Amniotic fluid phospholipid profile determined by two-dimensional thin-layer chromatography as index of fetal lung maturation.

A phospholipid profile, the main features of which were the lecithin/sphingomyelin (L/S) ratio and the presence or absence of phosphatidylglycerol (PG), was determined in amniotic fluid from 188 patients. There was a mature profile (L/S ratio of at least 2 . 0 and detectable PG) in 145 patients, including seven insulin-dependent diabetics, and noe of their babies developed respiratory distress syndrome (RDS). The L/S ratio was less than 2 . 0 and PG absent in 12 patients, nine of whose babies developed RDS, whereas only three small babies (delivered between 28 and 35 weeks because of fulminant pre-eclampsia or severe abruptio placentae) out of 31 developed RDS when the L/S ratio was less than 2 . 0 but PG was present. When amniotic fluid was collected from the vagina only one out of 69 babies developed RDS when PG was present (regardless of the L/S ratio), while all of seven babies developed RDS when PG was absent. It is concluded that the amniotic fluid phospholipid profile, particularly the presence or absence of PG, gives an accurate assessment of fetal lung maturation. The profile may prove a useful adjunct to the management of high-risk pregnancies, especially after premature membrane rupture and perhaps also when the mother is diabetic.     

Glial origin of rapidly adhering amniotic fluid cells.

Rapidly adhering cells (RA cells) from the amniotic fluid of a pregnancy with fetal anencephaly were investigated by immunofluorescence assay with an antiserum against glial cells. After 24 hours'' cultivation a high proportion of the cells showed positive glial-specific fluorescence, whereas no staining was seen in cells from samples of normal amniotic fluid. At the 24th week the mother was delivered of a stillborn infant with anencephaly. Immunofluorescence staining of RA cells with glial-specific antiserum may be used for the differential diagnosis of fetal abnormalities associated with a high alpha-fetoprotein concentration in amniotic fluid.     

The urinary excretion of pregnanediol during pregnancy determined by gas-liquid chromatography. II. Its relation with other parameters controling pregnancy (author's transl)]

The relation between the urinary pregnanediol determined by gas-liquid chromatography during pregnancy, with ultrasonic findings (biparietal diameter, thickness and echorrefringency of the placenta and the appreciated quantity of amniotic fluid), with biochemical parameters of control of pregnancy (beta-glucoronidase, total and thermostable alkaline phosphatase), with amnioscopic findings, and with several studied parameters of the amniotic fluid by amniocentesis, were studied. A good relation exists with the parameters which denote placentary insufficiency and fetal risk (irregularity in the homogeneity of the placenta, elevation of the total and thermostable alkaline phosphatase, positive amnioscopy) and also with those that are related with fetal maturity (biparietal diameter, shake test, organge cells, creatinine in amniotic fluid, etc.). These results indicate that the determination of urinary pregnanediol can be one of the tests controlling the normal and pathological pregnancies, being useful in the detection of fetal risk and the diagnosis of the intrauterine fetal maturity.     

The possibility of respiratory distress syndrome prevention of premature born children

Pregnant women and premature born children were classified into four groups. In each group there were thirty of them. The first group included the pregnant women with premature rupture of membranes and amniotic fluid effluxed for 72 hours before the delivery. The second group included the pregnant women with amniotic fluid effluxing less then 72 hours before the delivery. The third group included the pregnant women who were given corticosteroids. The forth group was a control group formed by those pregnant women (and their premature born children) whose amniotic fluid did not efflux long and those who weren't given corticosteroids during pregnancy. In all groups of pregnant women we observed: median age of pregnant women, the duration of pregnancy and mode of delivery (vaginal or cesarean section). In groups of premature born children we also observed: newborn birth weight, Apgar score in the first minute after delivery, Apgar score in the fifth minute after delivery, pH of the blood of umbilical cord, L/S ratio of amniotic fluid (lecithin-sphingomyelin ratio), RDS (neonatologist valuation in any degree of RDS developed et newborn child). Symptoms of RDS include tachypnoea, chest wall retraction and cyanosis and a zground glass' appearance of the chest on X-ray. Histopatological examinations of placentas compared the frequency of inflammatory or noninflammatory changes, also in all groups. No significant difference was found among groups of pregnant women for the following factors: the age of pregnant women, the duration of pregnancy and mode of delivery. No significant difference was found among the groups of children for the following factors: newborn birth weight, Apgar score in the fifth minute after delivery, blood pH of umbilical cord, L/S ratio of amniotic fluid. Significant difference was found among groups for the following factors: Apgar score in the first minute after delivery, the frequency of RDS and hystology of placentas. The prevention of premature delivery is the most important. All the pregnant women with symptoms of the premature delivery must be transported to the centers with the well developed unites of intensive neonatal care ("transport in utero").     

