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An Integrated Proteomics and Genomics Approach to Identify Essential Protein Kinases During Human Trophoblast Development.

In the developing human placenta, three subtypes of trophoblast cells, cytotrophoblasts (CTBs), extravillous trophoblasts (EVTs), and syncytiotrophoblasts (STBs), mediate critical functions essential for a successful pregnancy. CTBs constitute the stem/progenitor compartment and differentiate into STBs and EVTs within the floating and anchoring villi, respectively. STBs establish the maternal-fetal exchange interface and secrete human chorionic gonadotropin (hCG), a hormone vital for the maintenance of early pregnancy. EVTs anchor the maternal endometrium and invade the uterine tissue to remodel maternal cells, supporting implantation and progression of pregnancy. In this study, we used human trophoblast stem cells (hTSCs) as a model system and performed quantitative, label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile the proteome and phosphoproteome in TSC stem state (analogous to undifferentiated CTBs) and following their differentiation to STBs and EVTs. Through a multiomics approach, we integrated our proteomics data with global gene expression profiles to correlate cell-type specific gene and protein expression during human trophoblast development. We also identified global phosphoproteome and analyzed kinases that are specifically active in hTSC stem state, as well as in differentiated STBs and EVTs. We experimentally validated specific kinases, such as BUB1B, PAK6, PKYMT1, and TNIK, that are essential for maintaining the hTSC stem-state. Additionally, atypical protein kinase C isoforms PKCζ are essential for STB development, whereas PTK2B, SRC, TRIO, and LYN are important for EVT development. Our findings highlight key kinases uniquely required for specific stages of trophoblast development during human placentation and suggest that pharmacological inhibition of these kinases could negatively impact the placentation process during pregnancy.

Humans

Aversive prenatal stimulation: effects on behavioral, biochemical, and somatic ontogeny in the rat.

Electric foot shock administered to pregnant rats altered the ontogeny of spontaneous motor activity in their pups. Prenatally stimulated (PMS) offspring were more active than controls on Days 1-10 but less active during the 3rd postpartum week. The age of peak activity, a major developmental landmark, occurred in PMS pups around 10 days of age; in controls maximum activity was not seen until the 3rd week. This effect was independent of the gender of the offspring and the timing of the gestational stimulation. Its appearance in both cross-fostered and fostered pups indicated the prenatal origin of the effect. The maturation of spontaneous alternation behavior and several reflexes and the appearance of physical features were not affected by prenatal stimulation. Moreover, both PMS and control groups exhibited an age-related increase in brain concentrations of norepinephrine, serotonin, and 5-hydroxyindoleacetic acid. These findings indicate that spontaneous motor activity is uniquely sensitive to PMS, and as far as can be determined here, PMS produces no generalized alteration in behavioral and physical ontogeny.

Age Factors

Transplacental passage of diazepam following intravenous injection immediately prior to operative vaginal delivery.

The early phase of diaplacental transfer of diazepam was studied in 39 women given the drug as a basic anaesthetic for operative vaginal delivery indicated by prolonged second stage of labour (9 cases), breech delivery (19 cases) and intrauterine hypoxia (11 cases). A total dose of 30 mg diazepam (Valium Roche) was injected intravenously over a period of 15 sec umbilical cord blood was collected immediately after delivery. Diazepam was extracted with diethyl ether and determined by gas chromatography. The concentration of diazepam in cord blood increased from greater than 5-250 ng/ml at 57-60 seconds to 48-1861 ng/ml at 90-100 seconds after completion of the intravenous injection. Thereafter a plateau seemed to be reached but the interindividual variation was still great with values ranging from 45-3034 ng/ml up to 360 seconds. Judged by Apgar score and the clinical course the neonates seemed to be unaffected by the medication administered to the mother.

Anesthesia, Obstetrical

Feto-maternal concentrations of diazepam and W-demethyldiazepam after intra-amniotic diazepam injection.

Ten milligrams of diazepam were injected intraamniotically in 8 mothers prior to therapeutic abortion between 12 and 19 weeks. The diazepam concentrations in the maternal plasma were comparable to those found after the same intramuscular diazepam dose to the mother. The concentration of diazepam in the amniotic fluid 12 to 18 hours after the injection was no longer significantly higher than in the maternal plasma. The concentrations of diazepam in the fetal plasma, liver and brain were comparable to the concentrations resulting from a 10 mg intramuscular diazepam dose to the mother about 2 hours before legal abortion. The feto-maternal ratio of diazepam was of same magnitude as after the intramuscular application to the mother. The results indicate that the disappearance of diazepam from the amniotic fluid in this stage of pregnancy occurs extraplacentally, through the mambranes into the uterine circulation. In the treatment of a fetus with drugs having properties similar to diazepam, intra-amniotic administration is no better than intramuscular administration to the mother.

Adolescent

Assessment of uteroplacental hemodynamics in complicated pregnancy.

