Proteomic profile determination of autosomal aneuploidies by mass spectrometry on amniotic fluids

Background  

Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time-consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance of women with normal results can be performed by molecular analysis of uncultured foetal cells. In the present study, we developed a proteomic fingerprinting approach coupled with a statistical classification method to improve diagnosis of aneuploidies, including trisomies 13, 18, and 21, in amniotic fluid samples.     

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.     

Free Amino-acid Concentrations in Fetal Fluids

The pattern of free amino-acid concentrations in maternal venous plasma, fetal umbilical arterial plasma, fetal urine, and amniotic fluid at 15 to 20 weeks'' gestation has been determined. Free amino-acid concentrations were greater in fetal plasma than in maternal plasma, amniotic fluid, or fetal urine.The ratios of amino-acid concentrations in fetal umbilical arterial plasma and urine indicate that the fetal kidney can effectively conserve amino-acids, possibly reaching an adult level of competence in this respect.There was little correlation between amino-acid concentrations in the fluids analysed with the exception of that between amniotic fluid and fetal urine.     

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.     

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.     

Computer model of fetal-maternal heat exchange in sheep

We constructed and used a mathematical model of maternal-fetal heat exchange in the sheep to explore the effects of changes in certain parameters on steady-state fetal temperatures and to determine whether the fetus in the model has any potential to control its own temperature. The model took into account both fetal and placental heat production and exchange of heat in the placenta, across the fetal skin, via amniotic fluid, and through the uterine wall. The maternal ewe was assumed to be a constant temperature heat sink. Changes in placental or fetal heat production were calculated to change the ratio of heat exiting across the placenta or fetal skin significantly but to have little effect on fetal core temperature, e.g., a rise of only 0.8 degrees C was predicted after a twofold increase in fetal heat production. Fetal placental blood flow was calculated to affect fetal temperature the most of any flow, a reduction to zero causing fetal temperature to rise 5.0 degrees C. Changes in heat conductances between fetal skin and amniotic fluid, or between amniotic fluid and uterine wall, had minimal effect on fetal temperature. From the model calculations here and because heat exchange within the sheep placenta has previously been calculated to be extremely efficient, we conclude that the fetal sheep has little ability to control its temperature by changes in heat dissipated through extraplacental pathways. Thus the model predicts an effective heat clamp that closely links fetal to maternal temperature.     

Absorption of amniotic fluid by amniochorion in sheep

Swallowing of amniotic fluid and lung fluid inflow were eliminated in 10 chronically instrumented fetuses. The urachus was ligated, and fetal was urine drained to the outside. At the beginning and the end of 21 experiments of 66 +/- 5 (SE) h duration, all amniotic fluid was temporarily drained to the outside for volume measurement and sampling. Amniotic fluid osmolalities and oncotic pressures were experimentally controlled. Amniochorionic absorption of amniotic fluid depended strongly on the osmolality difference between amniotic fluid and fetal plasma (P < 0.001), but at zero osmolality difference there still was a mean absorption rate of 23.8 +/- 4.7 (SE) ml/h (P < 0.001). Absorption was unaffected by the protein concentration difference between amniotic fluid and fetal plasma, but infused bovine albumin in the amniotic fluid was absorbed at a rate of 1.8 8 +/- 0.4 g/h (P < 0.001), corresponding to a volume flow of fluid of 33.8 8 +/- 6.1 ml/h (P < 0.001). Fluid absorption in the amniochorion is driven in part by crystalloid osmotic pressure, but about 25 ml/h is absorbed by a path that is permeable to protein. That path has the physiological characteristics of lymphatic drainage, although no anatomic basis is known to exist for a lymphatic system in the amniochorion.     

羊水菌群的研究进展

传统观念认为胎儿在宫内发育的过程是无菌的。随着研究方法的快速发展,针对于微生物的研究方法从传统的培养到现代分子生物学检测的进步,研究者已经认识到胎盘、羊水中具有微生物的存在。目前的研究尚不能明确胎儿宫内胎盘、羊水的菌群来源及菌群转移的具体途径,但已有的研究提示羊水菌群最可能来源为母亲肠道、生殖道以及口腔。本文综述了研究者对羊水菌群认识的转变、羊水菌群的来源以及羊水菌群与早产的相关性。     

Effects of acute oligohydramnios on respiratory system of fetal sheep.