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.     

Predicting lung maturity in preterm rupture of membranes via lamellar bodies count from a vaginal pool: a cohort study

Background  

Amniocentesis is the accepted mode of attaining amniotic fluid to perform tests for fetal lung maturity. The purpose of this study was to validate a non-invasive fetal lung maturity test by counting lamellar bodies from a vaginal pool among women with preterm premature rupture of membranes.     

Surfactant in respiratory distress syndrome and lung injury

A deficiency in alveolar surfactant due to immaturity of alveolar type II epithelial cells causes respiratory distress syndrome (RDS). In contrast to animals, the fetal maturation of surfactant in human lungs takes place before term, exceptionally large quantities of surfactant accumulating in the amniotic fluid. The antenatal development of surfactant secretion is very variable but corresponds closely to the risk of RDS. The variation in SP-A and SP-B genes, race, sex and perinatal complications influence susceptibility to RDS. Surfactant therapy has improved the prognosis of RDS remarkably. Abnormalities in alveolar or airway surfactant characterize many lung and airway diseases. In the acute respiratory distress syndrome, deficiencies in surfactant components (phospholipids, SP-B, SP-A) are evident, and may be caused by pro-inflammatory cytokines (IL-1, TNF) that decrease surfactant components. The resultant atelectasis localizes the disease, possibly allowing healing (regeneration, increase in surfactant). In the immature fetus, cytokines accelerate the differentiation of surfactant, preventing RDS. After birth, however, persistent inflammation is associated with low SP-A and chronic lung disease. A future challenge is to understand how to inhibit or redirect the inflammatory response from tissue destruction and poor growth towards normal lung development and regeneration.     

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.     

Standardization criteria for the study of fetal lung maturity by means of gas-liquid chromatography of the fatty acid content of the amniotic fluid]

Gas-chromatographic analysis of the fatty acids (P/S ratio) in 10 samples of amniotic fluid and 10 samples of the pellets obtained after centrifugation of amniotic fluid at 3500 X g for 60 minutes were carried out to evaluate the effects of contaminants that might be present in amniotic fluid. The P/S ratio is used as an index of the degree of maturity of the fetal or neonatal lung. We propose a standard procedure of centrifugation for 60 minutes at 3500 X g followed by extraction and gas-chromatography as a rapid, valid way to measure the P/S ratio.     

过期妊娠并发羊水过少70例分析

目的 探讨过期妊娠合并羊水对围产儿的影响。方法 将２３３例过期妊娠的孕妇分为羊水过少组７０例及不适量组１５３例,动态观察两 组４０￣４３孕周１２ｈ尿Ｅ／Ｃ值、Ｂ超检测羊水量,产后胎盘检查,胎儿宫内窘迫,新生儿窒息剖宫产及围产儿死亡发生率,两组进行分析比较。结果 不过少组４０￣４１孕周即开始出现不量减少及胎盘功能下降,产时胎儿窘迫,羊水杂,Ａｐｇａｒ评分以及剖宫产率均地羊水适量组（Ｐ〈０．０５,Ｐ〈     

胎膜早破后羊水残余量与宫内感染和新生儿发病率的相关性分析

目的：探讨胎膜早破后残余羊水量与宫内感染和新生儿发病率的相关性。方法：根据产妇胎膜早破后残余羊水指数（Amnitic Fluid Index,AFI）的监测结果,将本组120例产妇分为三组：羊水量正常组82例;羊水量较少组20例;羊水量过少组18例。所有产妇入院后行胎膜早破常规处理,回顾分析并统计三组产妇宫内感染率,胎儿窘迫率和新生儿发病率。结果：在宫内感染发生率上,三组比较,差异具有显著性,羊水量较少组、羊水量过少组宫内感染的发生率明显高于羊水量正常组（P〈0．01）。在胎儿窘迫发生率上,三组比较,差异无统计学意义（P〉0．05）。在新生儿发病率上,三组比较,差异具有显著性,羊水量较少组、羊水量过少组新生儿的发病率明显高于正常纽（P〈0．01）。Logistic回归分析发现羊水量、产妇基础性疾病和分娩孕周为孕产妇宫内感染和新生儿发病的关键影响因素。结论：胎膜早破后残余羊水量过少与宫内感染和新生儿发病=翠的升高密切相关,产妇于胎膜早破后应加强残余羊水量的监测,以采取有效措施降低宫内感染和新生儿发病的发生率。     

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.     

