Amniotic fluid concentration of 5-hydroxyeicosatetraenoic acid is increased in human parturition at term

5-hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if spontaneous human labor at term is associated with changes in the concentration of this metabolite in amniotic fluid. Fluid was retrieved from 36 women not in labor and from 30 women in active labor at term. 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE of women in labor was significantly greater than that of women not in labor (3538 pg/ml vs. 1977 pg/ml, respectively; p = 0.05). This observation is consistent with activation of the lipoxygenase pathway of arachidonic acid metabolism during spontaneous human parturition at term.     

5-Hydroxyeicosatetraenoic acid and human parturition

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonic acid (AA) metabolite derived from the lipoxygenase pathway which is capable of inducing uterine contractions. The purpose of this study was to determine a). whether 5-HETE concentrations in amniotic fluid increase before or after the onset of labor and b). whether acetylsalicylic acid (ASA) could modulate the production of 5-HETE by human amnion cells. 5-HETE concentrations are increased in amniotic fluid before the onset of labor. Furthermore, ASA treatment as expected inhibited PGE₂, but also significantly increased 5-HETE production by amnion cells. 5-HETE concentrations on average increased by greater than 2.5 fold (p < 0.001) in amniotic fluid prior to spontaneous labor when compared with samples obtained from the same patients earlier in gestation and therefore may be important in mechanisms regulating the onset of labor. ASA provokes an increase in 5-HETE biosynthesis by amnion cells: control media 2.60 ± 1.5, ASA treatment alone 5.17 ± 0.20, IL-1β alone 6.39 ± 2.1, and ASA + IL-1β 8.95 ± 1.2 (mean ± SEM) picograms per microgram protein per 16 hours. These findings may explain in part why cyclooxygenase inhibitors are not always successful in treating women with preterm labor.     

Arachidonic acid in amniotic fluid after extra-amniotic instillation of rivanol for midtrimester abortion

Free arachidonic acid was measured by gas liquid chromatography in amniotic fluid in 13 women during induction of midtrimester abortion. The abortions were induced by extraamniotic instillation of 0.1% Rivanol. Serial sampling of amniotic fluid were performed through a trans-abdominal catheter up to 22 hrs. Free, total arachidonic acid showed a significant increase from 26±8 ng/ml to 293±102 ng/ml at 22 hrs. The percentage of free arachidonic acid in total free fatty acids increased significantly from 2.2±0.5 to 6.1±1.6% during the same time. The C results suggest a selective release of arachidonic acid during Rivanol-induced abortion.     

Lysozyme in amniotic fluid during rivanol-induced second trimester abortion.

The concentrations of lysozyme and free arachidonic acid in amniotic fluid were determined in eight patients during induction of second trimester abortion. Abortion was induced by extraamniotic instillation of 150 ml 0.1% Rivanol. Lysozyme increased in all patients during the induction reaching significancy after 60% of the induction-abortion time. This suggests a leakage of lysosomal enzymes from the fetal membranes. Free arachidonic acid relative to total free fatty acids increased parallell to lysozyme.     

Lysozyme in amniotic fluid during Rivanol-induced second trimester abortion

The concentrations of lysozyme and free arachidonic acid in amniotic fluid were determined in eight patients during induction of second trimester abortion. Abortion was induced by extraamniotic instillation of 150 ml 0.1% Rivanol. Lysozyme increased in all patients during the induction reaching significancy after 60% of the induction-abortion time. This suggests a leakage of lysosomal enzymes from the fetal membranes. Free arachidonic acid relative to total free fatty acids increased parallell to lysozyme.     

Differential sialylation regulates the apoptotic activity of glycodelin A

Glycodelin A (GdA), a dimeric lipocalin, expressed by the uterine endometrium, is an immunomodulatory agent and induces apoptosis in T-cells. In this study we demonstrate that two populations of GdA with subtle differences in their net ionic charge are present in the amniotic fluid and that, apoptotic activity is exhibited only by the population with more sialic acid residues. Significantly, removal of sialic acid residues from the active populations of GdA abrogates the activity of the molecule, suggesting that the extent of sialylation might be a factor regulating the activity of GdA.     

