Reduced expression of the epidermal growth factor signaling system in preeclampsia.

INTRODUCTION: The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. METHODS: Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. RESULTS: Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). DISCUSSION: Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia.

Free fetal DNA levels in patients at risk of preterm labour.

OBJECTIVES: Our main goals were to evaluate the capability of ffDNA to increase the accuracy in prediction of preterm labour by cervical length and to explore potential mechanisms of disease associated with this pathology. METHODS: Fifty-six women, with male fetus, with cervical length assessment at between 22 and 24 weeks were included in the study and divided in 1) Short cervix (<15 mm) delivered at term (T = 20); 2) Short cervix delivered before 37 weeks (PT = 14); and 3) Patients who delivered at term with normal cervical length (N = 22). Maternal plasma samples were collected between 22 and 24 weeks of gestational age. PCR using primers against DYS14 gene were used to quantified ffDNA in plasma samples. Statistical analysis was done using ANOVA test and spearman´s correlation. RESULTS: The median gestational age at delivery for short cervix groups was 26 + 1 for PT and 39 + 3 for T. The control group delivered at a median gestational age of 39 + 6 weeks. ffDNA was detectable in all cases. There was no significant difference between the 3 groups. Similarly, no significant correlation was observed between ffDNA and gestational age at delivery (r = -0.23; p = 0.07). CONCLUSIONS: ffDNA does not increase the accuracy of short cervix at between 22 and 24 weeks for the prediction of preterm labour.

Review: Differential placental macrovascular and microvascular endothelial dysfunction in gestational diabetes.

Human endothelial dysfunction is a common feature in many diseases of pregnancy, such as gestational diabetes (GD). Metabolic changes include abnormal synthesis of nitric oxide (NO) and abnormal membrane transport of l-arginine and adenosine in primary cultures of human umbilical vein (HUVEC, macrovascular) and placental microvillus (hPMEC, microvascular) endothelial cells. These alterations are associated with modifications in the expression and activity of endothelial (eNOS) and inducible (iNOS) NO synthases, respectively, an effect that is maintained at least up to passage 5 in culture. HUVEC and hPMEC exhibit expression and activity of the human cationic amino acid transporter 1 (hCAT-1), equilibrative nucleoside transporters 1 (hENT1) and hENT2, as well as the corresponding SLC7A1, SLC29A1 and SLC29A2 gene promoter activities. Altered gene expression results from increased NO level, protein kinase C, mitogen-activated protein kinases, and hCHOP-C/EBPα transcription factor activation. Reduced ENT-mediated adenosine transport in GD is associated with stimulation of the l-arginine/NO pathway, and mainly due to reduced expression and activity of hENT1. In addition, hENT2 activity seems able to restore the reduced adenosine transport in GD. Additionally, insulin exerts a differential modulation of endothelial cells from macrocirculation compared with microcirculation, possibly due to expression of different insulin receptor isoforms. It is suggested that a common functional characteristic leading to changes in the bioavailability of adenosine and metabolism of l-arginine is evidenced by human fetal micro and macrovascular endothelium in GD.

Adiponectin multimers in maternal plasma.

OBJECTIVE: Adiponectin is an anti-diabetic, anti-atherogenic, anti-inflammatory, and angiogenic adipokine that circulates in oligomeric complexes including: low molecular weight (LMW) trimers, medium molecular weight (MMW) hexamers, and high molecular weight (HMW) isoforms. The aim of this study was to determine whether there are changes in adiponectin multimers in pregnancy and as a function of maternal weight. STUDY DESIGN: In this cross-sectional study, plasma concentrations of total, HMW, MMW, and LMW adiponectin were determined in women included in three groups: (1) normal pregnant women of normal body mass index (BMI) (n = 466), (2) overweight pregnant women (BMI >or=25; n = 257), and (3) non-pregnant women of normal weight (n = 40). Blood samples were collected once from each woman between 11 and 42 weeks of gestation. Plasma adiponectin multimer concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Non-parametric statistics were used for analysis. RESULTS: (1) The median HMW adiponectin concentration and the median HMW/total adiponectin ratio were significantly higher, and the median LMW adiponectin concentration was significantly lower in pregnant women than in non-pregnant women. (2) Among pregnant women, the median plasma concentration of total, HMW, and MMW adiponectin was significantly higher in normal weight women than in overweight patients. (3) Maternal HMW was the most prevalent adiponectin multimer regardless of gestational age or BMI status. (4) There were no significant differences in the median concentration of total, MMW, and LMW adiponectin and their relative distribution with advancing gestation. CONCLUSION: Human pregnancy is characterized by quantitative and qualitative changes in adiponectin multimers, especially the most active isoform, HMW adiponectin.

