Concentrations of altrenogest in plasma of mares and foals and in allantoic and amniotic fluid at parturition

Treatment with the progestin altrenogest is widely used in pregnant mares. The fact that foals born from healthy mares treated with altrenogest until term suffered from neonatal problems raises the question of direct effects of altrenogest on vital functions in the neonate. We have therefore investigated altrenogest concentrations in maternal and neonatal blood plasma and in fetal fluids. Pregnant mares were treated with altrenogest orally once daily (0,088 mg/kg bodyweight, n = 7) or left untreated (n = 8) from 280 d of gestation until foaling. Altrenogest concentration was determined in plasma of the mares, their foals and in amniotic and allantoic fluid. The concentration of altrenogest in plasma from treated mares (2.6 ± 1.0 ng/mL) was significantly lower than in plasma from their foals immediately after birth (5.6 ± 1.9 ng/mL; p < 0.05), but was significantly higher than in their fetal fluids (amniotic fluid: 0.4 ± 0.1 ng/mL; p < 0.05; allantoic fluid: 3.0 ± 1.5 ng/mL). Altrenogest was undetectable in maternal and fetal plasma and fetal fluids of control pregnancies at all times. Altrenogest concentration in plasma of foals from treated mares was strongly correlated to the altrenogest concentration in plasma of their dams (r = 0.938, p < 0.001) and in amniotic (r = 0.886, p < 0.001) and allantoic fluid (r = 0.562, p < 0.05). A significant decrease in altrenogest concentration between the time periods 0-15 min, 30-120 min, and 180-360 min after parturition was seen in the plasma from foals born to altrenogest-treated mares. In conclusion, our data demonstrate that altrenogest reaches the equine fetus at high concentrations.     

Bovine fetal fluid cells in vitro: Fate and fetal sex prediction accuracy

Summary The in vitro fate of bovine fetal fluid cells and the efficiency of fetal sex prediction from cultures of these cells are studied using aspirates from live animals and pregnant uteri collected from the slaughterhouse. Over 70% of bovine amniotic fluid samples aspirated from pregnant uteri at the time of slaughter yielded cultures adequate for chromosome analysis, whereas only 10% of allantoic fluid samples produced growth of cells satisfactory for chromosome analysis. Fetal sexing accuracy was 100% in all samples studied. Seven readily recognizable cell types were noted in cultures of fetal fluid cells obtained at various stages of gestation. In a majority of cases, the in vitro morphology of cells from both fetal cavities was similar to that observed in primary human amniotic fluid cell cultures.     

Investigations into the rheological characteristics of bovine amniotic fluid

The rheological characteristics of bovine amniotic fluid have been studied at different shear rates. The viscosity of bovine amniotic fluid at 20°C was found to increase with time at a constant low shear rate during the measurement. Additionally, the viscosity was observed to decrease with increasing shear rate, indicating that a shear thinning behaviour of the fluid was occurring. The log-log plot of shear stress versus shear rate yielded a straight line, consistent with non-Newtonian behaviour of the fluid and characteristic of pseudoplastic liquids. The data of shear stress versus shear rate could be represented by a power law model. The treatment of amniotic fluid with cetylpyridinium chloride (CPC) resulted in the precipitation of a mixture of components, including complex sulphated polysaccharides and extracellular proteoglycans, with the viscosity of the resulting liquid similar to that of water at 20°C. In addition, the viscosity of the CPC-pretreated fluid did not increase with time at constant shear rate and remained constant with the increase in shear rate. The apparent increase in viscosity with time and the shear thinning behaviour of the amniotic fluid can thus be attributed to pseudoplastic liquid behaviour associated with the presence of structurally complex polysaccharides and extracellular proteoglycans. The implications of this fluid viscosity behaviour are discussed in terms of their impact on the operation of packed or expanded (fluidized) chromatographic bed systems when amniotic fluid biofeed-stocks are used as a source of commercially important proteins.     

