Estradiol decreased UA GTPase activity compared with untreated ovariectomized animals. ACh increased cGMP in pregnant but not nonpregnant UAs. Pretreatment of nonpregnant UAs with CTX increased ACh-induced cGMP levels similar to pregnancy. Thus pregnancy and estradiol decrease the GTPase activity of a CTX-sensitive G protein in UAs, increasing receptor-dependent cGMP release. This alteration in receptor-mediated G protein coupling in UAs may contribute to the characteristic cardiovascular adaptation to pregnancy.

Use of McRoberts' position during delivery and increase in pushing efficiency.

McRoberts' position is used during the second stage of labour to facilitate delivery of the fetal shoulders. Few clinical studies have been done to measure its efficacy. We measured intrauterine pressure in 22 women in term labour, after the vertex reached 3+ station, in the dorsal lithotomy position. Patients pushed with legs either in stirrups or hyperflexed by 1358 (McRoberts' position). Maternal valsalva transiently increased the expulsive force by 32% over naturally occurring contractions. Use of McRoberts' position almost doubled the intrauterine pressure developed by contractions alone (from 1653 mm Hg s to 3262 mm Hg s [97%]).

Marked variation in responses to long-term nitric oxide inhibition during pregnancy in outbred rats from two different colonies.

OBJECTIVE: Some but not all studies have shown that long-term nitric oxide synthase inhibition during pregnancy induces symptoms similar to those of preeclampsia that include hypertension, proteinuria, and intrauterine growth restriction. This study was undertaken to compare the effects of long-term nitric oxide synthase inhibition during pregnancy on blood pressure and fetal weight between Sprague-Dawley rats from outbred colonies of two different suppliers. STUDY DESIGN: Osmotic minipumps were inserted on day 10 or day 17 of pregnancy in Sprague-Dawley rats obtained from Charles River Laboratories, Inc, Wilmington, Mass, or Harlan Sprague Dawley, Inc, Indianapolis, Ind. The pumps were set to deliver vehicle only (control group) or N omega-nitro-L -arginine methyl ester (a nitric oxide synthase inhibitor) at a rate of 50 mg/d until postpartum day 7. Systolic blood pressures were measured daily with the tail-cuff method. Neonatal weights and survival were recorded. RESULTS: N omega-nitro-L -arginine methyl ester infusion initiated on gestational day 10 increased blood pressure relative to control levels in all rats studied. Harlan rats were much more sensitive to the hypertensive effect of N omega-nitro-L -arginine methyl ester. When N omega-nitro-L -arginine methyl ester infusion was initiated on gestational day 17, blood pressure increased only in Harlan rats. Pups born to Harlan rats treated with N omega-nitro-L -arginine methyl ester had lower birth weights and a higher stillbirth rate than did pups of Charles River rats. The degree of hypertension was significantly correlated with the deleterious effects of N omega-nitro-L -arginine methyl ester on the fetuses. CONCLUSION: Within the same strain of rats the effects of long-term nitric oxide synthase inhibition on blood pressure and fetal outcome depended on the original animal colony, with animals from Harlan Sprague Dawley being more sensitive than those from Charles River Laboratories. This difference in response between animals from different suppliers is most likely caused by genetic differences inbred into the strain. In addition to explaining some of the reported inconsistencies between laboratories, these results may also provide insights into the genetic basis of preeclampsia.

Natriuretic peptide-induced relaxation of myometrium from the pregnant guinea pig is not mediated by guanylate cyclase activation.

We tested both relaxation and cGMP generation by atrial (ANP), brain (BNP), and C-type natriuretic peptide (CNP) in oxytocin-stimulated myometrium from near-term pregnant guinea pigs to investigate the ability and mechanism of natriuretic peptides to inhibit myometrial contractility. Myometrial strips were contracted by 10(-8) M oxytocin, and relaxation to the cumulative addition (10(-9)-10(-6) M) of the natriuretic peptides measured. Maximal relaxation to BNP was significantly greater than to ANP (52 versus 32% respectively; p < 0.05), whereas CNP failed to produce relaxation. However, the increase in cGMP produced by BNP (10(-7) M) was significantly less than that produced by ANP (10(-7) M) (4.5 versus 7.0 times basal; p < 0.05); CNP did not increase myometrial cGMP. Anantin, a competitive blocker of the guanylate cyclase A receptor, significantly reduced the increase in cGMP produced by ANP and BNP, but had no effect on relaxation induced by either peptide. Rp-8-Br-cGMP, an inhibitor of the cGMP-dependent protein kinase, did not alter BNP-induced relaxation. The atrial natriuretic peptide-fragment 4-23 amide, a natriuretic peptide clearance receptor agonist, failed to inhibit oxytocin-stimulated myometrial contraction. We conclude that natriuretic peptide induced relaxation of oxytocin-stimulated myometrium from the pregnant guinea pig is not mediated by either guanylate cyclase A or B activation, is independent of the cGMP pathway, and does not involve clearance receptor activation. Our results suggest that natriuretic peptide-induced relaxation of pregnant myometrium is mediated via a novel mechanism.

Chorion releases a factor that inhibits oxytocin-stimulated myometrial contractility in the pregnant guinea pig.

