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.     

Gestational profiles of rat placental lactogen-II (rPL-II) and growth hormone (GH) in maternal and fetal serum, amniotic fluid, and placental tissue.

Rat placental lactogen-II (rPL-II) and growth hormone (rGH) in maternal and fetal serum, amniotic fluid, and placental tissue were measured by a homologous radioimmunoassay during the last half of pregnancy. rPL-II appeared first in maternal circulation and the placental tissue on day 11 of pregnancy. The maternal serum rPL-II concentration increased progressively and reached the peak value (684 +/- 76 ng/ml) on day 19, and declined thereafter up to term. rPL-II content in the tissue had a similar pattern to the maternal serum profile of rPL-II, while its concentration in the tissue increased dramatically on day 12 and remained high until day 19. Fetal serum rPL-II was detected on days 17 and 18, though its concentration was much lower (ranged between 3-10 ng/ml) than that of maternal serum. rPL-II in amniotic fluid was also detectable only on days 12-14 of pregnancy, and the peak value on day 13 was 22% of the maternal serum rPL-II concentration. The rGH concentration increased gradually as pregnancy advanced with a decline on the day before parturition. Although rGH in fetal serum increased on day 20 with a decline on the following day, it was slightly detectable in amniotic fluid on the last two days of pregnancy. The molecular profile of rPL-II in amniotic fluid and maternal serum of day 13 pregnant rats were examined by Western blotting. Anti-rPL-II serum detected two proteins with molecular weights (mol wt) of 19.5K and 20.5K in amniotic fluid and one protein with a mol wt of 20.5K in maternal serum under nonreducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Cultivation of chorionic cells in studying the fetal karyotype in the 1st trimester of pregnancy

Primary culture of chorion cells obtained during the first trimester of pregnancy (7-9 weeks) through artificial abortion or by transcervical biopsy of the chorion tissue is an object for cytogenetic examination of fetus cells. The application of this cell culture for prenatal diagnosis of human hereditary diseases has a considerable advantage in time as compared with a routine method of using amniotic fluid cell culture during the second trimester of pregnancy.     

Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation

During fusion of the mammalian secondary palate, it has been suggested that palatal medial edge epithelial (MEE) cells disappear by means of apoptosis, epithelial-mesenchymal transformation (EMT) and epithelial cell migration. However, it is widely believed that MEE cells never differentiate unless palatal shelves make contact and the midline epithelial seam is formed. In order to clarify the potential of MEE cells to differentiate, we cultured single (unpaired) palatal shelves of ICR mouse fetuses by using suspension and static culture methods with two kinds of gas-mixtures. We thereby found that MEE cells can disappear throughout the medial edge even without contact and adhesion to the opposing MEE in suspension culture with 95% O2/5% CO2. Careful examination of MEE cell behavior in the culture revealed that apoptosis, EMT, and epithelial cell migration all occurred at various stages of MEE cell disappearance, including the transient formation and disappearance of epithelial triangles and islets. In contrast, MEE cells showed poor differentiation in static culture in a CO2 incubator. Furthermore, mouse and human amniotic fluids were found to prevent MEE cell differentiation in the cultured single palatal shelf, although paired palatal shelves fused successfully even in the presence of amniotic fluid. We therefore conclude that terminal differentiation of MEE cells is not necessarily dependent on palatal shelf contact and midline epithelial seam formation, but such MEE cell differentiation appears to be prevented in utero by amniotic fluid unless palatal shelves make close contact and the midline epithelial seam is formed.     

Amniotic Fluid Cells; Prenatal Sex Prediction and Culture

Sex chromatin studies were carried out on small amounts of amniotic fluid obtained by amniocentesis or from intact amniotic sacs removed at hysterotomy. Provided that satisfactory preparations were obtained the accuracy of fetal sexing was 87%. Nevertheless, in the management of a pregnancy in which there is a risk of a serious X-linked recessive disorder, repeat amniocentesis may be necessary to ensure satisfactory specimens.Of 90 samples of fluid cultured, satisfactory growth was obtained in 49; the success rate was not increased by the addition of stimulants to the culture medium. It is suggested that between the 13th and the 16th week of pregnancy is the optimum time for amniocentesis to obtain cells for culture, since sufficient cells are then present in a small volume of fluid and therapeutic abortion would still be possible once the results were available.     

Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes.

In pemphigoid gestationis (PG), autoantibodies target collagen XVII, a hemidesmosomal transmembrane protein, which is an important element in cutaneous epithelial adhesion and signalling. We report that collagen XVII is expressed in the first trimester and term syncytial and cytotrophoblastic cells of normal placenta and in epithelial cells of amniotic membrane. Immunoelectron microscopy confirmed the localization of collagen XVII to the hemidesmosomes of amniotic epithelium. Examination of three PG placentas showed mild villitis, but there were no differences between collagen XVII expression levels or immunostaining signals as compared to normal placenta. Collagen XVII expression was also detected in cultured extravillous trophoblast HTR-8/SVneo cells, where collagen XVII expression was upregulated by PMA and TGF-beta1. Interestingly, the presence of Col15, the cell migration domain of collagen XVII, induced the migration of HTR-8/SVneo cells in transmigration assay. Analysis of amniotic fluid samples at different gestational weeks revealed that a large quantity of collagen XVII ectodomain was shed into amniotic fluid throughout pregnancy. Biochemical and immunoblotting analysis indicated that the ectodomain in amniotic fluid is structurally very similar to the ectodomain produced by cultured keratinocytes. Cultured cells from amniotic fluid samples also expressed collagen XVII. Our results suggest that collagen XVII may contribute to the invasion of extravillous trophoblasts during placental development and is also required for the integrity of amniotic basement membrane. Although the exact pathomechanism of PG is still largely unknown, the clinical symptoms of PG are initiated after the expression of collagen XVII in placenta during the first trimester of pregnancy.     

