Immunoassay for the determination of surfactant apoprotein (SP-A) in human amniotic fluid: comparison with other indices of assessing foetal lung maturity.

The concentration of the major surfactant-associated protein SP-A (28-36 kDa) was determined in 73 amniotic fluid samples obtained from normal (n = 40) and complicated (n = 33) pregnancies. Lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol (PG) levels were also determined in all the samples by one-dimensional step-wise thin-layer chromatography. An enzyme-linked immunosorbent assay was used to determine human lung surfactant apoprotein SP-A. The amount of SP-A in human amniotic fluid increased as a function of gestational age from 8 mg l-1 at 36 weeks to 11.75 mg l-1 at 40-41 weeks of gestation. There was a significant difference (p less than 0.01) in amniotic fluid SP-A concentration from female (9.93 +/- 0.60 micrograms ml-1) compared to male (9.10 +/- 0.52 micrograms ml-1) foetuses. In amniotic fluid samples obtained from a group of complicated pregnancies, SP-A levels were significantly lower than in the normal group when adjusted for gestational age and sex of the foetus (p less than 0.05).     

Studies on the effects of betamethasone, L-carnitine, and betamethasone-L-carnitine combinations on the dipalmitoyl phosphatidylcholine content and phosphatidylcholine species composition in foetal rat lungs

Administration of L-carnitine or betamethasone to pregnant rats failed to increase either the total phospholipid or dipalmitoylphosphatidylcholine (DPPC) contents in foetal rat lungs on the 20th day of gestation, compared to controls. The combined administration of betamethasone (0.3 mg/kg) and L-carnitine (80 mg/kg) resulted in a pronounced increase of dipalmitoylphosphatidylcholine (7.8 +/- 2.5 mg/g dry weight) compared with the control group (5.4 +/- 1.8 mg/g dry weight), and compared with the groups receiving betamethasone (5.9 +/- 1.9 mg/g dry weight) or L-carnitine (5.6 +/- 1.5 mg/g dry weight) alone. The proportion of dipalmitoylphosphatidylcholine in the phosphatidylcholine species increased from 20.9 +/- 2.1% in the foetal lungs of the control group to 22.6 +/- 5.0% in the L-carnitine group, to 24.3 +/- 3.3% (p less than 0.01) in the betamethasone-L-carnitine (20 mg/kg) group, to 25.2 +/- 3.5% (p less than 0.01) in the betamethasone group, to 27.1 +/- 2.6% (p less than 0.01) in the betamethasone-L-carnitine (40 mg/kg) group, and to 28.4 +/- 3.7% (p less than 0.01) in the betamethasone-L-carnitine (80 mg/kg) group, while the palmitic acid portion in the phosphatidylcholine fatty acids was nearly unchanged. A pronounced increase of palmitoyl-myristoyl phosphatidylcholine (PC-30), the second disaturated phosphatidylcholine species present in lungs in significant amounts beside dipalmitoylphosphatidylcholine, was noted only in betamethasone treated animals. Furthermore, after betamethasone and betamethasone-L-carnitine treatment, a significant diminution (p less than 0.01) of the proportion of palmitoyl-palmitoleyl phosphatidylcholine (16 : 0/16 : 1-PC) in the phosphatidylcholine species was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration and physicochemical characterisation of unsaturated cobalamin binding proteins in amniotic fluid

