Cytogenetic studies of amniotic fluid taken before the 15th week of pregnancy for earlier prenatal diagnosis: A report of 114 consecutive cases

One hundred and fourteen samples of amniotic fluid taken before 15 weeks of gestation were cultured for cytogenetic studies. The results of culturing these early amniotic fluid (EAF) samples were compared with the results of culturing 114 standard amniotic fluid (SAP) samples taken after 15 weeks of gestation matched for maternal age and received in the laboratory within the same week. Cell culture was successful in all 114 of the EAF samples and in 111 SAP samples. There was no significant difference in the days to harvesting and days to reporting in the two groups. Three samples of SAP failed to grow and two EAF samples produced tetraploid karyotypes, so that in these five cases amniocentesis had to be repeated. These problems were attributed to toxicity of a fungicide used in the culture medium. Pseudomosaicism was noted in two EAF samples and one SAP sample; and maternal cell contamination was noted in one EAF and one SAP sample. Thus, culturing and karyotyping cells harvested from EAF and SAP are similar, indicating that EAF samples from 12–14-week pregnancies could be used for prenatal diagnosis.     

True trisomy 15 mosaicism,detected by amniocentesis at 12 weeks of gestation and fetal echocardiography

A case of mosaicism of trisomy 15, with two-thirds of the cells trisomic, was detected at 12 weeks of gestation in amniotic fluid cell cultures obtained with the filtration technique. Ultrasound examination at 13 weeks showed a nodule protruding into the amniotic cavity which was speculated to be remnants of a co-twin, causing the trisomic cell line. At 20 weeks of gestation, a malformation scan (level III) was normal, but supplementary fetal echocardiography revealed a severe cardiac defect (mitral atresia and a ventricular septal defect). Fetal lymphocytes obtained by cordocentesis showed trisomy 15 mosaicism, but only in 5 per cent of the mitoses. After termination, the same percentage of trisomy 15 mosaicism was found in cells from skin and tendon as in the original early amniocentesis. No sign of earlier twinning was found in the placenta or membranes. We conclude that mosaicism in early amniotic fluid obtained by the filter technique in this case reflected the true karyotype accurately and that supplementary echocardiography added significantly to the interpretation of the clinical implications.     

Discrepant karyotypes after second- and third-trimester combined placentacentesis/amniocentesis

Cytogenetic data are presented from a total of 1306 consecutive pregnancies with successful diagnosis obtained from both chorionic villi after short-time culture (CVS-SC) and amniotic fluid cell cultures (AC); samples had been taken simultaneously at combined placentacentesis (placental biopsy) and amniocentesis during the second (92·8 per cent) and third (7·2 per cent) trimesters. Concordant results were obtained in 1218 pregnancies with a normal karyotype and in 62 pregnancies with an aberrant fetal karyotype. Discrepant, i. e. false-positive and false-negative, results were found in 26 cases (2 per cent). From these data the accuracy of CVS-SC, defined as the proportion of all correct diagnoses, is calculated to be 98 per cent. Three non-mosaic and 14 mosaic false-positive results obtained after CVS-SC could not be confirmed by AC. Related to 1235 true normal fetal karyotypes, the specificity of CVS-SC, i.e. the proportion of normal karyotypes correctly diagnosed, amounts to 98·6 per cent. In nine pregnancies, an aberrant fetal karyotype detected after AC was missed by CVS-SC. The sensitivity of CVS-SC, i.e. the proportion of abnormal fetuses correctly diagnosed (62 out of 71), amounts to 87·3 per cent in our study group.     

Rapid karyotyping in prenatal diagnosis: A comparative study of the ‘pipette method’ and the ‘in situ’ technique for chromosome harvesting

Rapid karyotyping in the second and third trimesters has important implications for the management of pregnancies at risk. From September 1985 to March 1992, 735 amniotic fluid samples sent to our laboratory for rapid karyotyping from 64 different diagnostic centres of the Federal Republic of Germany were included in a comparative study on harvesting for chromosome analysis using the ‘pipette method’ or the ‘in situ’ technique. The average time between preparation of the amniotic fluid and verbal notification of the analysed karyotype was 5·41 days. The ‘pipette method’ needed on average 4·65 days, and the ‘in situ’ technique 5·97 days. In comparison with other more invasive techniques available for rapid karyotyping such as cordocentesis and placental biopsy, amniocentesis and subsequent chromosome harvesting using the ‘pipette method’ and/or the ‘in situ’ technique proved very useful and efficient. The overall incidence of chromosome aberrations was 15·3 per cent. The high rate of structural chromosome aberrations and uncommon aneuploidies found in our investigation (12 per cent) indicates that for rapid karyotyping in the second and third trimesters, conventional cytogenetic techniques cannot be replaced by faster techniques based on fluorescent in situ hybridization on interphase cells in the near future.     