Cryopreservation does not alter karyotype, multipotency, or NANOG/SOX2 gene expression of amniotic fluid mesenchymal stem cells

Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.     

Serum-free cryopreservation of human amniotic epithelial cells before and after isolation from their natural scaffold

Amniotic epithelial cells are a promising source for stem cell-based therapy through their potential capacity to differentiate into the cell lineages of all three germ layers. Long-term preservation is necessary to have a ready-to-use source of stem cells, when required. Reduced differentiation capability, decrease of viability and use of fetal bovine serum (FBS) are three drawbacks of clinical application of cryopreserved stem cells. In this study, we used human amniotic fluid instead of animal serum, and evaluated viability and multipotency of amniotic epithelial cells after cryopreservation in suspension and compared with those cryopreserved on their natural scaffold (in situ cryopreservation). There was no significant difference in viability of the cells cryopreserved in amniotic fluid and FBS. Also, the same results were achieved for expression of pluripotency marker OCT-4 when FBS was replaced by amniotic fluid in the samples with the same cryoprotectant. The cells cryopreserved in presence of scaffold had a higher level of viability compared to the cells cryopreserved in suspension. Although, the number of the cells expressed OCT-4 significantly decreased within cryopreservation in suspension, no decrease in expression of OCT-4 was observed when the cells cryopreserved with their natural scaffold. Upon culturing of post-thawed cells in specific lineage differentiating mediums, the markers of neuronal, hepatic, cardiomyocytic and pancreatic were found in differentiated cells. These results show that replacement of FBS by amniotic fluid and in situ cryopreservation of amniotic epithelial cells is an effective approach to overcome limitations related to long-term preservation including differentiation during cryopreservation and decrease of viability.     

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.     

Amniotic fluid cell morphology in early antenatal prediction of abortion and low birth weight.

The morphology of rapidly adherent (RA) amniotic fluid cells was examined in 201 pregnant women referred for amniocentesis because of two sequential high serum alpha-fetoprotein (AFP) concentrations. Out of 43 amniotic fluid samples containing increased amounts of AFP, 42 had neural or peritoneal cells predominating among the RA cells, the outcome being an infant with a neural-tube defect or exomphalos. In the other case with a raised amniotic fluid AFP concentration but only anterior placental cells the infant was normal. In 25 amniotic fluid samples containing normal amounts of AFP distinctive new patterns of RA cells were observed, termed fetal distress cells. These pregnancies resulted in five spontaneous abortions and 20 infants with birth weights under 2500 g. Fetal distress cells were not detected in any of the remaining 133 samples. One pregnancy was terminated because of a chromosomal abnormality, and there were seven twin pairs not recognised on ultrasonography before amniocentesis. The remaining 125 pregnancies went to term, resulting in infants with birth weights exceeding 2500 g. The results suggest that RA-cell morphology will prove to be of value in the early antenatal prediction of spontaneous abortion and low birth weight.     

Reduced fetal, placental, and amniotic fluid PTHrP in the growth-restricted spontaneously hypertensive rat

Evidence implicates pivotal roles for parathyroid hormone-related protein (PTHrP) in stimulating cell growth and differentiation, placental calcium transport, and placental vasodilatation. As spontaneously hypertensive rat (SHR) fetuses are growth restricted compared with those of its normotensive control, the Wistar Kyoto (WKY) rat, we examined intrauterine PTHrP and total and ionic calcium concentrations in these rats. Fetal plasma PTHrP concentrations, but not total calcium concentrations, were lower in the SHR compared with WKY (P < 0.05). SHR placental concentrations of PTHrP were lower than in WKY (P < 0.03) and failed to show the increase observed in WKY near term (P < 0.05). PTHrP concentrations in amniotic fluid from SHR were not raised near term and were lower compared with WKY (P < 0.0005). The increased ionic calcium concentrations in amniotic fluid in the WKY near term (P < 0.05) were not detected in the SHR. Thus SHR fetal plasma, placental, and amniotic fluid PTHrP concentrations were reduced and associated with fetal growth restriction. We suggest that PTHrP may play a role in the etiology of both growth restriction during pregnancy and hypertension later in life.     