Indirect evidence for the presence of intrinsic factor in amniotic fluid has been provided recently, using either a radioisotope binding assay or a radioimmunoassay. We have determined the unsaturated cobalamin binding capacity and the physicochemical properties of the 3 cobalamin binding proteins in 59 amniotic fluids using radioisotope binding assay, gel filtration and isoelectrofocussing. A good correlation with gestational age was found for the total unsaturated Cbl binding capacity (r = 0.735) and for the concentration of unsaturated haptocorrin (r = 0.746), but not for the concentration of unsaturated intrinsic factor (r = 0.003). When their binding capacities were expressed as a percentage of the total unsaturated Cbl binding capacity, the percentage of intrinsic factor, transcobalamin II and transcobalamin III (the less acidic fraction of haptocorrin) decreased and the percentage of haptocorrin increased in function of gestational age. The physicochemical properties of intrinsic factor in amniotic fluid were close to those in gastric juice: the molecular mass was estimated to 49,200 +/- 4,900 Da (n = 24) in Sephacryl S 300 gel filtration, the cobalamin-protein complex was resolved in 2-6 isoproteins isoelectric at a pH range of 4.6-5.8 and with a mean isoelectric point of 5.18 +/- 0.16 (n = 5) in isoelectrofocusing and it crossreacted with anti-intrinsic factor autoantibodies (from a Biermer anaemia serum). Amniotic fluid collected at 13 wk of gestational age was found to contain intrinsic factor and haptocorrin with less acidic isoproteins than those usually observed in gastric juice and serum. This could indicate that sialic acid is less involved in the composition of the carbohydrate core of cobalamin binding glycoproteins in this period of the gestational age than later on and that cobalamin binding proteins have mainly a foetal origin.     

Is there a relationship between fetal weight and amniotic fluid index?

OBJECTIVE: To establish whether there is a relationship between the amniotic fluid index and estimated fetal weight in the third trimester. The presence of a relationship would require adjustment of amniotic fluid index to take account of estimated fetal weight with potential improvement in its prediction of adverse perinatal outcomes. METHODS: Paired measurements of amniotic fluid index and estimated fetal weight from 274 low-risk pregnancies enrolled in a longitudinal study of fetal growth. Measurements were made at fortnightly intervals from 30 weeks' gestation until delivery. A relationship between amniotic fluid index and estimated fetal weight was sought at gestational age week intervals of 30-32, 33-35, 36-38 and 39-41. RESULTS: One thousand and three pairs of measurements of amniotic fluid index and estimated fetal weight were available for analysis. Mean amniotic fluid index decreased towards term as expected. There was no correlation between amniotic fluid index and estimated fetal weight. Furthermore, there was no correlation between amniotic fluid index and estimated fetal weight at any of the gestational age intervals. CONCLUSIONS: There is no clinically relevant correlation between amniotic fluid index and estimated fetal weight. It should remain clinical practice to take account of gestational age when interpreting amniotic fluid index but it is not necessary to make adjustments for estimated fetal weight.     

Measurement, Characterization, and Source of Somatostatin-like Immunoreactivity in Human Amniotic Fluid

Somatostatin-like immunoreactivity (SLI) is widely distributed in tissues and biological fluids. To determine whether SLI is also present in amniotic fluid, samples obtained by amniocentesis from 30 normal and 27 abnormal pregnancies were studied by radioimmunoassay. Direct incubation of [(125)I-Tyr(1)]tetradecapeptide somatostatin (SRIF) with amniotic fluid resulted in 89% tracer degradation. Damage was reduced to <5% when samples were acidified and boiled before the assay. With this technique, SLI was detectable in all normal amniotic fluid samples; the mean level at 15-20 wk of gestation (320+/-55 pg/ml, n = 15) being 4.5 times higher than the mean at 32-43 wk (70+/-12 pg/ml, n = 15) (P < 0.001). In cases of preeclampsia (n = 6), gestational diabetes (n = 5), anencephaly (n = 1), and meningomyelocele (n = 1), SLI values were in the normal range, but in one juvenile diabetic and one patient with chronic renal failure, SLI was undetectable (<10 pg/ml). In a pair of monochorionic diamniotic twins, SLI levels were very different (33 and 197 pg/ml), which suggests that fetal factors are more important than materno-placental ones in determining amniotic fluid SLI. Serial dilutions of amniotic fluid showed parallelism with standard SRIF. When concentrates of pooled amniotic fluid were chromatographed on Sephadex G-25 columns, all SLI eluted in the void volume ahead of SRIF even after treatment with 8 M urea and dithiothreitol. This "big" SLI incubated in amniotic fluid showed 100% stability over 24 h at 37 degrees C, whereas SRIF was rapidly inactivated (t((1/2)) congruent with 7 min). Extracts of placenta and fetal membranes contained no SLI, but small amounts (6-20% of total amniotic fluid SLI) were found in cells from fresh fluid. Radioimmunoassay of SLI in extracts of seven paired cord arterial and venous plasma samples showed no arteriovenous gradient consistent with fetal origin of cord blood SLI. It is concluded that (a) amniotic fluid contains SLI which is of fetal origin and (b) normal levels vary with gestational age. The SLI has a higher molecular weight (>/=5,000) and is more stable in amniotic fluid than SRIF.     