Changes in acethylcholinesterase activity and total protein in the developing fetal brain

Summary Pre-natal changes in the physiological development of the porcine conceptus indexed by acetylcholinesterase (AChE) activity and total protein content of the fetal brain and amniotic fluid were determined from 4 to 12 weeks of gestation at intervals of two weeks. Marked brain and body development was observed between four and six weeks of gestation. AChE activity in the amniotic fluid declined non-significantly with gestation length while fetal brain AChE activity increased with advancing gestation. Total protein levels in both the amniotic fluid and fetal brain were relatively steady and no significant changes were observed. Changes in AChE activity of the fetal brain may therefore be related to growth changes in the fetus.     

Intestinal and renal origin of trehalase activity in rabbit amniotic fluid

To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term.     

A practical method to analyze the phospholipids in the amniotic fluid using HPTLC

An operative method using HPTLC which makes possible the determination of nine phospholipids in the amniotic fluid individually is described: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid and cardiolipine. Since a simple and reliable method for clinical practice was sought, the working techniques, materials, chromatographic solvents and staining reagents were chosen accordingly. The standardization method was made by using standard phospholipids. This method was tested with 34 samples of amniotic fluid. A discussion of the practical application in the determination of some ratios of fetal pulmonary maturity is made.     

Perinatal asphyxia pathophysiology in pig and human: a review

In utero fetuses are evidently exposed to several factors that cause an interruption of the oxygen flow through the umbilical cord causing asphyxia leading to hypoxia and metabolic acidosis. These conditions are important causes of intra-partum and neonatal mortality. The main objective of this review is to provide current information regarding the pathophysiology of asphyxia in piglets around parturition; the physiological mechanisms invoked by affected piglets to compensate perinatal hypoxemia are discussed. This review also addresses some similarities and differences of asphyxia between piglets and other mammals, including human neonates. Metabolic acidosis and hypoxia are sequela to asphyxia and can cause profound health effects in postnatal performance because of an abnormal suckling, a reduced absorption of colostrum and inadequate passive transfer of neonatal immunity. Acidosis also cause hypothermia, increased mortality and reduced survival in neonates. One of the first deleterious effects of intrauterine hypoxia is the expulsion of meconium into the amniotic sac leading to meconium staining of the skin, and in severe cases, meconium aspiration into the lungs. Even though there have been technological changes and improvements in husbandry, piglet mortality due to asphyxia remains a major problem. One potential alternative to reduce neonatal mortality in pigs is the monitoring of fetal stress during birth and the implemention of strategies such as the Apgar score, that is often used in human pediatrics. It is also important to consider the physiological, behavioral and biochemical changes that take place during parturition which subsequently impact the vitality, maturity and development of neonatal pigs. Understanding the pathophysiology of fetal hypoxia should help practitioners and farmers implement more effective delivery techniques aimed at reducing neonatal mortality and improving postnatal performance.     

Bovine amniotic and allantoic fluids for the culture of murine embryos

This study evaluated bovine amniotic and allantoic fluids as culture media for two-cell murine embryos to the hatched blastocyst stage. Amniotic and allantoic fluids were collected from four 70-d periods of pregnancy and pooled from at least five different animals. In Experiment 1 (n = 470) the fluids were frozen twice. Treatments consisted of twice frozen amniotic or allantoic fluid from each pregnancy period, Whitten's medium and fetal calf serum. The later two media were controls. Twice-frozen amniotic fluid <70 d pregnancy period, fetal calf serum and Whitten's medium supported the development of embryos to the hatched blastocyst stage. Whitten's medium was superior to twice-frozen amniotic fluid <70 d pregnancy period or fetal calf serum (P<0.01). Biochemical analysis showed lower glucose in amniotic and allantoic fluids than in Whitten's medium. Experiment 2 (n = 425) was performed to evaluate the effect of glucose supplementation to amniotic fluid. No benefit of glucose supplementation of the amniotic fluid was observed. In Experiment 3 (n = 432), the fluids were transported nonfrozen on ice. Treatments consisted of nonfrozen amniotic fluid <70 d pregnancy period; nonfrozen amniotic fluid <70 d pregnancy period + glucose), nonfrozen allantoic fluid <70 d pregnancy period; and Whitten's medium. The percentages of embryos developing to hatched blastocyst stage were 66.6, 56.5, 57.4 and 63.9% respectively, for each of the four treatments. No differences were found between any two treatments (P<0.05). In Experiment 4 (n = 231) the fluids were stored at -20 degrees C for 15 d. Whitten's medium was superior to amniotic or allantoic fluid <70 d pregnancy period in sustaining embryo development (P<0.05). In conclusion, these data indicate that nonfrozen bovine amniotic or allantoic fluid <70 d pregnancy period can support the development of murine embryos to the hatched blastocyst stage comparable to culture in Whitten's medium. Glucose supplementation of the amniotic fluid offered no advantage, and freezing of fluids had an adverse effect on in vitro embryo development.     