Circadian uterine activity in the pregnant rhesus macaque: do prostaglandins play a role?

Eight rhesus macaques between 127 and 132 days of gestation had catheters implanted into maternal femoral vessels and the amniotic fluid cavity and were placed in a vest-and-tether system for chronic catheter maintenance. Uterine activity was continuously recorded, and paired maternal arterial blood and amniotic fluid samples were collected at 0900 h (AM) and 2100 h (PM) until delivery and analyzed for prostaglandin metabolites (PGFM and PGEM-II). A circadian pattern in uterine contractility was observed, with peak activity occurring between 1900 and 0100 h (p less than 0.001). No significant AM-PM differences were observed in maternal plasma PGFM (240 +/- 24 AM vs. 273 +/- 35 PM) or PGEM-II (537 +/- 41 AM vs. 484 +/- 34 PM) or amniotic fluid PGFM (360 +/- 72 AM vs. 287 +/- 70 PM) or PGEM-II (1626 +/- 383 AM vs. 1771 +/- 431 PM). All values represent mean +/- SEM, pg/ml. Additional samples were collected at 3-h intervals for 24 h at selected times during the study. This more intensive sampling protocol also failed to reveal any significant time trends in maternal plasma or amniotic fluid prostaglandins. Despite the lack of AM-PM differences, amniotic fluid PGFM and PGEM-II increased significantly as delivery approached (p less than 0.01). It appears that circadian uterine activity is not related to changes in maternal plasma or amniotic fluid prostaglandins. Although prostaglandins are responsible for the progression of labor, other factors may be involved in the generation of uterine activity rhythms prior to the initiation of labor.     

Catecholamines and uterine activity rhythms in the pregnant rhesus macaque.

This study was designed to examine the relationship between uterine contractile rhythms with maternal plasma and amniotic fluid catecholamine concentrations in the pregnant rhesus macaque. Six chronically catheterized rhesus macaques were maintained in a vest and tether system and exposed to a 12L:12D cycle. Continuous uterine activity recordings demonstrated a contractile pattern with peak activity at 2200 h (p less than 0.05). Paired maternal plasma and amniotic fluid samples were collected at 3-h intervals for 24 h between Days 131 and 148 of gestation. Samples were analyzed for norepinephrine, epinephrine, and dopamine by HPLC. Maximum plasma concentrations across the 24-h periods for norepinephrine (633 +/- 230; mean pg/ml +/- SEM) and dopamine (378 +/- 110) were observed at 2100 h and epinephrine (408 +/- 95) at 1200 h, but these values were not significant. The maximum amniotic fluid values were 378 +/- 126, 267 +/- 190, and 556 +/- 87 pg/ml for norepinephrine, epinephrine and dopamine, respectively. However, concentrations across 24 h did not differ. Neither maternal plasma nor amniotic fluid catecholamine concentrations were correlated with uterine activity rhythms. Therefore, we conclude that the nocturnal uterine activity in the rhesus macaque is not related to maternal arterial or amniotic fluid catecholamine concentrations.     

Fetal surfactant as a source of arachidonate in human amniotic fluid

The factors responsible for the onset of labor in women are not well understood but it is clear that parturition is associated with increased production of prostanoids and release of arachidonic acid by intrauterine tissues. Pulmonary surfactant is secreted from the fetal lung into the amniotic fluid where its concentration increases toward term. In this paper we have shown that the ability of fetal surfactant to stimulate prostaglandin production by amnion cells is greatly enhanced by pre-incubating surfactant with amniotic fluid. This is due to the release of fatty acids, including arachidonate, from the lipids of fetal surfactant by the sequential action of phospholipase C and diglyceride lipase. Thus, in addition to providing the amnion with a source of arachidonate derived from the intracellular transfer of arachidonate from surfactant phosphatidylcholine to phosphatidylethanolamine and phosphatidylinositol in amnion cells, fetal surfactant also contributes to the pool of free arachidonate in amniotic fluid.     