Examination of the uteroplacental hemodynamics was performed on 109 occasions in women admitted to the hospital in the third trimester because of various complications of pregnancy. The radioactive tracer 113m In was injected intravenously and the build-up and equilibrium times were measured over the placenta, myometrium and heart. The placental build-up times were most informative, with prolonged values being recorded in about one quarter of these cases. When those extending beyond three minutes were further studied, they were usually found to be associated with severe complications of pregnancy. This did not obtain in cases with shorter placental build-up times.

Adult

Changes in liver tyrosine aminotransferase after acute and chronic administration of morphine in the rat.

Acute administration of Morphine (20 mg/kg/s.c.) in the rat results in a rise of liver tyrosine aminotransferase (TAT) expressed as mumoles of p-hydroxyphenylpyruvate/100 mg/h. With chronic administration, a tolerance develops to this enzymatic effect. TAT induction is not evident in pregnant rats, given the narcotic, in which enzyme levels are already initially high. After delivery TAT returns to normal levels and it is possible to show both induction and tolerance developing to morphine. Enzyme activity in fetal livers is much lower than that of adult animals: after maternal administration of morphine only a modest TAT increase is seen which is not, however, statistically significant. TAT activity is fully evident in livers of offspring, with much higher mean levels in newborn rats from morphine-treated animals, as a possible consequence of morphine deprivation. In this latter group of newborn rats narcotic administration causes TAT activity to return to levels as high as those of naive animals. On the other hand, morphine administration to the prole of naive rats results in an induction of liver TAT.

Animals

Transfer in vitro of three benzodiazepines across the human placenta.

A comparative study of the placental transfer to the foetus of three benzodiazepines was performed using a dual perfusion system of the human placental lobule. A transport fraction was calculated for each benzodiazepine and was compared with reference substances. Relative to antipyrine, the transport fraction of diazepam was 85%, and that of nordiazepam was 84%. The transport fraction of clorazepate represented only 20% of that of tritiated water. The relatively high transfer of diazepam and nordiazepam can be attributed to their high lipid solubility, and the lower transfer of clorazepate is due to its polar nature. It is suggested that in certain instances this benzodiazepine may be of especial value to obstetricians.

Anti-Anxiety Agents

Pharmacokinetics of the placental transfer and distribution of clorazepate and its metabolite nordiazepam in the feto-placental unit and in the neonate.

Clorazepate 20 mg was given i.m. to 49 mothers during the first stage of labour. The elimination of the drug was studied in 27 newborns produced by these mothers. The same dose was given to 13 women who underwent amniocentesis and to 7 women who were breast-feeding. "Total nordiazepam", i.e. the sum of clorazepate and its metabolite nordiazepam, was determined by gas-liquid chromatography in maternal blood, umbilical cord blood (both arterial and venous), amniotic fluid and in milk. Clorazepate was found to cross the placental barrier slowly, but nordiazepam was transferred more rapidly. Nordiazepam was found in the milk and in the blood of neonates after breast-feeding had started.

Adult

Effect of propranolol infusion on the umbilical and uterine circulations of pregnant sheep.

Propranolol was infused intravenously for 60 minutes to five ewes (4 mug per kilogram per minute) or five fetal sheep (10 mug per kilogram per minute). The umbilical blood flow was significantly decreased by 18 per cent from control at 60 minutes with either maternal or fetal propranolol infusion. Uterine blood flow and maternal and fetal mean arterial pressure did not significantly change. Maternal and fetal heart rates decreased 18 and 9 per cent from control, respectively, during maternal propranolol infusion. With propranolol to the fetus, fetal heart rate decreased 15 per cent and maternal heart rate did not change. During all infusion, maternal and fetal arterial pH, PCO2 and PO2 remained within normal physiologic limits.

Acid-Base Equilibrium

Factors determining human chorion laeve permeability in vitro.

An increased mean diffusion permeability across human chorion laeve in vitro was measured for meperidine (D = 5.26 x 10(-6) cm.2 sec.-1) and diazepam (D = 4.51 X 10(-6) cm.2 sec.-1). These values corresponded to large chloroform-buffer partition coefficients (49 and 29) measured for these two compounds. Diffusion permeability values of 3.98 and 2.18 x 10-6 cm.2 sec.-1 measured for urea and glucose corresponded to their relative insolubility in lipid, as indicated by chloroform-buffer partition coefficients of 0.05 and 0.0004, respectively. An increase in placental permeability in vitro to the weak organic acid 5,5-dimethyl, 2,4-oxazalidinedione at lower pH's corresponded to an increase in the fat-soluble nonionized fraction of this compound. These data support the concept that this tissue is most permeable to compounds of relatively small molecular size and/or with a high level of lipid solubility. The large diffusion permeability values measured for meperidine and diazepam suggest that these compounds will diffuse rapidly between mother and fetus at a maximal rate limited only by uterine blood flow.

Cell Membrane Permeability

Fetal breathing and adaptation to maternal hemorrhage in the sheep.