Increased amniotic vasopressin levels in experimentally growth-retarded rat fetuses

Arginine-vasopressin (AVP) and oxytocin are neuropeptides that are not only released as hormones into the peripheral circulation, but are also involved in central processes, e.g., in brain development. Earlier experiments suggested an inverse relationship between amniotic AVP and fetal growth. To see whether increased peptide levels reflect fetal growth retardation, and to determine cause and effect of this relationship, AVP and oxytocin content were determined in amniotic fluid of growth-retarded fetuses by radioimmunoassay. Growth retardation was established either by intraperitoneal administration of methylazoxymethanol to the mother, or by undernourishment of the mother. Elevated amniotic AVP levels were found in the methylazoxymethanol-treated and undernourished rats, partly concomitant with smaller amount of amniotic fluid. Amniotic AVP levels were inversely related to fetal body weight, while a similar trend was found for fetal brain weight. In addition, a positive correlation was found between fetal body weight and amniotic oxytocin in control rats.     

Soluble Leukocyte-Associated Ig-Like Receptor-1 in Amniotic Fluid Is of Fetal Origin and Positively Associates with Lung Compliance

The soluble form of the inhibitory immune receptor leukocyte-Associated Ig-like Receptor-1 (sLAIR-1) is present in plasma, urine and synovial fluid and correlates to inflammation. We and others previously showed inflammatory protein expression in normal amniotic fluid at term. We hypothesized that sLAIR-1 is present in amniotic fluid during term parturition and is related to fetal lung function development. sLAIR-1 was detectable in all amniotic fluid samples (n=355) collected during term spontaneous deliveries. First, potential intra-uterine origins of amniotic fluid sLAIR-1 were explored. Although LAIR-1 was expressed on the surface of amniotic fluid neutrophils, LAIR-1 was not secreted upon ex vivo neutrophil stimulation with LPS, or PMA/ionomycin. Cord blood concentrations of sLAIR-1 were fourfold lower than and not related to amniotic fluid concentrations and placentas showed no or only sporadic LAIR-1 positive cells. Similarly, in post-mortem lung tissue of term neonates that died of non-pulmonary disorders LAIR-1 positive cells were absent or only sporadically present. In fetal urine samples, however, sLAIR-1 levels were even higher than in amniotic fluid and correlated with amniotic fluid sLAIR-1 concentrations. Second, the potential relevance of amniotic fluid sLAIR-1 was studied. sLAIR-1 concentrations had low correlation to amniotic fluid cytokines. We measured neonatal lung function in a convenient subset of 152 infants, using the single occlusion technique, at a median age of 34 days (IQR 30-39). The amniotic fluid concentration of sLAIR-1 was independently correlated to airway compliance (ρ=0.29, P=.001). Taken together, we show the consistent presence of sLAIR-1 in amniotic fluid, which originates from fetal urine. Concentrations of sLAIR-1 in amniotic fluid during term deliveries are independent from levels of other soluble immune mediators. The positive association between concentrations of amniotic fluid sLAIR-1 and neonatal lung compliance suggests that amniotic fluid sLAIR-1 may be useful as a novel independent marker of neonatal lung maturation.     

Fetal-maternal transfer of [H3]-6 beta-hydroxycortisol in the pregnant ewe

Distribution and fetomaternal transfer of 6 beta-hydroxycortisol (6 beta-OHF) was studied using serial sampling following injection of tritium labelled 6 beta-OHF into various fluid compartments in the chronically cannulated unaesthesized pregnant ewe. There was a rapid transfer of 6 beta-OHF from the fetal circulation into amniotic fluid and maternal blood. In contrast, the maternal----fetal transfer of this steroid metabolite was considerably less. The sequence of appearance of 6 beta-OHF in fetal blood and amniotic fluid following injection into maternal blood suggests that this steroid is first transferred across the placenta to fetal blood before gaining entry into the amniotic fluid space. The half-lives of 6 beta-OHF after initial equilibration in maternal plasma, fetal plasma and amniotic fluid were 2.0 h, 5.1 h and 8.9 h respectively. The amniotic sac appears to contain a relatively static pool of 6 beta-OHF and may act as a "trap" for 6 beta-OHF in the ovine conceptus.