Abnormal pregnancy sonogram and chromosomal anomalies: Four years' experience with rapid karyotyping

Over a four-year period, 140 pregnancies with different malformations detected by ultrasound were examined cytogenetically. Gestational age ranged from 13 to 36 weeks. Twenty-six fetuses (18.6 per cent) had abnormal karyotypes, including trisomies, triploidy, monosomy X, and structural anomalies. Similar malformations were found in fetuses with different chromosomal anomalies, indicating that the types of malformations are not specific for particular chromosomal anomalies. Chromosomal analysis was performed on amniotic fluid culture and by direct karyotyping of placental biopsies. Direct karyotyping is suggested to be the most rapid approach, especially if sonographic anomalies are detected close to the 24th week of gestation, shortly before delivery, and in cases of significant oligohydramnios.     

Evaluation of amniotic fluid cell filtration: An experimental approach to early amniocentesis

Prior to a prospective application of amniotic fluid (AF) cell filtration to early amniocentesis, we tested the technique on a surplus from mid-trimester samples. By using the same sample size of 5 ml in experiments with a filter and in routine diagnostic procedures (control), we evaluated an optimal filter system. The prolonged culture time of filtered cells and the reduced number of clones are most probably due to mechanical stress (filtration pressure), whereas loss of the cells by adhesion to the filter system, and an AF-free culture medium (growth factors) are suggested to be less important. The AF cells are very sensitive to mechanical stress. Slow filtration (⩽3 ml AF/min) through filters with a high porosity and the largest possible pore size should be preferred. A mixed cellulose ester filter membrane with a pore size of 5·0 μm proved to be the most efficient, allowing harvest of the filtered cells after only a slight prolongation of the culture time (+2·4 days) compared with unfiltered aliquots. A filter set with a bypass connected by three-way taps allows cell filtration during either aspiration or reinjection of the AF. Cell filtration after amniocentesis and consecutive reverse flushing of the membrane with the appropriate amount of culture medium proved to be the best with regard to easy handling and reducing the risk of bacterial contamination.     

Simultaneous placentacentesis and amniocentesis for prenatal karyotyping: Report on 250 cases

The efficacy and risks of simultaneous transabdominal chorionic villus biopsy (placentacentesis) and amniocentesis in the second and third trimesters were evaluated in 250 singleton pregnancies. The major indications were advanced maternal age (36·0 per cent), abnormal ultrasound findings (23·2 per cent), and low maternal AFP value (17·6 per cent). Nine abnormal karyotypes were found in placental tissue (3·6 per cent). The karyotypes of placental and amniotic cells were different in three cases, including two cases of false-positive mosaicism (08 per cent) and one case of a false-negative result (0·4 per cent) obtained by placental karyotyping. The problem of discrepant karyotypes in embryonic and extra-embryonic tissue does not seem to be restricted to the first trimester. The post-procedure fetal loss rate was estimated as approximately 1·8 per cent. We conclude that the procedure presented here combines the advantages of rapid karyotyping (placentacentesis) and high diagnostic reliability (amniocentesis). It does not seem to be necessary to restrict its use to late presentations and suspicious ultrasound findings.     

Stimulation of human amniotic fluid cell proliferation and colony formation by cell plating on a naturally produced extracellular matrix

Human amniotic fluid cells exhibit a higher cloning efficiency and rate of cell proliferation when maintained on dishes coated with a naturally produced extracellular matrix (ECM) in comparison with the regular tissue culture plastic. In 22 out of 31 amniotic fluid samples there was by plating the cells on ECM a 2–6 fold increase in number and size of colonies and in the cell density per colony as detected by actual staining and viewing of each colony. These effects yielded, in 21 of 41 additional samples, a reduction ranging from 2–8 days, in the culture time elapsing between amniocentesis and the first harvesting of cells for chromosomal analysis. An even greater effect was obtained with primary cells that failed to attach to plastic surfaces and stayed floating in the medium but did attach and proliferate when seeded on ECM. Cells that were left firmly attached to ECM after the first trypsinization and harvesting of cells for chromosomal analysis yielded colonies ready for second karyotyping in less than half the time required for cells maintained on plastic. Studies with secondary cultures of human amniotic fluid cells have demonstrated a 5–10 fold decrease in serum requirement of cells cultured on ECM as compared with plastic. Addition of fibroblast growth factor (FGF) to the cultures further potentiated the effects of ECM. The ECM induced stimulation of cell attachment and proliferation was not associated with any chromosomal anomalies, nor did it interfere with the handling procedure. ECM coated dishes may be useful to reduce the time interval between amniocentesis and diagnosis, in particular when the amniotic cells exhibit an exceedingly slow rate of proliferation on plastic or when large quantities of cells are required for enzymatic studies.     