Cell morphology in long-term cultures of normal and abnormal amniotic fluids

Summary The cell morphology of long-term cultures of amniotic fluid cells from 10 fetuses with a neural tube defect (NTD) and three with omphalocele was examined and compared to 30 long-term cultures of normal amniotic fluids as well as a long-term culture of human fetal brain. Cultures from the amniotic fluids of the fetuses with NTD and omphalocele showed cells with the same general characteristics as normal amniotic fluid cells. However, the cultures of amniotic fluid cells from NTD pregnancies had an additional cell type also seen in fetal brain culture. This was a neuroblast-like cell, with small rounded refractile morphology and long branching processes forming clusters of varying sizes which lay on top of large flat cells. These neuroblast-like cells diminished in number with time in culture and were not present in subcultures. Their possible neuronal origin is discussed.     

Enhancement of human amniotic cell growth by ficoll-paque gradient fractionation

Summary Ficoll-Paque isopycnic centrifugation was used as a preparative procdure for amniotic fluid (AF) cells prior to tissue culture. This technique serves to reduce contaminating erythrocytes and also enhances cell growth or mitotic indices. The technique described in this report yields three subfractions designatedaas a turbid interphase layer (F-2), a middle cell layer (F-3), and a bottom pellet (F-4). The middle cell layer (F-3) demonstrated better cell growth and higher mitotic index than any of the other fractions or control unfractionated amniotic fluid cells. The use of Ficoll-Paque isopycnic preparative centrifugation of amniotic fluid cells is a valuable adjunct in cell culture for cytogenetic analysis. This may be especially true when amniotic fluid contains large numbers of erythrocytes. Hsiao-chen Chang was supported by National Research Service Award 1 F32 AM HD 05887-01 from the National Institute of Arthritis, Metabolism, and Digestive Diseases; U. S. Public Health Service, National Institutes of Health. This work was supported in part by USPHS Human Biochemical Genetics Program (G177 17702-9) and the State of California, Department of Public Health, Prenatal Diagnosis Program (79-00016).     

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.     

Protection of human induced pluripotent stem cells against shear stress in suspension culture by Bingham plastic fluid

Suspension culture is an important method used in the industrial preparation of pluripotent stem cells (PSCs), for regenerative therapy and drug screening. Generally, a suspension culture requires agitation to keep PSC aggregates suspended and to promote mass transfer, but agitation also causes cell damage. In this study, we investigated the use of a Bingham plastic fluid, supplemented with a polysaccharide-based polymer, to preserve PSCs from cell damage in suspension culture. Rheometric analysis showed that the culture medium gained yield stress and became a Bingham plastic fluid, after supplementation with the polymer FP003. A growth/death analysis revealed that 2 days of aggregate formation and 2 days of suspension in the Bingham plastic medium improved cell growth and prevented cell death. After the initial aggregation step, whereas strong agitation (120 rpm) of a conventional culture medium resulted in massive cell death, in the Bingham plastic fluid we obtained the same growth as the normal culture with optimal agitation (90 rpm). This indicates that Bingham plastic fluid protected cells from shear stress in suspension culture and could be used to enhance their robustness when developing a large-scale.     

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.     

Amniotic fluid levels of tumor necrosis factor-alpha and soluble tumor necrosis factor receptor 1 before and after the onset of labor in normal pregnancies.

Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells and promotes the regulation of trophoblast growth and invasion. Tumor necrosis factor receptor 1 (TNF-R1) is a receptor for TNF-alpha, and soluble TNF-R1 (sTNF-R1) is present in amniotic fluid after receptor shedding. We evaluated whether amniotic fluid TNF-alpha and sTNF-R1 levels during labor differed from those before the onset of labor in normal pregnancies. This study enrolled 34 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these subjects, 17 went into labor and had subsequent term deliveries (the labor group), and the other 17 underwent cesarean section without labor (the nonlabor group). The average gestational age at entry was 38-39 weeks of gestation. Maternal ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the TNF-alpha and sTNF-R1 levels were determined by the ELISA method. Each of these levels was compared between the two groups. There was a significant increase in amniotic fluid TNF-alpha levels in the labor group. However, amniotic fluid sTNF-R1 levels did not differ significantly between the two groups. Amniotic fluid TNF-alpha may promote the onset of labor at term and/or term labor contributing to subsequent delivery may induce the production and secretion of TNF-alpha into the amniotic cavity. There was no pregnancy-associated increase in receptor shedding or cell apoptosis at the onset of labor.     

Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells

1. 1. The specific activity of the lysosomal hydrolases in cultured skin fibroblasts varies according to the phase of growth in culture.

2. 2. Diagnosis of heterozygous genotypes for lysosomal enzyme deficiency diseases is unreliable with cultured fibroblasts, at least partly because of the growth curve-associated variations in specific activity.

3. 3. Fluctuations in specific activity during the beginning of the growth curve in vitro can be avoided by initiating cultures with cells which are in the early log phase of growth.

4. 4. Primary amniotic fluid cell cultures show no relationship between length of time in culture and lysosomal enzyme specific activity.

5. 5. Secondary amniotic fluid cell cultures exhibit growth curve-related variations in lysosomal enzyme specific activity as they assume fibroblast-like growth kinetics.

6. 6. Prenatal and postnatal diagnosis on cultured amniotic fluid cells and fibroblasts requires the use of appropriate controls which are matched for stage of growth and length of time after the last trypsinization.