Intrinsic factor in human amniotic fluid as determined by radioimmunoassay

The intrinsic factor (IF) concentration in 55 human amniotic fluid specimens was determined by radioimmunoassay (RIA). The antiserum was produced by immunizing rabbits with the cobalamin-IF complex isolated from human gastric juice. The median concentration of IF was 0.17 nmol/l and the extreme values less than 0.07-2.51 nmol/l. Three specimens with a clearly elevated level (0.96, 1.11 and 2.51 nmol/l) were observed. The highest value was associated with a fetal malformation, viz. obstruction of the proximal gut. There was no evident correlation between the concentration of IF in amniotic fluid and gestational age.     

Prostate-specific antigen in amniotic fluid of normal and abnormal pregnancies

Objective: To examine if prostate-specific antigen (PSA) is present in amniotic fluid or maternal serum during pregnancy and if its presence is associated with fetal abnormalities.

Methods: Samples tested included amniotic fluids from 853 pregnant women for whom amniocentesis was performed; 312 nonpregnant women who donated blood; 259 pregnant women who donated blood at various gestational ages. Amniotic fluid or serum PSA was measured with an ultrasensitive time-resolved immunofluorometric procedure. 372 pregnancies were studied for the presence of genotypic or phenotypic fetal abnormalities.

Results: PSA was present in most amniotic fluids; the median PSA concentration increased from gestational week 11 to 22 and stabilized thereafter until delivery. The most prominent PSA concentration change occurred during gestational weeks 13–14. Pregnant women had significantly higher serum PSA concentrations than nonpregnant women; the pattern of serum fSA concentration change during pregnancy was similar to that of amniotic fluid; however, serum PSA concentrations were lower by a factor of 20–40. No association existed between amniotic fluid F'SA and maternal age, gender of fetus, or length of abstinence of mother from sexual intercourse. After gestational week 15, fetuses with trisomy 21 or 18, anencephaly, or renal disorders were associated with low amniotic fluid PSA levels.

Conclusion: Our data suggest that PSA may play a role in fetal development, especially at gestational ages between 13–20 weeks. The diagnostic usefulness of PSA in identifying fetal abnormalities remains to be determined.     

Concanavalin A binding of alpha-fetoprotein in amniotic fluid as an aid in the diagnosis of neural tube defects

Concanavalin A nonreactive alpha-fetoprotein was determined in samples of amniotic fluid from 16 abnormal pregnancies complicated by anencephaly (7), open spina bifida (6), intra-uterine death (1), anencephaly with exomphalos (1), or open spina bifida with exomphalos (1), and in amniotic fluid from 50 normal pregnancies with gestational age between 13 and 24 weeks. In all 16 cases with fetal malformations, the proportion of nonreactive alpha-fetoprotein was significantly decreased (median 5.3%) as compared with amniotic fluid from pregnancies with a normal outcome (median 39.7%). The results confirm that this measurement is useful in the diagnosis of neural tube defects, especially when the concentration of alpha-fetoprotein in amniotic fluid is normal or only slightly above normal and gestational age is uncertain.     