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.     

Arachidonic acid in uterine phospholipid during early pregnancy and following hormone treatment

Evidence that prostaglandins are involved in intercellular communication during blastocyst implantation suggested that development and loss of uterine sensitivity to deciduogenic stimuli during early pregnancy might depend upon changes in uterine capacity to mobilize arachidonic acid from phospholipid. We measured levels of arachidonic acid in lipid fractions on Day 6 of pregnancy in uterine segments containing implantation sites, in uterine segments between implantation sites, and in luminal epithelial cells after a deciduogenic stimulus. Arachidonic acid in uterine phospholipid was depleted at implantation sites. With an intrauterine deciduogenic stimulus of hormonally primed ovariectomized rat uteri, the arachidonic acid content of the luminal epithelium decreased. When the fatty acid composition of the luminal epithelium was examined during pseudopregnancy and after progestin-estrogen treatment, however, no changes in arachidonic acid composition and content were observed. These data suggest that during blastocyst implantation, luminal epithelial cells at implantation sites mobilize arachidonic acid from phospholipid for prostaglandin synthesis, but that uterine sensitivity and the capacity to synthesize prostaglandins in response to the blastocyst does not depend upon changes in arachidonic acid levels in uterine phospholipid.     

Measurement of arachidonic acid release from permeabilised myometrial cells of guinea pig uterus.

A technique has been developed for prelabelling and permeabilisation of guinea pig uterine myocytes to enable measurement of arachidonic acid release/phospholipase A2 activity in cells with intact membranes. Intact cells were prelabelled with [3H]inositol or [3H]arachidonic acid for measurement of phospholipase C and A2 respectively. In intact cells 10 nM endothelin-1 or 1 microM bradykinin stimulated both inositol polyphosphate and arachidonic acid release, whilst 1 microM oxytocin, arginine vasopressin or histamine were without effect. In Streptolysin-O permeabilised myometrial cells calcium-stimulation of inositol polyphosphate and arachidonic acid release was detected between 10 microM and 1 mM free calcium. The patterns of inositol polyphosphate and arachidonic acid release were broadly similar. Responses to 1 mM calcium were not detected in intact cells not treated with Streptolysin-O. For arachidonic acid release the K0.5 for calcium activation was about 7 microM, a level above that normally likely to be found in the uterine myocyte. Hence it is concluded that unless there are high local concentrations of calcium close to the plasma membrane, calcium is unlikely alone to be the primary regulator of arachidonic acid release and phospholipase A2.     