The use of inversion mode and 3D manual segmentation in volume measurement of fetal fluid-filled structures: comparison with Virtual Organ Computer-aided AnaLysis (VOCAL).

OBJECTIVE: Volume measurements by three-dimensional (3D) ultrasonography are considered more accurate than those performed by two-dimensional (2D) ultrasonography. The purpose of this study was to compare the agreement of three techniques, as well as the inter- and intraobserver agreements for volume measurements of fetal fluid-filled structures. METHODS: Fifty 3D volume datasets of fetal stomachs and bladders were explored. Volume measurements were performed independently by two observers using: (1) Virtual Organ Computer-aided AnaLysis (VOCAL); (2) inversion mode; and (3) 'manual segmentation'. Reliability was evaluated using intraclass correlation coefficient (ICC), and Bland-Altman plots were generated to examine bias and agreement. The time required to complete the measurements was compared using Student's t-test or the Wilcoxon Signed Rank Test, and P-values < 0.025 or < 0.05 were considered statistically significant. RESULTS: All volume datasets could be measured using the three techniques. A high degree of reliability was observed between: (1) VOCAL and inversion mode (ICC, 0.995; 95% CI, 0.992-0.997); (2) VOCAL and manual segmentation (ICC, 0.997; 95% CI, 0.995-0.998); and (3) inversion mode and manual segmentation (ICC, 0.995; 95% CI, 0.992-0.997). There was good agreement between VOCAL and inversion mode (mean, - 2.4%; 95% limits of agreement, - 20.1 to 15.3%), VOCAL and manual segmentation (mean, - 8.3%; 95% limits of agreement, - 28.8 to 12.2%) as well as between inversion mode and manual segmentation (mean, 5.9%, 95% limits of agreement: - 14.3 to 26%). Manual segmentation and inversion mode measurements were obtained significantly faster than those by VOCAL. CONCLUSIONS: Volume measurements of fetal fluid-filled structures of relatively regular shape with inversion mode and manual segmentation are feasible. Both techniques have good agreement with VOCAL and are significantly faster than VOCAL. Inversion mode is a reliable method for volume calculations of fluid-filled organs, whereas manual segmentation can be used when volume measurements by VOCAL or inversion mode are technically difficult to obtain, such as solid structures with poorly defined borders as the volume dataset is rotated, like the uterine cervix.

Clinical significance of the presence of amniotic fluid 'sludge' in asymptomatic patients at high risk for spontaneous preterm delivery.