It was postulated that chorion releases a substance necessary for the maintenance of uterine quiescence during pregnancy. A decrease in the release of this substance at the end of the pregnancy would be necessary for normal myometrial activation. This hypothesis was tested by demonstrating the ability of chorion to inhibit oxytocin-stimulated myometrial contractility in vitro. Tissues were obtained from timed pregnant Duncan-Hartley guinea pigs either at pre-term or near-term gestation. Myometrial strips were placed in organ baths for isometric tension measurement and contractions stimulated by oxytocin (10(-8) mol/l). Fetal membranes or conditioned medium from chorion were added directly to the organ bath. Near-term chorion and chorion conditioned-medium decreased oxytocin-stimulated contractile activity to 39% and 49% respectively. Neither pre-term nor near-term amnion reduced oxytocin-stimulated myometrial contractile activity. Relaxation induced by pre-term chorion was greater than near-term chorion (23% and 41% of the oxytocin-induced basal level respectively; P < 0.05). Further, chorion-induced relaxation was independent of the gestational age of the myometrium. Human chorion from a term, not-in-labour woman also inhibited oxytocin-stimulated guinea pig myometrial contractility. It was concluded that the chorion releases a substance or substances that reduce oxytocin-stimulated myometrial contractility and may be involved in the maintenance of uterine quiescence during pregnancy.

BCL-2 is involved in preventing oxidant-induced cell death and in decreasing oxygen radical production.

It has been hypothesized that programmed cell death is mediated, in part, through the formation of free radicals via oxidative pathways. Furthermore, it has been proposed that BCL-2 acts to inhibit cell death by interfering with the production of oxygen-derived free radicals induced by a wide variety of stimuli. In order to examine the antioxidant function of BCL-2, we transfected mouse epidermal cells JB6 clone 41 with the expression vector pD5-Neo-BCL-2 and studied the effect of BCL-2 overexpression on oxidant-induced cell death and on the production of reactive oxygen species. Compared to Neo control cells, BCL-2-expressing cells are more resistant to the killing and growth retardation induced by hydrogen peroxide, superoxide, or by the oxygen radical-generating quinone-containing compounds menadione, diaziquone and adriamycin. The latter compounds generate reactive oxygen species during bioreductive metabolism. In addition, the exposed cells die by necrosis rather than apoptosis. Hydroxyl radical levels generated by the quinone-containing agents were low in BCL-2-expressing JB6 cells compared to control Neo cells. BCL-2, however, does not change the activities of the major cellular antioxidant enzymes superoxide dismutase, catalase or glutathione peroxidase. On the other hand, the glutathione concentrations increased in BCL-2 overexpressing cells after oxidative challenge, while the opposite was true for control cells. Thus, our results suggest that BCL-2 inhibition of oxidant-induced cell death is mediated, at least in part, through an antioxidant pathway, and that this pathway involves glutathione.

Differential alterations in responsiveness in particulate and soluble guanylate cyclases in pregnant guinea pig myometrium.

OBJECTIVE: The mechanism underlying myometrial quiescence during pregnancy is unknown. Our group has previously shown that during pregnancy myometrial cyclic guanosine monophosphate content rises to several hundred times the nonpregnant levels, only to abruptly decline days before the onset of labor. Cyclic guanosine monophosphate plays an integral role in the relaxation of smooth muscle. The aim of this investigation was therefore to determine the effects of pregnancy on both soluble and particulate guanylate cyclase enzymatic activities and messenger ribonucleic acid expressions. STUDY DESIGN: Myometrium was obtained from randomly cycling adult nonpregnant guinea pigs and near-term (50-60 days' gestation) pregnant guinea pigs of similar chronologic age. Subcellular fractions were prepared by differential ultracentrifugation. Guanylate cyclase activity was determined by the conversion of guanosine triphosphate to cyclic guanosine monophosphate under basal or stimulated conditions in either the soluble guanylate cyclase or particulate guanylate cyclase fraction. A nitric oxide donor, S-nitroso- N-penacillamine, was used to activate soluble guanylate cyclase (n = 10 animals in each group). Several natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) and uroguanylin were used to stimulate the different particulate guanylate cyclase isoforms guanylate cyclase A, guanylate cyclase B, and guanylate cyclase C, respectively, in pregnant (n = 8) and nonpregnant (n = 6) animals. Cyclic guanosine monophosphate content was measured by radioimmunoassay, and enzymatic activity was expressed as picomoles of cyclic guanosine monophosphate per milligram of protein per minute. Total guanylate cyclase represented the sum of soluble guanylate cyclase and particulate guanylate cyclase activities for a tissue. To investigate whether the observed changes in guanylate cyclase activity were paralleled by changes in receptor expression, messenger ribonucleic acid levels of the genes for guanylate cyclase A and guanylate cyclase B isoforms were quantified by ribonuclease protection assay (n = 5 animals in each group). RESULTS: Under basal conditions particulate guanylate cyclase represented 78% (nonpregnant state) to 88% (during pregnancy) of the total guanylate cyclase activity in the guinea pig myometrium. Pregnancy further reduced myometrial soluble guanylate cyclase (both basal and stimulated by nitric oxide) relative to the nonpregnant state. Pregnancy selectively increased atrial natriuretic peptide-stimulated particulate guanylate cyclase activity (attributed to guanylate cyclase A), although it did not change basal myometrial particulate guanylate cyclase activity in general. Guanylate cyclase B (particulate guanylate cyclase stimulated by C-type natriuretic peptide) and guanylate cyclase C (particulate guanylate cyclase stimulated by uroguanylin) activities were unaltered by pregnancy. The selective increase in responsiveness of particulate guanylate cyclase to atrial natriuretic peptide during pregnancy was not paralleled by an increased in level of messenger ribonucleic acid for the gene for guanylate cyclase A. CONCLUSION: Pregnancy reduced the in vitro responsiveness of the myometrial soluble guanylate cyclase to nitric oxide while increasing the responsiveness of the particulate isoform to atrial natriuretic peptide and brain natriuretic peptide through a mechanism independent of any change in receptor expression.