THE NATURE AND ORIGIN OF THE SOLUBLE PROTEIN IN HUMAN AMNIOTIC FLUID

1. Amniotic fluid surrounds the human fetus and is separated from the uterus by the amnion, chorion and placenta. The ability to obtain samples of amniotic fluid from women by a simple procedure has encouraged studies on the nature and origin of the fluid, and on its use for the diagnosis of a variety of clinical conditions. The fluid contains cells, which are of fetal origin, and can be grown in a tissue culture. Cyto-genetic and biochemical analyses can therefore be used to detect chromosomal aberrations and inborn errors of metabolism in the fetus. 2. The supernatant of amniotic fluid contains many of the solutes typical of extracellular fluid. In particular, it contains a wide range of proteins and those which are of fetal origin are likely to be of use in the prenatal diagnosis of fetal disease. This review examines the nature and origin of the soluble protein in amniotic fluid, and discusses the diagnostic uses of the proteins which are of fetal origin. 3. In other mammals, the arrangement of the fetal membranes is different from that in man, and these differences are reflected by changes in the nature of the amniotic fluid. Thus data from other animals have little applicability to man. 4. Electrophoresis and immunoelectrophoresis have established that the major proteins in amniotic fluid are also present in maternal and fetal sera. Their concentrations in the fluid are influenced by their molecular weight and proteins larger than about 2.5 times 10⁶ may be excluded. Towards term, phenotyping studies show that a number of serum proteins in amniotic fluid are of maternal origin. In the case of group-specific component (Gc) this has been shown to be so throughout pregnancy. Such proteins must enter the fluid by diffusing across either the chorion or the chorionic plate and then the amnion. 5. It has been previously claimed that various serum proteins in amniotic fluid are of fetal origin. For albumin and IgG there are data that strongly support a maternal origin. The evidence on the origin of insulin is inconclusive. The concentration of β₂-microglobulin in amniotic fluid exceeds that in maternal serum and is probably too high also for fetal serum to be its major source. It has a wide tissue distribution and probably enters the fluid from surrounding structures. 6. Alpha-fetoprotein in amniotic fluid is of fetal origin as it is present in maternal serum at far lower concentrations. It is found in fetal serum, urine and yolk sac, but it is not clear how it enters the amniotic fluid of normal fetuses. The concentrations of Gc and alpha-fetoprotein have been measured in amniotic fluid and in their sera of origin. The relative concentration of Gc in amniotic fluid was found to be much greater than that of alpha-fetoprotein and the concentration gradients of these marker proteins can be compared with data for other proteins. In this way further evidence has been obtained that the albumin, α₁,-antitrypsin and transferrin in amniotic fluid are mainly of maternal origin throughout pregnancy. 7. Immunological studies have shown that at least three proteins of non-serum origin are present in amniotic fluid and they have also been located in the amnion and uterine decidua. 8. The enzymes present in amniotic fluid are summarized. Many lysosomal enzymes are clearly of fetal origin since they show altered specific activities in the appropriate cases where the fetus is affected with an inborn error of metabolism. For other enzymes, analysis of specific activity gradients can help to decide the extent to which an enzyme is of serum origin, although this will not exclude the possibility of a maternal (uterine) contribution. The results of such analyses suggest that, relative to the serum protein in amniotic fluid, the greatest concentrations of the minor non-serum proteins in the fluid occurs between thirteen and eighteen weeks of pregnancy and also towards term. 9. Some inborn errors of metabolism may be diagnosed prenatally by measuring the specific activity of the respective enzyme in amniotic fluid. However, the presence of different enzymes with similar substrate specificities has prevented this in Pompe's disease. 10. In cases where the fetus is affected with anencephaly or spina bifida there is an increase in the concentration of alpha-fetoprotein in the amniotic fluid. This has provided a way of detecting these diseases early enough to allow termination of pregnancy. 11. The discovery of new proteins in fetal serum and in the tissues surrounding the amniotic cavity would seem to provide the best chance of extending the uses of amniotic fluid into the other areas of prenatal medicine.     

Citrullinemia: prenatal diagnosis of an affected fetus.

We monitored a pregnancy in a family at risk for citrullinemia due to argininosuccinic acid (ASA) synthetase deficiency. ASA synthetase activity in cultured epithelioid amniotic fluid cells from the fetus at risk was less than 2% of control epithelioid amniotic fluid cell activity. An increased concentration of citrulline was found in the at-risk amniotic fluid (0.14 mumol/ml) as compared with fluid from six controls and one at-risk but unaffected pregnancy (trace). The pregnancy was terminated, and the in utero diagnosis was confirmed by assay of ASA synthetase activity in cultured fetal skin fibroblasts (4.4% of control activity). In addition, all five fetal tissues studied had significant accumulation of citrulline, whereas control fetal tissues had none. These data provide evidence that, if precise control is maintained over tissue culture variables, citrullinemia can be diagnosed successfully in utero by microassay of ASA synthetase activity in cultured amniotic fluid cells. They also suggest that amniotic fluid citrulline concentrations provide strong adjunctive evidence for this prenatal diagnosis.     