Correct prenatal diagnosis of a hurler fetus where amniotic fluid cell cultures were of maternal origin

Contamination of amniotic fluid cell cultures by maternal cells can be expected to lead to misdiagnosis of fetal genotype in 0·1 to 0·5/100 cultures, when assays are carried out directly on cultured cells. Chemical analysis of the cell-free amniotic fluid supernatant may overcome this source of error and has the added advantages of speed and independence from amniotic cell culture failure. We describe a pregnancy at risk for Hurler's disease where amniotic cells cultured at amniocentesis had a female karyotype and an α-iduronidase activity towards both phenyl and 4-methylumbelliferyl substrates at the lower end of the normal range, suggesting a heterozygous fetus. An affected fetus was predicted, however, because of a high concentration of dermatan sulphate in the amniotic fluid. The discrepancy between these findings was shown to be due to maternal cell contamination of amniotic fluid cell cultures by the birth of a male infant with Hurler's disease.     

A study of chromosomal aberrations in amniotic fluid cell cultures

This paper represents the analysis of 1916 routine amniotic fluid specimens harvested by an in situ fixation technique in a prospective study with regard to cultural chromosome anomalies. Excluding constitutional abnormalities, 2·9 per cent of 19432 cells analysed showed some form of chromosome anomaly, terminal deletions (57 percent) and chromatid/chromosome breaks and gaps (18 per cent) being the most frequent, followed by interchange aberrations (13 per cent) and trisomy (5 per cent). No case was found of more than one colony from the same culture showing the same anomaly without it being present in other cultures from the same fluid. The wholly abnormal colonies had a surplus of trisomies and from the mathematical considerations presented one may infer that these are likely to reflect the presence of abnormal cells in the amniotic fluid. Partly abnormal colonies appeared at a frequency that would correspond to virtual absence of selection against chromosomally abnormal cells when cultured in vitro. The aberrations found were similar to those seen as single cell anomalies, except for chromatid breaks and exchanges. The data suggest a basic preferential induction of trisomy for chromosomes 2,18,21, and the Y-chromosome. Structural aberrations showed a marked clustering of breakpoints around the centromeres. The frequency of mutant cells was low (1·4 × 10⁻³) before culture was initiated. At harvest, the frequency of abnormal cells was much higher (3 × 10⁻²) corresponding to 3 × 10⁻³ mutations per cell per generation accumulating over approximately ten generations in vitro.     

Amniotic fluid-AFP in Down syndrome and other chromosome abnormalities

80·2 Per cent of 111 Down syndrome pregnancies had amniotic fluid (AF) alpha fetoprotein (AFP) levels on or below the median and 10·8 per cent at or below 0·5 MoM compared with 41·9 and 1·4 per cent of controls. These differences were even more striking when the gestational age was < 18 weeks compared with ⩾ 18 weeks. No such association was seen for other chromosome abnormalities including trisomy 18,45,X and mosaics, 47,XXY, 47,XXX, and other structural abnormalities and triploidy, even when high levels due to defects such as omphalocele and cystic hygroma were excluded. All cases of trisomy 13 and 80 per cent with 47,XYY had AF-AFP levels above the median. Selection of cases for karyotyping by a low level of AF-AFP would clearly fail to detect aneuploidies other than Down syndrome and is not recommended. A possible weak association between low maternal serum (MS) and AF-AFPs in Down syndrome was most evident at < 18 weeks, suggesting that MS screening between 16 and 18 weeks may be the most informative time.     

Early amniocentesis and fetal nuchal translucency in women requesting karyotyping for advanced maternal age

Fetal nuchal translucency was measured at 11–14 weeks' gestation in 97 pregnancies referred for early amniocentesis for advanced maternal age. The nuchal translucency was abnormal in 11 fetuses and the fetal karyotype was abnormal in five of these 11 cases. The karyotype was normal in 86 cases with normal nuchal translucency. The culture failure and miscarriage rates associated with early amniocentesis were 3·3 per cent and 2·2 per cent respectively. Amniotic fluid leakage occurred in 6 per cent of cases. In women requesting fetal karyotyping for advanced maternal age without additional biochemical screening, fetal nuchal translucency should be measured at 11–14 weeks. If the nuchal thickness is ≥ 3 mm, a first-trimester diagnostic procedure is indicated; however, if it is <3 mm, amniocentesis should be delayed until 16 weeks' gestation.     