OBJECTIVES: To determine the clinical significance of the presence of amniotic fluid (AF) 'sludge' among asymptomatic patients at high risk for spontaneous preterm delivery. METHODS: This retrospective case-control study included 281 patients with (n = 66) or without (n = 215) AF 'sludge', who underwent transvaginal ultrasound examination between 13 and 29 completed weeks of gestation. Patients with threatened preterm labor, multiple gestation, fetal anomalies, placenta previa or uterine contractions were excluded. RESULTS: The prevalence of AF 'sludge' in the study population was 23.5% (66/281). The rates of spontaneous preterm delivery at < 28 weeks, < 32 weeks, < 35 weeks and < 37 weeks of gestation were 14.7% (29/197), 21.3% (46/216), 28.7% (62/216) and 42.1% (91/216), respectively. Patients with 'sludge' had: (1) a higher rate of spontaneous preterm delivery at < 28 weeks (46.5% (20/43) vs. 5.8% (9/154); P < 0.001), < 32 weeks (55.6% (25/45) vs. 12.3% (21/171); P < 0.001) and < 35 weeks (62.2% (28/45) vs. 19.9% (34/171); P < 0.001); (2) a higher frequency of clinical chorioamnionitis (15.2% (10/66) vs. 5.1% (11/215); P = 0.007), histologic chorioamnionitis (61.5% (40/65) vs. 28% (54/193); P < 0.001) and funisitis (32.3% (21/65) vs. 19.2% (37/193); P = 0.03); (3) a higher frequency of preterm prelabor rupture of membranes (PROM) (39.4% (26/66) vs. 13.5% (29/215); P < 0.001), lower gestational age at preterm PROM (median 24.7 (interquartile range (IQR), 22.3-28.1) weeks vs. 32.3 (IQR, 27.7-34.8) weeks; P < 0.001); and (4) shorter median ultrasound-to-delivery interval ('sludge' positive 127 days (95% CI, 120-134 days) vs. 'sludge' negative 161 days (95% CI, 153-169 days); P < 0.001) and ultrasound-to-preterm PROM interval ('sludge' positive 23 days (95% CI, 7-39 days) vs. 'sludge' negative 57 days (95% CI, 38-77 days); P = 0.003) than those without 'sludge'. AF 'sludge' was an independent explanatory variable for the occurrence of spontaneous preterm delivery at < 28 weeks, < 32 weeks and < 35 weeks, preterm PROM, microbial invasion of the amniotic cavity (MIAC) and histologic chorioamnionitis. Moreover, the combination of a cervical length < 25 mm and 'sludge' conferred an odds ratio of 14.8 and 9.9 for spontaneous preterm delivery at < 28 weeks and < 32 weeks, respectively. CONCLUSIONS: AF 'sludge' is an independent risk factor for spontaneous preterm delivery, preterm PROM, MIAC and histologic chorioamnionitis in asymptomatic patients at high risk for spontaneous preterm delivery. Furthermore, the combination of 'sludge' and a short cervix confers a higher risk for spontaneous preterm delivery at < 28 weeks and < 32 weeks than a short cervix alone.

Pleural and pericardial effusion: a potential ultrasonographic marker for the prenatal differential diagnosis between congenital diaphragmatic eventration and congenital diaphragmatic hernia.

OBJECTIVES: To determine whether or not the presence of pleural and/or pericardial effusion can be used prenatally as an ultrasonographic marker for the differential diagnosis between diaphragmatic eventration and diaphragmatic hernia. METHODS: We present two case reports of non-isolated diaphragmatic eventration associated with pleural and/or pericardial effusion. Additionally, we reviewed the literature for all cases of congenital diaphragmatic hernia (CDH) and diaphragmatic eventration that met the following criteria: (1) prenatal diagnosis of a diaphragmatic defect and (2) definitive diagnosis by autopsy or surgery. The frequencies of pleural effusion, pericardial effusion and hydrops were compared between the two conditions using Fisher's exact test. A subanalysis was conducted of cases with isolated diaphragmatic defects (i.e. diaphragmatic defects not associated with hydrops and other major structural or chromosomal anomalies). RESULTS: A higher proportion of fetuses with diaphragmatic eventration had associated pleural and pericardial effusions compared with fetuses with diaphragmatic hernia (58% (7/12) vs. 3.7% (14/382), respectively, P < 0.001). This observation remained true when only cases of diaphragmatic defects not associated with hydrops and other major structural or chromosomal anomalies were compared (29% (2/7) with eventration vs. 2.2% (4/178) with CDH, P < 0.02). CONCLUSIONS: The presence of pleural and/or pericardial effusion in patients with diaphragmatic defects should raise the possibility of a congenital diaphragmatic eventration. This information is clinically important for management and counseling because the prognosis and treatment for CDH and congenital diaphragmatic eventration are different. Published by John Wiley & Sons, Ltd.

Surfactant protein-A mRNA expression by human fetal membranes is increased in histological chorioamnionitis but not in spontaneous labour at term.