Prolonged oligohydramnios, or a lack of amniotic fluid, is associated with pulmonary hypoplasia and subsequent perinatal morbidity, but it is unclear whether short-term or acute oligohydramnios has any effect on the fetal respiratory system. To investigate the acute effects of removal of amniotic fluid, we studied nine chronically catheterized fetal sheep at 122-127 days gestation. During a control period, we measured the volume of fluid in the fetal potential airways and air spaces (VL), production rate of that fluid, incidence and amplitude of fetal breathing movements, tracheal pressures, and fetal plasma concentrations of cortisol, epinephrine, and norepinephrine. We then drained the amniotic fluid for a short period of time [24-48 h, 30.0 +/- 4.0 (SE) h] and repeated the above measurements. The volume of fluid drained for the initial studies was 1,004 +/- 236 ml. Acute oligohydramnios decreased VL from 35.4 +/- 2.9 ml/kg during control to 22.0 +/- 1.6 after oligohydramnios (P less than 0.004). Acute oligohydramnios did not affect the fetal lung fluid production rate, fetal breathing movements, or any of the other measured variables. Seven repeat studies were performed in six of the fetuses after reaccumulation of the amniotic fluid at 130-138 days, and in four of these studies the lung volume also decreased, although the overall mean for the repeat studies was not significantly different (27.0 +/- 5.2 ml/kg for control vs. 25.5 +/- 5.5 ml/kg for oligohydramnios). Again, none of the other measured variables were altered by oligohydramnios in the repeat studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Metabolic response to starvation in late pregnant rats. II. Fetal response

Effect of prolonged maternal fasting on the fetal liver and heart glycogen and triglyceride content and on concentration of glucose, urea, uric acid and alpha amino-nitrogen in the amniotic fluid has been studied in rats. The animals were divided into four groups: fed (control), fasted for one day (from 20 to 21 day of pregnancy), fasted for two days (from 19 to 21 day) and fasted for three days (from 18 to 21 day). Maternal fasting for two and three days resulted in reduction in fetal growth. The fetal liver glycogen content was reduced already after one day of fasting, stabilized after two days and then further decreased after three days. The fetal heart glycogen content was reduced only after three days of fasting. The fetal liver triglyceride content increased gradually during the first two days of fasting and then stabilized. The content of triglycerides in the heart was elevated after two and three days of food deprivation. The amniotic fluid glucose concentration decreased after one day of fasting and then stabilized. Fasting did not effect the concentration of the nitrogenous compounds in the amniotic fluid. It is concluded that maternal fasting affects markedly metabolism of energy substrates stored in the fetal liver and the heart and the composition of the amniotic fluid.     

Temperature responses following ventilation of the fetal sheep in utero

The mammalian fetus produces significant quantities of heat. This passes to the mother principally through the placenta and to a lesser extent via a pathway comprising the skin, amniotic fluid, and uterine wall. To assess the importance of the lesser pathway, temperature responses were recorded in 7 near-term fetal sheep after intrauterine ventilation with oxygen, after snaring the umbilical cord to block the placental route, and following fetal death. Four distinguishing characteristics of responses were observed: fetal temperature rose 0.10 +/- 0.03 (SEM) degrees C after oxygenation; it rose progressively an additional 0.9 +/- 0.1 degrees C during the 90-min interval after cord snaring; amniotic fluid temperature rose slowly until it was about midway between fetal and maternal temperature; and after fetal death, fetal amniotic fluid temperatures fell slowly. In a simple mathematical model with constant parameters these results could not be explained fully. It was necessary to assume that heat production rose with increased oxygenation and elevated body temperature and that ventilation increased heat transfer through the amniotic fluid, as would occur if chest wall movement were stirring the fluid. Using the model, the value for heat conductance from fetal skin to amniotic fluid was estimated to be 10.5 watts degrees C-1 under basal conditions.     

The origin and fate of hyaluronan in amniotic fluid

The mechanisms which regulate the steady-state concentration and molecular weight of hyaluronan in the amniotic fluid of sheep at different gestational ages have been investigated. An attempt to trace the origin of the polysaccharide has been made by analyses of various fetal fluids (amniotic fluid, allantoic fluid, tracheal fluid, urine, and serum). The fate has been studied by injection of radioactively labelled hyaluronan into the amniotic cavity and following the tracer in fetal tissues and fluids. The concentration of hyaluronan in amniotic fluid varies considerably but is in the order of 5 mg/l at mid-pregnancy and decreases to 1 mg/l in late pregnancy. The polysaccharide has a Mr-distribution with a weight-average in the order of 10(6) at 10 to 13 weeks of gestation which decreases to 10(5) closer to term. Calculations show that urine contributes 0.1 and 0.5 mg of low-molecular (Mr = 10(4) hyaluronan per day in mid- and late pregnancy, respectively, and the lung 10-20% of that amount in the form of high-molecular weight polymer (Mr greater than 10(6). The hyaluronan disappears from the amniotic cavity by bulk flow due to fetal swallowing. It is taken up and degraded in the fetal intestine. Molecules of Mr = 10(3) can pass the intestinal barrier. Calculations show that about 0.5 mg and 1.0 mg of hyaluronan is eliminated per day from the amniotic fluid at 12 and 17 weeks of gestation, respectively. Thus, the higher rate of elimination and the relatively high urinary contribution in more mature fetuses explain the low concentration and Mr of amniotic hyaluronan in late gestation, whereas a slower elimination combined with a relatively larger contribution of high molecular weight hyaluronan both from lung and urine and possibly from other sources are responsible for the higher concentration and Mr of the compound in early pregnancy.     

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.     