Acetylcholinesterase and butyrylcholinesterase measurement in the pre-natal detection of neural tube defects and other fetal malformations

Acetylcholinesterase activity in amniotic fluid was measured at 30 degree C by a reaction rate method employing acetyl-beta-methyl thiocholine as substrate and ethopropazine as a selective inhibitor of butyrylcholinesterase. This assay proved more specific than previously reported methods. Activity was greater in five cases of anencephaly (4.8-9.7 U/l) and nine cases of spinal bifida (5.1-8.6 U/l) than in 50 pregnancies with normal outcome (mean activity 2.0 +/- 0.9 (S.D.) U/l). There was no overlap between results from normal and neural-tube-defect groups, and the results showed no significant correlation with gestational age. Butyrylcholinesterase activity in amniotic fluid was measured using butyrylthiocholine as substrate. In accordance with previous reports, levels were elevated in pregnancies affected by neural tube defects. The ratio butyrylcholinesterase/acetylcholinesterase activity showed similar values for anencephalic, spina bifida and normal pregnancies; however, the two cases of exomphalos investigated could be clearly distinguished from all other groups on this basis.     

Tetranectin in amniotic fluid, maternal serum and fetal fluids

The concentration of the newly discovered protein tetranectin has been measured in different fetal and maternal compartments. In amniotic fluid a significant, positive correlation between the tetranectin concentration and gestational age was found (a mean of 0.2 mg l-1 at week 14 to a mean of 0.5 mg l-1 at week 21). In maternal serum a slight negative correlation was found between tetranectin concentration and gestational week (a mean of 6.17 mg l-1 at week 14 to a mean of 5.79 mg l-1 at week 21). In-term cord blood collected at delivery a mean level of 6.0 mg l-1 was found, and no difference was found between arterial- and venous-blood tetranectin concentration. In fetal serum the overall mean level was 2.6 mg l-1 and a significant positive correlation between tetranectin concentration and gestational age was found. The mean level was 1.1 mg l-1 in fetal cerebrospinal fluid and no correlation to gestational age was found. Fetal tetranectin may, therefore, be correlated to fetal maturation.     

Human serum Zn-alpha2-glycoprotein in amniotic fluid

Human serum Zn-alpha2-glycoprotein (Zn-alpha2-GP) was found to be present in the amniotic fluid in the mean concentration of 0.98 +/- 0.40 mg/100 ml, which represents about one-tenth of its concentration in the maternal serum (9.65 +/- 1.18 mg/100 ml). Its concentration in the amniotic fluid was proportional to the amniotic fluid total protein and very approximately to the maternal serum Zn-alpha2-GP. The relationship between the maternal serum Zn-alpha2GP and the maternal serum total protein as well as between the amniotic fluid total protein and the maternal serum total protein was found to be not significant. The amniotic fluid Zn-alpha2-GP as well as the amniotic fluid total protein showed some increase during gestation to reach the highest values at the end of the second trimester. At present both the origin and significance of the amniotic fluid Zn-alpha2-GP are not known.     

Amniotic fluid palmitic acid concentrations and prediction of fetal lung maturity.

A method for rapid determination of total esterified palmitic acid concentration (TEPAC) in amniotic fluid is described. The correlation coefficient between the TEPAC and the lecithin concentration was 0.93 in 123 samples of amniotic fluid obtained during the last trimester. The respiratory distress syndrome (RDS) occurred in 73% of the cases studied with TEPAC less than mmol/l in predelivery samples. The RDS was not observed with higher concentrations except in cases of maternal diabetes mellitus. The predictive value of total esterified fatty acid studies was confirmed to the concentration of palmitic acid recorded, and no specific fatty acid distributions or ratios were reliable in identifying RDS or estimating gestational age.     

脑钠肽联合速度向量成像技术评估先天性心脏病胎儿心功能的临床价值

目的探讨羊水脑钠肽（BNP）联合速度向量成像技术（VVI）评估先天性心脏病胎儿心功能的临床价值。 方法应用ELISA方法测定正常对照组304例胎儿与先天性心脏病组47例胎儿羊水中BNP浓度,分析两组胎儿羊水中BNP浓度的差异及其与孕周的关系,同时联合运用速度向量成像技术对胎儿心功能进行评估。 结果先天性心脏病组胎儿羊水中BNP浓度值高于正常对照组,两组胎儿羊水中BNP浓度值比较差异有统计学意义（F ＝ 4.032,P<0.05）,且先天性心脏病组胎儿羊水中BNP浓度值随着羊水穿刺孕周增加而升高（r ＝ 0.289,P<0.05）,同时先天性心脏病组胎儿左心室心肌整体运动速度、应变、应变率较正常对照组胎儿降低,差异有统计学意义（P<0.05）。 结论先天性心脏病组胎儿左心室心肌整体运动参数下降,羊水中BNP浓度增高,且与孕周呈正相关性,羊水BNP浓度测定值可作为产前评价胎儿心功能潜在损害的一个生物学指标。     

Cervical length assessment in women with idiopathic polyhydramnios.