Surfactant protein-A (SP-A) is produced by the fetal lung, participates in innate immunity, and has been proposed to play a role in the initiation of parturition in mice. Amniotic fluid SP-A concentration increases as a function of gestational age, and SP-A protein has been demonstrated in human chorioamniotic membranes. This study was conducted to determine whether parturition at term, gestational age and chorioamnionitis in preterm delivery (PTD) are associated with changes in the expression of SP-A in the chorioamniotic membranes. Chorioamniotic membranes were obtained from women at term and women with PTD (n=58). SP-A mRNA and protein expression was detected in amniotic epithelial cells, chorionic trophoblasts and macrophages by in situ hybridization and immunohistochemistry. Quantitative real-time reverse transcription-PCR demonstrated predominant expression of SP-A1 mRNA, whose expression was 17.4-fold higher in patients with PTD with chorioamnionitis (n=15) than in those without (n=13) (p=0.018). While no difference was observed in SP-A1 mRNA expression in the chorioamniotic membranes of women at term not in labour (n=16) and those in labour (n=14) (p=0.87), the expression in term membranes was higher than that of membranes from women with PTD without chorioamnionitis (p=0.003). Analysis of JAR choriocarcinoma cells demonstrated SP-A1 mRNA expression that was up-regulated following lipopolysaccharide treatment. Furthermore, monocytic cell lines (THP-1 and U937) and peripheral blood monocytes (CD14+/CD115+) obtained from pregnant women also expressed SP-A1 mRNA and protein, suggesting the presence of autocrine/paracrine activation in vivo. Interestingly, a mid-trimester amniotic fluid sample obtained from a case of tracheal atresia contained SP-A (3.13 microg/ml), indicating the presence of SP-A of extrapulmonary origin. These findings suggest not only that SP-A expression is a part of the innate immune response deployed during chorioamniotic inflammation, but also that chorioamniotic membranes are a source of SP-A in the amniotic fluid with advancing gestation.

Distribution of CD14+ and CD68+ macrophages in the placental bed and basal plate of women with preeclampsia and preterm labor.

OBJECTIVE: Macrophages play a key role in implantation, placentation and parturition. Yet, whether or not the number of macrophages at the fetomaternal interface (basal plate of the placenta and placental bed) is altered in women with preeclampsia is the subject of controversy. The purpose of this study was to compare the immunoreactivity and distribution patterns of CD14 and CD68 positive macrophages in both the basal plate and placental bed from preeclamptic and non-preeclamptic pregnancies. METHODS: A cross-sectional study was conducted. Paraffin embedded sections of placental tissues and placental bed biopsies were obtained from patients with early onset preeclampsia (n=10) and from those with preterm labor/delivery (n=10) without preeclampsia matched for gestational age. Double immunohistochemistry using antibodies to CD14 and CD68 was performed, and the density of double or single positive cells in the basal plate and placental bed was evaluated. Non-parametric statistics were used for analysis. RESULTS: 1) A unique subset of CD14-/CD68+ cells was identified. The cells in question were present at a higher level in the decidua than in the myometrial segment of the placental bed (p<0.01); 2) The density and proportion of CD14+/CD68+ cells (double positive cells) were significantly higher in the myometrial segment than in the basal plate (p=0.0003); and 3) There were no significant differences in the density and patterns of immunopositive macrophages in the basal plate, the decidua, and the myometrium between women with preeclampsia and those with preterm labor/delivery (p>0.05). CONCLUSION: The macrophages at the fetomaternal interface can be dichotomized by CD14 and CD68 immunoreactivity. A gradient of CD14+/CD68+ macrophages was demonstrated between the superficial myometrium and the basal plate regardless of the etiology of preterm birth (preeclampsia or spontaneous preterm labor). The biological function of single positive (CD14-/CD68+) and double positive (CD14+/CD68+) macrophages at the fetomaternal interface remains to be established. The overall findings also suggest that the discrepancies in the literature are due to the varying markers used to detect macrophages and in the anatomical plane of the fetomaternal junction analyzed.

The prevalence and clinical significance of amniotic fluid 'sludge' in patients with preterm labor and intact membranes.