The modulation of prostaglandin synthesis by peritoneal fluids

Biological fluids from several sources (e.g. blood, fetal urine, amniotic fluid) have been shown to contain factors that modulate prostaglandin (PG) synthesis. In this study, we investigated the possibility that peritoneal fluid contains substances that may regulate PG synthesis. Peritoneal fluids were obtained from women undergoing diagnostic laparoscopy for infertility. Fluids from women without evident pelvic pathology were incubated with prostaglandin synthase prepared from bull seminal vesicles in the presence of excess arachidonic acid, and the production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha was quantified by specific radioimmunoassay. The untreated fluids inhibited potently the synthesis of PGE2 but such inhibitory activity was not extractable by chloroform:methanol. An ultrafiltrate of the fluid containing molecules smaller than 10,000 Daltons stimulated PGF2 alpha synthesis but this activity was also lost after extraction. The extracted fluid did, however, stimulate the synthesis of prostacyclin (as reflected by 6-keto-PGF1 alpha).     

Occurrence of soluble glycosyltransferases in human amniotic fluid

Human amniotic fluid obtained by amniocentesis during the third trimester of pregnancy was found to contain glycosyltransferases for the transfer of galactose, N-acetylgalactosamine, N-acetylglucosamine and sialic acid from their nucleotide derivatives to various exogenous protein and small molecular weight acceptors. The specific activity of the galactosyl- and N-acetylgalactosaminyl transferases was found to be 30 to 40 times higher in amniotic fluid as compared to serum. The specific activity of N-acetylglucosaminyl- and sialyl transferases was only 3 to 6 fold higher in amniotic fluid.     

Endogenous stimulant of prostaglandin endoperoxide synthase activity in human amniotic fluid

In this study we describe the discovery and characterization of a substance in human amniotic fluid that stimulates prostaglandin biosynthesis by a microsome-enriched preparation of bovine seminal vesicles. The stimulatory activity is not retained substantially upon anisotropic ultrafiltration through a filter with a molecular weight exclusion limit of 500. Stimulation of prostaglandin biosynthesis by this substance is time- and concentration-dependent; maximal stimulation of approx. 200% being observed within 20 min of commencing incubation with 1 ml-equivalent of stimulant fraction. Stimulatory activity is demonstrable both in the presence of reduced glutathione (1.3 mM) and L-tryptophan (20 mM), either separately or combined, and in the presence of exogenous arachidonic acid (5-120 microM). In the absence of added cofactors, the stimulatory substance increases the rates of biosynthesis of prostaglandin E2 and prostaglandin F2 alpha to equal extents. The amount of stimulatory substance added to incubations is correlated positively with increased oxygen consumption during incubations. The stimulatory substance is stable to heating at 100 degrees C for 10 min but is inactivated substantially (to less than 20% of original activity) by treatment with pronase. It is concluded that human amniotic fluid contains a substance of relatively low molecular weight, which is proteinaceous in character, that stimulates prostaglandin endoperoxide synthase activity.     

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.     

Proteolytic enzymes in human fetal membranes and amniotic fluid. A comparison of normal and premature ruptured membranes

The amniotic and chorionic membranes obtained at term and term amniotic fluid contain a soluble protease activity which cleaves [14C]-labeled globin at acid pH. In contrast, a salt extract of the pellet fraction obtained from the fetal membranes displays only negligible protease activities at the pH range of 4-8. Specific activities of the proteases in the soluble and salt-extractable fractions of fetal membranes which were intact before onset of labor were not significantly different from the respective activities in cases of premature rupture of fetal membranes (PROM). However, the protease activity of the amniotic fluid was found to increase with advancing gestational age and to reach maximal activity at term. A heat-sensitive and nondializable protease inhibitory activity was found in term amniotic fluid. This inhibitory activity acted on the cytosolic protease of amniotic membranes from control and PROM cases, but not on the soluble protease of chorionic membranes, and had a similar potency in fluids from PROM cases or fluids collected at term. These results do not support a role for fetal membrane proteases, amniotic fluid proteases, or amniotic fluid protease inhibitory activities in the etiology of PROM. However, the observed changes in amniotic fluid protease activity with fetal age suggest a physiological role for the enzyme in normal fetal development.