OBJECTIVE: The aims of the study were to determine cervical length among patients with polyhydramnios and to assess the relationship between the severity of polyhydramnios, cervical length and gestational age at delivery. PATIENTS AND METHODS: A prospective study was designed including 92 consecutive singleton pregnancies with polyhydramnios between 24 and 40 weeks' gestation. Cervical length was measured using transvaginal sonography. Polyhydramnios was defined when amniotic fluid index (AFI) was equal to or greater than 20 cm. A single sonologist performed all the examinations of the cervical length and the AFI. RESULTS: The median cervical length and AFI were 37.5 (range, 7-52) mm and 28.8 (range, 20-43) cm, respectively. A significant gradual shortening of the cervical length was observed with advancing gestational age (P=0.027). No significant association was found between AFI and cervical length (P=0.24). A cut-off of 15 mm (n=5) was associated with a significantly lower gestational age at delivery (30+/-2.6 weeks vs. 37.2+/-4.2 weeks, respectively, P<0.001). CONCLUSIONS: Women with polyhydramnios have a gradual shortening of cervical length with advancing gestational age. However, this finding is not related to the severity of polyhydramnios.     

Effect of amniotic fluid volume on ultrasonic fetal weight estimation.

We evaluated the relationship between the amniotic fluid index and the accuracy of fetal weight estimation using ultrasonography. Six hundred and sixty-four patients, between 20 and 42 weeks' gestational age, who were delivered within 1 week of a sonographic examination were studied. Five formulas for estimating fetal weight also were studied. Systematic and random errors in predicting birth weight were analyzed relative to birth weight and amniotic fluid index. The accuracy of sonographic fetal weight estimation was independent of amniotic fluid index across all gestational ages and birth weights. Each of the five formulas had similar error percentages, and no significant differences were detected. Predicted fetal weight was significantly underestimated with each of the formulas studied, a finding that was also independent of birth weight and amniotic fluid index. We conclude that ultrasonography can be used reliably to estimate fetal weight in patients with altered amniotic fluid volumes.     

The thromboplastic activity of lung surfactant in amniotic fluid and its application to prenatal assessment of fetal lung maturity

Based on the fact that both tissue thromboplastin and lung surfactant show lamellar structures under the electron microscope and belong chemically to lipoprotein, the thromboplastic activity of lung surfactant in amniotic fluid was studied by measuring plasma recalcification time. The results obtained were as follows (1) The surfactant fractions isolated from amniotic fluid and rabbit or pig lung showed the thromboplastic activity with dose response. (2) The thromboplastic activity of amniotic fluid increased with advancing gestational age. (3) It was found that the thromboplastic activity determined by plasma recalcification time was parallel with the surfactant concentration of amniotic fluid. (4) The shortening rate of plasma recalcification time in amniotic fluid could estimate well the risk of RDS, and the critical value for RDS was assumed to be about 33%.     

Evaluation of two assays of functional surfactant in amniotic fluid: surface-tension lowering ability and the foam stability index test

We characterize two assays of total amniotic fluid surfactant that are based on function: the surface-tension lowering ability of extracts of amniotic fluid lipid (I) and the foam stability index test (II). I is determined on chloroform extracts of amniotic fluid. II is defined as the highest ethanol volume fraction of an amniotic fluid-ethanol mixture that will permit a stable foam to form after 30 s of vigorous shaking. The relationship of I to disaturated phosphatidylcholine concentrations (after osmium tetroxide treatment of the amniotic fluid lipid extract) is in the expected theoretical form of a hyperbolic function. The relation between values for II and disaturated phosphatidylcholine concentrations showed a consistent bias, suggesting that components other than disaturated phosphatidylcholine contribute to stable foam formation. Phosphatidylclycerol concentrations did not appear to account for this bias. The relation between I to II values suggest that both assays measure total surfactant. I, II, and concentration of disaturated phosphatidyl choline are all excellent indicators of fetal pulmonary maturity. From a practical standpoint, the foam stability index test is the most efficient approach to routine assessment of fetal pulmonary status.     