OBJECTIVE: To determine the prevalence and clinical significance of amniotic fluid (AF) 'sludge' observed during transvaginal ultrasound examination of the cervix in patients with preterm labor and intact membranes, and in those with uncomplicated pregnancies. METHODS: This retrospective study included patients with preterm labor and intact membranes (n = 84) and those with uncomplicated term pregnancies (n = 298). The outcome variables included the occurrence of documented microbial invasion of the amniotic cavity (MIAC), histological chorioamnionitis, examination-to-delivery interval, admission to the neonatal intensive care unit (NICU), a composite neonatal morbidity, perinatal death, and delivery within 48 h, 7 days, and < 35 weeks and < 32 weeks. Statistical analysis included Chi-square test, stepwise logistic regression analysis and survival analysis. RESULTS: The prevalence of AF 'sludge' was 1% (3/298) in patients with uncomplicated term pregnancies and 22.6% (19/84) in those with preterm labor and intact membranes. Among patients with preterm labor and intact membranes: (1) cervical length < or = 15 mm was present in 58.3% (49/84) of the patients; (2) the prevalence of MIAC and histological chorioamnionitis was 12.1% (7/58) and 32.9% (25/76), respectively; (3) the rate of spontaneous preterm delivery within 48 h, 7 days, and < 32 weeks and < 35 weeks of gestation was 13.6% (8/59), 28.8% (17/59), 39.5% (17/43) and 50.8% (30/59), respectively; (4) patients with AF 'sludge' had a higher frequency of positive AF cultures [33.3% (6/18) vs. 2.5% (1/40), P = 0.003] and histological chorioamnionitis [77.8% (14/18) vs. 19% (11/58), P < 0.001] than those without AF 'sludge'; (5) a higher proportion of neonates born to patients with AF 'sludge' was admitted to the NICU [64.3% (9/14) vs. 12.9% (8/62), P < 0.01], had a composite neonatal morbidity [36.8% (7/19) vs. 13.8% (9/65), P = 0.04] and died in the perinatal period [36.8% (7/19) vs. 4.6% (3/65), P = 0.001] than those born to women without 'sludge'; (6) a higher proportion of patients with AF 'sludge' had spontaneous delivery within 48 h [42.9% (6/14) vs. 4.4% (2/45), P = 0.001], within 7 days [71.4% (10/14) vs. 15.6% (7/45), P < 0.001], < 32 weeks [75% (9/12) vs. 25.8% (8/31), P = 0.005] and < 35 weeks [92.9% (13/14) vs. 37.8% (17/45), P < 0.001] than those without AF 'sludge'; and (7) patients with AF 'sludge' had a shorter examination-to-delivery interval than those without AF 'sludge' [AF 'sludge' median, 1 (IQR, 1-5) days vs. no AF 'sludge' median, 33 (IQR, 18-58) days; P < 0.001]. CONCLUSION: The presence of AF 'sludge' in patients with preterm labor and intact membranes is a risk factor for MIAC, histological chorioamnionitis and impending preterm delivery.

Cerebrospinal fluid of HTLV-1 associated myelopathy patients induces axonal sproutings and Schwann cell proliferation in the rat sciatic nerve.

HTLV-1 (human T-cell leukemia virus type I) associated myelopathy (HAM) is a demyelinating disease. We showed that the CSF of patients and heated CSF of normal subjects induce a segmentary demyelination in rat nerves, and potentiate trypsin in vitro. Here we further characterize the neuropathy induced by the CSF of patients. Peroneal nerves injected 5-8 days before with native or heated CSF of patients, besides extensive demyelination, presented proliferation of myelinating and nonmyelinating Schwann cells, axonal sprouting, fine fibres with a few turns of myelin, disarray of nonmedullated bundles, desmosome-like junctions, and coated pits and vesicles in Schwann cells and axons. The normal CSF was innocuous to the nerve in its native form, but after heating, it induced a neuropathy in all, similar to that elicited by the CSF of patients. Our findings indicate that the CSF of HAM patients contains a thermostable pathogen for nerves of the rat; a thermostable pathogen also occurs in the normal CSF although its activity is checked by endogenous thermolabile factors. We suggest that the pathogen present in the CSF of HAM patients participates in the disease.

Segmental demyelination induced by cerebrospinal fluid of progressive spastic paraparesis: correlation with altered proteolytic parameters.

Progressive spastic paraparesis (PSP) is a demyelinating disease of the central nervous system. We studied the ability of the cerebrospinal fluid (CSF) of patients to induce alterations in rat peroneal nerves, and to modify the proteolytic activity of trypsin in vitro. Subperineurial injection of native or heated CSF of patients induced segmental demyelination and other cytological alterations 5-7 days later, in the infiltrated zone, while proximal and distal regions were normal. The CSF of normal subjects did not induce demyelination, but upon heating, it did so. Trypsin was strongly inhibited by the normal CSF but upon heating, its inhibitory activity was replaced by a strong potentiation. In contrast, native and heated CSF of patients potentiated trypsin. Our findings indicate that (1) the normal CSF contains a thermostable factor that potentiates trypsin whose function is overruled by thermolabile protease inhibitors; (2) the CSF of PSP patients has a reduced inhibitory activity and a conserved ability to potentiate trypsin; and (3) the CSF is endowed with a pathogenic power that correlates with an unchecked potentiating activity. We propose that the imbalance of a protease system may play a role in the pathogenesis of PSP lesions.