Detection and enumeration of colonic mucosal cells in amniotic fluid using a colon epithelial-specific monoclonal antibody

Since its introduction, prenatal diagnosis of chromosomal and metabolic disorder by mid-trimester amniocentesis has relied upon the use of a mixture of fetal cells obtained from amniotic fluid. Little knowledge has been gained in the sorting of these cells for diagnosis of tissue-specific disorders. In an attempt to determine the contribution of fetal colonic mucosal cells to the overall amniocyte population, we used the colonic epithelial-specific monoclonal antibody (MC-Ab) 7E₁₂H₁₂, IgM isotype. Specimens of the small intestine, colon, buccal mucosa, kidney, urinary bladder, and umbilical cord were obtained from electively aborted normal fetuses of 12–28 weeks' gestation. All of these specimens were examined with 7E₁₂H₁₂ by the immunoperoxidase technique. The MC-Ab reacted with the colonic epithelial cells but not with any of the other tissues. In addition, 40 amniotic fluid samples obtained from women between 16 and 18 weeks of gestation, who underwent amniocentesis because of advanced maternal age, were tested using a fluorescent activated cell sorter. Among the amniotic fluid specimens examined, 18·4 ± 10·3 percent cells reacted with 7E₁₂H₁₂. Double immunofluorescence studies revealed that all Mc-Ab-stained cells contained secretory component, confirming that they were epithelial in origin. All fetuses whose amniotic fluid was analysed had normal karyotypes and amniotic fluid alpha-fetoprctein levels that were also normal. This study demonstrates that cell-specific Mc-Ab can be used to detect colon cells in the amniotic fluid and that colon cells contribute significant numbers in the mixture of amniotic fluid cells. This technique could be helpful in the prenatal diagnosis of disorders in which the flow of amniotic fluid through the fetal intestine is impaired, such as cystic fibrosis, imperforate anus, Hirschsprung aganglionic megacolon, and intestinal atresia.     

Abnormal amniotic fluid levels of CA 125 in second-trimester Down syndrome pregnancies

The aim of this study was to evaluate the concentration of CA 125 in second trimester amniotic fluid from Down syndrome pregnancies. CA 125 was measured in stored amniotic fluid samples from pregnancies of 14–19 weeks' gestation with and without Down syndrome fetuses. CA 125 levels were expressed in multiples of the median (MOM) for normal pregnancies of the same gestational age. Twenty-one pregnancies with Down syndrome fetuses and 63 unaffected controls matched for maternal age, gestational age, and duration of storage were studied. The median MOM values of the affected pregnancies were significantly higher than those of the controls (1·41 MOM versus 0·99 MOM). These findings show that there is an increased concentration of CA 125 in second-trimester amniotic fluid from Down syndrome pregnancies.     

Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by amniotic fluid steroid analysis

The concentration of 17OH-progesterone was measured in second trimester amniotic fluid samples from 12 mothers who previously had had an infant with congenital adrenal hyper-plasia due to 21-hydroxylase deficiency. In 4 affected pregnancies, the concentrations were more than 2 S.D. higher than those determined in 44 samples from normal pregnancies (mean ± S.D., 8·1 ± 2·4 nmol/1). The remaining 8 pregnancies were predicted to be unaffected based on the results of amniotic fluid concentrations within the normal range. In each instance, the infant was normal. The results indicate that measurement of amniotic fluid 17OH-progester-one concentrations during the second trimester is an accurate prenatal test for 21-hydroxylase deficiency. The results should be supplemented with determination of fetal sex by karyotype analysis on the amniotic fluid cells.     

Parental mosaic trisomy 21 detected following maternal cell contamination of an amniotic fluid specimen from a normal male pregnancy

We report a case of maternal mosaic trisomy 21 ascertained at prenatal diagnosis as a result of maternal cell contamination of an amniotic fluid sample. A 34 year old female was referred for karyotyping because of a previous trisomy 21 pregnancy. Chromosome analysis of primary in situ cultures showed a karyotype of 47,XX, + 21[6]/46,XY[32]/46,XX[2]. Molecular testing demonstrated maternal cell contamination of the amniotic fluid sample and G-banded karyotyping of maternal blood showed that 3/200 cells had trisomy 21, consistent with the mother being a Down syndrome mosaic. A normal male baby with a 46,XY chromosome complement was delivered at 30 weeks. This case emphasises the need for close collaboration between cytogenetic and molecular genetics laboratories in resolving unusual cases of mosaicism. Copyright © 2007 John Wiley & Sons, Ltd.     