Interference by endogenous glycerol in an enzymatic assay of phosphatidylglycerol in amniotic fluid

We investigated the possibility of interference by endogenous glycerol with the enzymatic measurement of phosphatidylglycerol in amniotic fluid. Phosphatidylglycerol is an important indicator of fetal lung maturity. The concentrations of glycerol and phosphatidylglycerol in amniotic fluid were measured by using a coupled enzymatic assay with and without phospholipase D (EC 3.1.4.4). The precision of the assay was acceptable (within-run CV = 1.2%, between-run CV = 4.8%). Endogenous glycerol content was demonstrated to be approximately 10-20 times that of phosphatidylglycerol. This high proportion of endogenous glycerol in amniotic fluid would preclude the accurate enzymatic determination of amniotic fluid phosphatidylglycerol unless the glycerol is first removed. Nor can the actual phosphatidylglycerol concentration be determined by subtracting the endogenous glycerol concentration from the total glycerol, which includes that glycerol derived from phosphatidylglycerol. With a usual range of 9 +/- 7 mumol/L, the error for a given phosphatidylglycerol measurement of +/- 6.6 mumol/L (+/- 2 SD) clearly is too high for this assay to be clinically useful. There was no correlation between concentration of endogenous glycerol or apparent phosphatidylglycerol in amniotic fluid and the lecithin/sphingomyelin ratio of the sample.     

PCR-SSP技术对羊水细胞ABO血型的基因鉴定

目的通过PCR-SSP基因技术检测胎儿羊水细胞ABO血型基因型,产前诊断胎儿ABO血型。方法选取了6名孕16 W以上的孕妇,抽取羊水细胞并进行分离,提取羊水细胞DNA,运用PCR-SSP技术分析其ABO血型基因型,并通过出生后的脐带血的血型鉴定进行确认。结果 6例羊水标本均通过PCR-SSP方法检测出了ABO血型的基因型;该6名胎儿的脐带血的ABO血型与羊水细胞的血型一致。结论 PCR-SSP技术可以准确地检测胎儿羊水细胞的ABO血型。     

The quantitation of oxalate in amniotic fluid by ion-chromatography

Calcium oxalate is the predominant constituent of most kidney stones. The rare genetic disorder, primary hyperoxaluria, is characterized by the continuous excessive synthesis and urinary excretion of oxalic acid, leading to stone formation and renal insufficiency. The earliest measurement of oxalate in suspected cases of primary hyperoxaluria is advantageous and would lend support for continued analysis and eventual confirmation of the disease. Therefore, we quantitated oxalate levels in amniotic fluid (AF) using medium pressure ion-exchange chromatography. The mean concentration of oxalate in amniotic fluid was 1.67 mg/l +/- 0.8 (SD); (range 0.64 to 5.11 mg/l). The mean oxalate/creatinine ratio (O/C) was 0.23 +/- 0.11 (SD); (range 0.07 to 0.53). This ratio is similar to that found in the urine of infants less than 1 year (0.19 +/- 0.10; n = 17). There was no significant difference between males and females in oxalate concentration or O/C ratio. Regression analysis showed no significant correlation of fetal age with oxalate, O/C or creatinine. Studies in 13 sets of di-amnionic twins showed no statistical difference in oxalate or O/C between twin A and B. This study demonstrates the ability to accurately quantitate oxalate in amniotic fluid by ion-chromatography, and suggests that this may have a potential application in the initial screening process for the prenatal detection of primary hyperoxaluria.