Oxytocin and vasopressin in the rat do not readily pass from the mother to the amniotic fluid in late pregnancy

In order to see whether the mother contributes to the vasopressin or oxytocin levels of amniotic fluid, these peptides were measured under conditions (1) in which the fetus lacks vasopressin (Brattleboro strain) and (2) where high maternal oxytocin and vasopressin plasma levels were induced by means of a controlled-delivery Accurel-collodion device. No vasopressin could be demonstrated in amniotic fluid of vasopressin-deficient fetuses present in a heterozygous (i.e., vasopressin-synthetizing mother). High peptide levels on the maternal side of Wistar rats generally failed to affect the amniotic fluid levels. The increase that was occasionally seen in amniotic vasopressin was probably due to fetal release concomitant with growth retardation. Amniotic vasopressin is derived from the fetus. Since amniotic fluid oxytocin is neither derived from the mother nor from the fetal brain, other fetal sources should be considered.     

Advances in the proteomics of amniotic fluid to detect biomarkers for chromosomal abnormalities and fetomaternal complications during pregnancy

Introduction: Amniotic fluid (AF) is a dynamic and complex mixture that reflects the physiological condition of developing fetus. In the last decade, proteomic analysis of AF for 16–18 weeks normal pregnancy has been done for the composition and functions of this fluid. Other body fluids such as urine, sweat, tears, etc. are being used for diagnosis of disease, but an insight into protein biomarkers of amniotic fluid can save the fetus and mother from future complications.

Areas covered: We have covered the proteomics of amniotic fluid done since 2000, in order to strengthen the establishment of these techniques as a recognized diagnostic tool in the field. After classifying the diseases based on chromosomal aneuploidies, gestational changes, and inflammation caused during pregnancy; we have focused on amniotic fluid to detect various complications during and post pregnancy and its effect on the fetomaternal relationship.

Expert comment: The main protein biomarkers responsible for various syndromes, diseases, and complications have been summarized. Major proteins identified for gestational conditions are IGFBP-1, fibrinogen, neutrophil defensins like calgranulins A and C, cathelicidin, APOA1, TRFE, etc. Validation of particular technique and establishing a single standardized biomarker for the diagnosis to avoid any overlapping for different diseases is required. After certain improvements, proteomics approach can be considered for diagnosis of diseases associated with fetal-maternal health.     

Amniotic fluid induces rapid epithelialization in the experimentally ruptured fetal mouse palate--implications for fetal wound healing

Cleft of the secondary palate is one of the most common congenital birth defects in humans. The primary cause of cleft palate formation is a failure of fusion of bilateral palatal shelves, but rupture of the once fused palate has also been suggested to take place in utero. The possibility of post-fusion rupture of the palate in humans has hardly been accepted, mainly because in all the cleft palate cases, the cleft palatal edge is always covered with intact epithelium. To verify whether the intrauterine environment of the fetus plays roles in wound healing when the once fused palate is torn apart, we artificially tore apart fetal mouse palates after fusion and cultivated them in culture medium with or without mouse or human amniotic fluid. We thereby found that the wounded palatal edge became completely covered with flattened epithelium after 36 hours in culture with amniotic fluid, but not in culture without amniotic fluid. Using histological and scanning electron microscopic analyses of the healing process, it was revealed that the epithelium covering the wound was almost exclusively derived from the adjacent nasal epithelium, but not from the oral epithelium. Such actions of amniotic fluid on the fetal wound were never simulated by exogenous epidermal growth factor (EGF), albumin, or both. In addition, the rapid epithelialization induced by amniotic fluid was not prevented by either PD168393 (an inhibitor of the EGF receptor-specific tyrosine kinase) or SB431542 (a specific inhibitor of TGFbeta receptor type I/ALK5). The present study provides new insights into the unique biological actions of amniotic fluid in the repair of injured fetal palate.     

The influence of experimentally produced oligohydramnios on lung growth and pulmonary surfactant content in fetal rabbits.

To study the effect of oligohydramnios on lung growth and biochemical lung development in fetal rabbits, amniotic fluid was drained through a tube inserted into the maternal peritoneal cavity on the 23 day of gestation. Littermate fetuses without an amniotic shunt were used as controls. The fetuses were delivered abdominally on the 28 day of gestation. In a total of 8 pregnant does, 17 fetuses underwent amniotic shunting and 22 fetuses were used as controls. The amniotic shunt produced a significant reduction in the amniotic fluid volume. There were no differences in the wet weights of the fetal body, liver or brain between the two groups. However, the amniotic shunt significantly decreased the wet weight of the fetal lung, fetal lung wet weight/body weight ratio, and protein concentration per lung as compared to the control fetuses. In the fetal liver and brain tissues, no changes were found in the concentrations of total phospholipids, phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC, the main component of lung surfactant) per g of wet tissue and per mg of protein. However, the lungs of the fetuses with amniotic shunts contained significantly more PC and DSPC, and the L/S ratio was higher than in the control fetuses. These results suggest that the oligohydramnios produced by an amniotic shunt causes pulmonary hypoplasia, but raises the pulmonary surfactant content of fetal rabbit lung.