Early amniocentesis at 11–14 weeks' gestation for the diagnosis of fetal chromosomal abnormality—a clinical evaluation

Early arnniocentesis between 11 and 14 weeks' gestation was offered to 110 women at risk of a chromosomally abnormal fetus due to maternal age. Four were found to be unsuitable for the procedure, and 106 early amniocenteses were performed. In 102 cases, clear amniotic fluid was obtained with a single tap. There were two dry taps and two bloodstained tapis; sampling was repeated in three of these cases before 15 weeks. In the fourth case, placental biopsy was performed at 16 weeks. Thus, we were able to obtain a satisfactory sample in all but three cases(2.8 percent). Karyotyping of cells harvested from the early amniotic fluid samples was successful in all the 105 cases. Cell culture from the initial samples revealed a normal karyotype in 99 cases, two balanced translocations, two tetraploid karyotypes, and two cases of pseudomosaicism. Of the 105 pregnancies successfully sampled, there have been two losses to date (1–8 per cent). Two further patients presented with premature rupture of membranes, both pregnancies having successful outcomes. Sixty-two babies have delivered to date, with four congenital anomalies. There were no respiratory problems. Twenty-nine pregnancies are continuing without known complications, and details are not yet available on the remaining 12. The results indicate that early arnniocentesis may replace the traditional test at 15–17 weeks.     

Amniotic fluid alpha-fetoprotein levels and prenatal diagnosis of autosomal trisomies

Pregnancies with fetal trisomy 21 have been associated with low amniotic fluid alpha-fetoprotein levels (AFAFP). This observation led to the suggestion that low AFAFP levels be used as a criterion for completion of a chromosomal analysis in patients who are not otherwise at increased risk for a fetal chromosome abnormality and in whom karyotyping might not have been completed for economic reasons. In order to assess the usefulness of such criteria, we reviewed the AFAFP levels of 90 cases of fetal trisomy 21, 23 cases of trisomy 18, and 10 cases of trisomy 13. These were compared with 2400 control samples with normal chromosome constitution. AFAFP levels were generally lower in pregnancies with trisomy 21, showing a median value of 0·72 MoM. However, 40 per cent of the trisomy 21 samples had AFAFP values greater than 0·8 MoM and 20 per cent were over 1·0 MoM. These data imply that over 50 per cent of Down syndrome cases might have been missed using a cut-off level of 0·70 MoM for completion of chromosome analysis. Using a higher cut-off level will leave only a small percentage of samples unkaryotyped. The distribution of AFP levels in trisomy 13 and 18 is no different from controls; we therefore believe that fetal karyotyping should be completed in every amniotic fluid sample obtained.     

Microculture of fetal blood for prenatal cytogenetic studies from blood-stained amniotic fluid

An alternative method to the culture of amniotic fluid cells for prenatal diagnosis of chromosome disorders is proposed. Microculture of fetal blood can be used when fetal blood is drawn at amniocentesis through accidental puncture of the placenta. An easy discrimination of fetal red cells, a good response of fetal lymphocytes to PHA and the possibility of identification of the fetal karyotype from the maternal one are the technical bases of this method. This technique offers some undoubted advantages: a reduced need for repeating amniocentesis because of a lack of growth of AF cells due to massive contamination with red cells; a result may be obtained sooner. Thirty-seven cases out of 1092 amniocenteses were processed in this way (3·4 per cent). In two cases no mitoses were obtained but in the others the diagnosis was confirmed by the results of AF cell culture and/or by the outcome of pregnancy.     

Genetic amniocentesis in biamniotic twin pregnancies by a single transabdominal insertion of the needle

We present a technique to aspirate amniotic fluid from both sacs in biamniotic twin pregnancies using a single abdominal insertion with a spinal needle. It was successful in 48 out of 55 cases of biamniotic twin pregnancies referred to our perinatal unit between 1985 and 1994. The single insertion technique was used when the inter-amniotic membrane was clearly evident and two separate free amniotic fluid pools could be reached by the operator with a single puncture. An adequate amount of amniotic fluid was sampled from both sacs to make a cytogenetic diagnosis in all cases. There were four fetuses with trisomy 21 in three twin pregnancies. In two cases, only one twin was affected whilst the co-twin was normal, so that a selective feticide was performed. No miscarriages due to genetic amniocentesis were reported. After 1990, all genetic amniocenteses in biamniotic twin pregnancies (except for one case due to late booking) were performed between 14 and 15 weeks of gestation and with all cases except one, it was possible to sample both twins by a single puncture. We suggest that early amniocentesis (14–15 weeks) by a single abdominal puncture could be a reliable and safe alternative to first-trimester chorionic villus sampling in twin pregnancies.