The effect of maternal hemorrhage in chronic preparations was studied on fetal lambs in the last month of gestation. Fourteen to 20 per cent of maternal blood was estimated to have been removed within 30 minutes, which resulted in a drop of 30 per cent of mean maternal arterial pressure. A fetal bradycardia started 28 +/- 13 minutes after the beginning of maternal hemorrhage. It lasted 30 +/- 15 minutes and was concomitant with a rise in fetal arterial pressure. It was followed by a long-lasting fetal tachycardia of 130 +/- 38 minutes and was corrected only by reinfusion of blood to the mother. The fetal blood gases demonstrated a mild asphyxia with a persistent metabolic acidemia until reinfusion of blood to the mother. Maternal and fetal plasma cortisol levels rose significantly at the end of the hemorrhage. Tracheal fluid flow did not change. Fetal breathing recorded 20 hours before and 24 hours after the experiment did not show consistent changes, but during fetal bradycardia there was no fetal breathing. Recent clinical investigations in this field have been made in the human fetus to estimate standards of fetal well being. These peculiar animal experiments do not show any significant improvement by recording fetal breathing over the recording of prelabor fetal heart rate.

Adaptation, Physiological

Effect of fenoterol (Th1165a) infusion on uterine and umbilical blood flow in pregnant sheep.

The effect of fenoterol (Th1165a) upon uterine artery blood flow (UtBF) and umbilical vein blood flow (UmBF) was investigated in near-term, nonlaboring chronic sheep preparations. During intravenous fenoterol infusions to the ewe in either incremental doses from 0.025 to 0.200 microng per kilogram per minute or constant infusions of 0.025 microng per kilogram per minute for 120 minutes. UtBF and UmBF did not change significantly. Dose-related maternal tachycardia, hyperglycemia, and relative acidemia occurred, but there were no significant changes in mean maternal and fetal arterial pressures or fetal heart rate. The simultaneous infusion of propranolol (2 microng per kilogram per minute) with fenoterol (0.200 microng per kilogram per minute) blocked the maternal tachycardia but resulted in a significant decrease in UmBF and a significant increase in UtBF. In all of the maternal infusions. UtBF significantly rose and plateaued up to 14 per cent above the control level during the 120 minute recovery period after infusion. A non-beta-adrenergic effect of fenoterol is suggested as the cause of this UtBF increase.

Animals

Effects of prolonged infusion of beta-adrenergic agonists on uterine and umbilical blood flow in pregnant sheep.

We have extended our evaluation of the effects of three beta-adrenergic agents in near-term pregnant sheep to include a period of infusion three times longer than previously studied. This extension has provided the following information: (1) Initial depression of uterine blood flow and mean arterial pressure associated with administration of ritodrine or salbutamol abate with time despite continued drug infusion; (2) the uterine hyperemia associated with salbutamol and fenoterol are drug-related rather than postinfusion-related phenomena; (3) increased uterine vascular resistance is found with ritodrine, and decreased uterine vascular resistance occurs with salbutamol and fenoterol.

Adrenergic beta-Agonists

Fetal respiration. A review.

During recent years respiratory movements by the human fetus have been rediscovered. The types of movements have been defined, and their dependence on some physiologic conditions has been described. Pharmacologic agents influence not only the occurrence of the movements but also their rate and depth. A tidal flow of fluid between lung and amnionic sac has been established, which may play an important role in lung development. Demonstration of this route for exchange of water and soluble substances identifies the lung as an organ very likely involved in exchange between fetal compartments. Additionally, the deposition in the lung of the particulate matter contained in amnionic fluid may not be proof of pathologic aspiration as previously thought.

Adrenocorticotropic Hormone

Effect of maternal diet on fetal hepatic lipogenesis.

The effects of: a, maternal diet; b, cyclic-3',5'-adenosinemonophosphate (cyclic AMP) and c, clofibrate on hepatic lipogenesis in fetal rats were studied. The experimental diets contained 22% protein, 40--50% carbohydrate, adequate vitamins, and minerals. In addition, the fat-containing diets were supplemented with either 15% corn oil, 25% corn oil, or 5% cholesterol + 10% oleic acid. In the clofibrate feeding studies, 0.3% (w/v) of the ethyl ester was added to a stock ration or to fat-free diet. Lipogenesis was measured in liver slices incubated with [2-14C]pyruvate, [1-14C]acetate, or 3H2O. In addition, activities of lipogenic enzymes were measured in cytosol fractions from liver homogenates. The effec-s of the experimental diets on liver composition were also examined. Lipogenic activity was higher in fetal than in maternal liver. When 15% corn oil was added to the maternal diet, fatty acid synthesis in fetal liver did not decrease as it did in maternal liver. Maternal fasting decreased fetal fatty acid synthesys by 50% when measured with 14C and less than 10% when measured with 3H2O. Although the addition of cholesterol to the maternal diet decreased cholesterol synthesis in maternal liver, no such decrease was observed in fetal liver. Changes in enzyme activities paralleled alterations in lipogenesis in maternal but not in fetal liver. Corn oil feeding or fasting increased the rate of transfer of linoleate from the dam to the fetus. However, accumulation of linoleate in fetal liver did not correlate with a decreased rate of fatty acid synthesis as it did in maternal liver. Maternal hepatic glycogen stores were depleted by fasting, but glycogen levels in fetal liver remained high under these conditions.

Acetates