Isolation of queuine from bovine amniotic fluid

A facile method for isolation of large quantities of queuine from bovine amniotic fluid is described. Queuine was sequentially purified by cation-exchange chromatography, adsorption chromatography on Sephadex G-25, and size-exclusion chromatography on Sephadex G-10. The queuine isolate was identified by its participation in the queuine-guanine tRNA transglycosylase reaction and comparisons with authentic queuine.     

CD117+ amniotic fluid stem cells

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

Chemical dilution and clearance studies to estimate amniotic fluid volume and amniotic fluid ingestion in normal baboon pregnancies

Amniotic fluid volume (AFV) and amniotic fluid ingestion rate (or fetal swallowing rate, FSR) were estimated by inulin and para-aminohippurate (PAH) dilution in 14 normal baboon pregnancies. Mean (± SE) AFV was significantly lower at 137–140 days of pregnancy (preterm) than at 173–178 days (term) (inulin: 326 ± 22.9 ml vs 483 ± 55.9 ml, P = 0.014; PHA:269 ± 39.4 ml vs 471 ± 39.4 ml, P = 0.002). In proportion to fetal weight, however, mean AFV was similar throughout the third trimester of pregnancy (inulin: 582 ± 40.9 ml/kg; PAH: 541 ± 39.8 ml/kg). Mean FSR was lower in preterm than in term animals when estimated by inulin dilution (587 ± 55.5 ml/day vs 784 ± 55.0 ml/day, P = 0.030) but not when estimated by PAH dilution (753 ± 65.7 ml/day vs 625 ± 50.6 ml/day). In proportion to their weights, however, preterm fetuses swallowed amniotic fluid more rapidly than term fetuses (inulin: 1,216 ± 117.6 ml/kg/day vs 840 ± 67.5 ml/kg/day, P = 0.025; PAH: 1,561 ± 142.9 ml/kg/day vs 682 ± 62.7 ml/kg/day, P < 0.001). Furthermore, our data suggest that the commonly accepted technique for estimating AFV may be based on inaccurate premises, that insulin may be a better marker than PAH to estimate AFV and FSR, and that needle aspiration of amniotic fluid does not appear to be an adequate technique to validate chemical dilution methods. Our data, however, provide estimates which indicate that the baboon is an appropriate animal model in which to seek refinements and validation of our techniques.     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

Biochemical characteristics of collagen produced by long term cultivated amniotic fluid cells

Summary The synthesis of the most common type I of collagen was proven in human amniotic fluid cell culture with fibroblast morphology in the cells as well as in the culture medium. Mesenchymal origin of long term cultivated amniotic fluid cells is indicated and the possibility of prenatal investigation of hereditary disorders of connective tissue is pointed out.     

Characterization of hCG regulation in cultured human amniotic fluid cells

Summary We showed previously that sodium butyrate stimulated human chorionic gonadotropin (hCG) measured by radioimmunoassay of medium from human second trimester amniotic fluid cell cultures, termed AF cells. We now find that stimulation of hCG in the presence of sodium butyrate takes as long as 20 h. When AF cells are preincubated with sodium butyrate, hCG levels increase in direct relation to length of the preincubation period. These findings suggest that elevation of hCG is not due merely to a release of hormone from the cells. Addition of cycloheximide or Actinomycin D inhibited protein synthesis and RNA synthesis, respectively, and prevented the stimulation of hCG by sodium butyrate. These results lend support for a mechanism of regulation involving protein and RNA synthesis, the increase in hCG levels being due to new synthesis of the hormone. Other agents reported to influence hCG production by different types of cell cultures include dibutyryl cyclic AMP, epidermal growth factor (EGF), methotrexate, and hydroxyurea. Dibutyryl cyclic AMP and EGF have no effect on hCG production in our AF cells: methotrexate causes a minimal increase, hydroxyurea causes a further increase, but sodium butyrate has the strongest stimulatory effect. We conclude that amniotic fluid cells in culture are susceptible to environmental agents capable of modulating synthesis of hCG by mechanisms involving synthesis of RNA and protein. Research supported by Grant HD 11379 from the National Institutes of Health.     

Composition of the fetal fluids in African antelopes

Twenty-four samples of amniotic and allantoic fetal fluids were collected from 15 African antelopes of 11 species and subspecies. Two samples were taken from delivered placentas and the rest were from animals that died during pregnancy or parturition. Data on the sex, development (crown-rump length) and age (trimester of pregnancy) of fetuses, fluid volumes, pH, and 18 biochemical parameters were obtained wherever conditions permitted. Collecting data on fetal fluids in zoo and wild animals may help evaluate both normal and pathological pregnancies.     

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid.

Intestinal lactase activity (with its associated cellobiase, 4-methylumbelliferyl-beta-galactosidase and -beta-glucosidase activities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66--85 days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-alpha-glucosidase showed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas alpha-galactosidase and N-acetyl-beta-hexosaminidase which were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of alpha-galactosidase and N-acety-beta-hexosaminidase activities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86--90 days of gestation and thereafter passes more and more into the amniotic sac.     

Relative amounts of sialic acid and fucose of amniotic fluid glycoconjugates in relation to pregnancy age

The present knowledge concerning the glycan structures and role of glycoconjugates derived from amniotic fluid is fragmentary and mainly focuses on the individual glycoproteins. The question has arisen as whether the general glycosylation pattern of amniotic fluid glycoconjugates can change with the progression of a normal pregnancy. In the present work we have described the dynamic, quantitative alterations in relative amounts of sialic acid and fucose linked by a variety of anomeric linkages to subterminal oligosaccharide structures of amniotic fluid glycoconjugates in relation to pregnancy age. The analysis was performed in the following groups of amniotic fluids derived from normal pregnancy by lectin dotting method: “2nd trimester” (14–19 weeks), “3rd trimester” (29–37 weeks), “perinatal period” (38–40 weeks) , “delivery at term” (39–41 weeks) and “post date pregnancy” (41–43 weeks). In the “3rd trimester” the amniotic fluid glycoconjugates contained higher relative amounts of glycans terminated by α2-6-linked sialic acid (p < 0.00002) and by α1-6 innermost fucose (p < 0.000001) than those in the 2nd trimester. In contrast, they showed the lower relative amount of fucose linked α1-3 (p < 0.02). At the perinatal period the relative amount of α2-6-linked sialic acid increased (p < 0.03), and it then decreased during delivery (p < 0.02) to the level found in the “3rd trimester” group. In the post date pregnancy all parameters studied increased. The sialyl- and fucosyl-glycotopes of the amniotic fluid glycoconjugates may play an critical role in growth and tissue remodeling of the foetus, as well as may might reflect maturation of a foetus. Additionally, a determination of the glycotope expressions might be helpful in prenatal diagnosis as predictor factors for well being of mother and child.     

Distribution of alpha 1-fetoprotein in fetal plasma, allantoic fluid, amniotic fluid and maternal plasma of cows.

Maximal concentrations of AFP, measured by RIA, were obtained in fetal plasma and amniotic and allantoic fluid between the 3rd and 4th month of gestation, with levels declining thereafter until term. AFP values in maternal plasma were unchanged. Throughout gestation, AFP values were higher in allantoic than in amniotic fluid and the ratio of allantoic fluid/amniotic fluid AFP was significantly correlated with gestational age.     

Detection ofBacteroides fragilis endotoxin in amniotic fluid by counterimmunoelectrophoresis

The ability of counter immunoelectrophoresis (CIE) to detectBacteroides fragilis endotoxin in amniotic fluid in small concentrations was evaluated. A method was developed which, in combination with ultrafiltration, permits detection ofB. fragilis endotoxin in amniotic fluid in a concentration of 40 ng/ml or more. The sensitivity threshold was reduced to 2 ng/ml by using a highly reactive IgG-fraction isolated from rabbit anti-B. fragilis IPL E 323 antiserum.     

CD117+ amniotic fluid stem cells: State of the art and future perspectives

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

Differentiation in human amniotic fluid cell cultures: Chorionic gonadotropin production

Summary Two of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the β-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast. Research supported by Grand HD 11379 from the National Institutes of Health.     

Proteins in the amniotic and allantoic fluids of fetal pigs

Seven proteins were identified in the amniotic and allantoic fluids of fetal pigs (Sus scrofa domesticus) using crossed immunoelectrophoresis: albumin, fetuin, transferrin, alpha-fetoprotein (AFP), alpha 1-acid glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin. The total protein concentrations were determined by the method of Bradford and individual protein concentrations by radial immunodiffusion or rocket immunoelectrophoresis. Transferrin and fetuin were the major proteins in amniotic fluid during the second trimester of gestation and together with AFP and albumin accounted for the majority of the total protein in amniotic, but not allantoic fluid.     

Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells （AFCs） are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.     

Peptidases from pig reproductive tract: purification and properties of aminopeptidases from uterine secretions, allantoic fluid, and amniotic fluid

In ovariectomized sows, aminopeptidase is secreted into the uterine lumen under the influence of progesterone. The enzyme also accumulates in allantoic and amniotic fluids of pregnant animals. We have purified the predominant form of this enzyme from uterine flushings, allantoic fluid, and amniotic fluid by the following steps: ammonium sulfate precipitation, Sepharose 6B chromatography, ion-exhange chromatography on diethylaminoethyl cellulose, and affinity chromatography usingl-leucylglycine immobilized on agarose. The overall procedure gave approximately 974-, 110-, and 230-fold purifications of the allantoic, uterine, and amniotic enzymes, respectively. The enzymes from all three sources are glycoproteins with pI's around 4 and molecular weights of about 480,000. They may be dissociated into six apparently identical subunits of molecular weight 80,000 as judged by sodium dodecyl sulfate gel electrophoresis. With l-leucyl-β-naphthylamide as substrate the pH optimum and apparent K_m value for each enzyme were 7.1 and 14 μm, respectively. However, the uterine and allantoic aminopeptidases exhibited V values of 0.35 μmol of substrate hydrolyzed/min/mg of protein, whereas the V for the amniotic enzyme was at least sixfold greater. The amniotic enzyme also differed from the other two in pH and temperature stability. The activity of all three enzymes was stimulated by Co²⁺ and inhibited by Cu²⁺, Fe³⁺, and chelating agents, while iodoacetate and mercaptoethanol had no effect on catalysis. The effect of Co²⁺ on the allantoic enzyme was investigated in further detail. The stimulation of peptidase activity by Co²⁺ was shown to be a complex process but consistent with Co²⁺ replacing another metal at the active site and at some other additional site on the enzyme. The function of the aminopeptidases in the pregnant uterus is unknown.     

Estimation of gestational age from study of amniotic fluid and clinical assessment.

Study of 108 samples of amniotic fluid obtained between 28 and 42 weeks'' gestation from 101 patients revealed that in normal pregnancies the creatinine concentration, lecithin/sphingomyelin (L/S) ratio and percentage of fat cells correlated better with the gestational age of the newborn--assessed by clinical criteria--than did the bilirubin and sodium concentrations. A creatinine concentration of 1.75 mg/dL or more, an L/S ratio of 4 or more and a fat cell percentage of 10 or more correlated significantly with a gestational age of 37 weeks or more. In abnormal pregnancies (those with obstetric or medical complications, or both) the mean creatinine concentration in the amniotic fluid was significantly less than expected for gestational age in fetal dysmaturity and greater than expected when the mother had diabetes. The mean L/S ratio in the amniotic fluid was elevated when the mother had hypertension or smoked and in cases of fetal dysmaturity or long interval between rupture of the membranes and delivery, whereas it was significantly lower than normal when the mother had diabetes. The mean bilirubin concentration in the amniotic fluid was significantly lower than normal when the mother had hypertension. When the mother had diabetes, maturity of the fetal lung, liver, skin and brain appeared to be delayed, according to the values for the amniotic fluid constituents.