Clinical significance of persistent amniotic fluid leakage after genetic amniocentesis

Chronic amniotic fluid leakage is a rare complication of genetic amniocentesis. Pregnancy outcomes in two such patients are presented and six previous cases reviewed. Although chorioamnionitis has not been reported, potentially serious complications may occur including an increased risk for pre-term delivery and fetal skeletal deformity. While conservative management of post-amniocentesis amniotic fluid leakage is advocated, patients should be advised of these risks.     

Short communications normal fetal growth despite persistent amniotic fluid leakage after genetic amniocentesis

Two women not only lost relatively large amounts of amniotic fluid immediately following genetic amniocentesis, but continued to lose fluid for the remainder of their pregnancies. Periodic ultrasonographic assessment confirmed normal fetal growth and presence of some amniotic fluid. Both women were delivered at term of normal offspring who showed no evidence of fetal deformations. Although amnionitis is a risk, cautious surveillance may permit continuation of pregnancies complicated by copious or persistent amniotic fluid leakage following genetic amniocentesis.     

First-trimester amniotic fluid and extraembryonic coelomic fluid acetylcholinesterase electrophoresis

Acetylcholinesterase (AChE) gel electrophoresis was performed on samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 38 women between 8 and 12 weeks of pregnancy. AChE was positive in 33 per cent (12/36) of the amniotic fluid samples; the percentage of positive results decreased as gestation advanced. AChE was positive in 32 per cent (9/28) of the extraembryonic coelomic fluid samples. In 81 per cent (21/26) of matched samples, the AChE results were identical in the two fluids. Amniotic fluid and extraembryonic coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation.     

Filtration and recirculation of early amniotic fluid. Evaluation of cell cultures from 100 diagnostic cases

Due to the low cell concentration, cultures from early amniotic fluid specimens usually require 2–3 weeks in culture prior to karyotyping. The purpose of this study was to evaluate the culture quality of amniotic fluid cells from early pregnancy, obtained by a new filter technique. The hypothetical advantage of the technique was that the increased cell yield might reduce the culture time before karyotyping. Culture quality was assessed by the number of colonies, the percentage of colonies containing mitoses in filter and control cultures, and the culture time. The setting was a consecutive clinical trial. One hundred samples were obtained from ongoing pregnancies at 11–14 weeks of gestation (mean 12·8 weeks). By circulating a mean of 26 ml of amniotic fluid through a cell filter system leading the cell-free fluid back to the amniotic cavity, the cell yield was increased in the sample of 7 ml corresponding to the dead space of the filter system. The culture results were compared with control cultures from 5 ml samples drawn from the same pregnancies prior to recirculation. The cultures from the first flushing of the filter system yielded 2·6 times more colonies and in total 4·2 times more colonies were found in the three cultures grown from each filter sample when compared with the control cultures. Moreover, the filter cultures showed significantly more colonies with mitoses. The mean culture time was 8·0 days for the filter cultures, from which the karyotypes were analysed. The controls would have needed more time in culture to fulfil the diagnostic criteria for karyotyping. One case of 47,XY, + 21 was found; the rest had normal karyotypes. We conclude that the filter technique improves the culture quality of early amniotic fluid samples and allows early arrest of the cultures.     

First trimester amniocentesis between the seventh and 13th weeks: Evaluation of the earliest possible genetic diagnosis

Amniocentesis performed at the 12th week and later gives reliable results. The procedure can be performed using regimens developed for mid-trimester amniotic fluid (AF) cells. Extension to the 10th–11th week is, in principle, feasible. However, the high cytogenetic failure rate is a difficulty and despite a high clone count, the culture time is prolonged. The problem of the relatively high loss of AF could be overcome by cell filtration techniques and replacement of the fluid. Because of the short turnover rate of the AF, this may be unnecessary or replacement with an isotonic solution may be sufficient. (Pseudo)mosaicism appears to occur more frequently in early than in late amniocentesis. As yet, data are too sparse to allow a comparison with chorionic villus sampling. There are no reliable follow-up data from which to estimate the abortion rate and the number of embryonic malformations.     

Reduction of sera requirements in amniotic fluid cell culture

Reduction in serum requirement for culture of primary human amniotic fluid cells can be achieved by the addition of 10 growth-promoting factors to the nutrient medium. This supplemented medium preserves cell types normally found in amniotic fluid cell cultures supplemented with 20–30 per cent fetal bovine serum. The volume of amniotic fluid required to initiate culture can be as little as 1 ml. Amniotic fluid samples contaminated with red blood cells with no visible clot also grow well in the low serum medium. Cell-free amniotic fluid combined with equal parts of supplemented medium is useful in initiating cell culture.     

Mosaicism or pseudomosaicism: The problem of hypermodal cells in amniotic fluid cell culture

A series of 2029 consecutive amniotic fluid specimens studied for prenatal genetic diagnosis were reviewed and reassessed so as to evaluate the frequency and clinical significance of hypermodal cells in amniotic fluid cell cultures. Hypermodal cells were defined as those with more than 46 chromosomes, and were characterized by an additional structurally normal or structurally abnormal chromosome. Of 2029 specimens, 47 (2.31 per cent) contained a total of 167 hypermodal cells. True fetal mosaicism was detected in three cases (0.14 per cent). All had hypermodal cells in more than one culture flask or colony which contained the same aberrant chromosome complement. In all but one case the babies were normal when only one cell was hypermodal, or when several cells were hypermodal but present in only one colony or one culture vessel. One case had an extra No. 20 chromosome in one cell. Although the child had multiple anomalies, they were not characteristic of trisomy 20, and subsequent chromosomal study on the baby postnatally revealed a 46,XX karyotype. The in situ coverslip technique is recommended as the preferred method for prenatal diagnosis, and it is useful as an aid in differentiating true mosaicism from pseudomosaicism.     

Introduction of early amniocentesis to routine prenatal diagnosis

With growing awareness of the problems associated with prenatal cytogenetic diagnoses after CVS, attempts have been made to provide early amniocentesis as an alternative to CVS. Since 1990, at our clinic the gestational age limit for routine diagnostic amniocentesis has been successively lowered, first to 14 and then to 13 weeks of gestation. Thus, 811 prenatal diagnoses were performed after early amniocentesis at 13 weeks (n = 217) and at 14 weeks of gestation (n = 594). No problems were encountered. Culture failure was never observed in the early samples. Using the criteria ‘number of colonies’ and ‘culture duration until harvest’, early samples taken at 14 weeks did not differ significantly from the controls after standard amniocentesis performed at 15 and 16 weeks, respectively, whereas a minority of samples taken at 13 weeks experienced some delay in culturing. However, in each group at least 85 per cent of samples led to a diagnosis fulfilling our standard criteria of a safe diagnosis (at least 20 metaphases of at least five colonies from at least one primary culture after trypsinization) within 15 days. Some differences between the different groups can be recognized: culture duration of less than 11 days tends to be increasing after standard amniocentesis, whereas long culture duration (more than 20 days) is more often associated with early amniocentesis. However, this trend is only minimal and did not result in an increased risk of missing a diagnosis.     

A simple method for preparing prometaphase chromosomes from amniotic fluid cell cultures

A simple method for preparing prometaphase chromosomes from amniotic fluid cell cultures is described. The technique is based upon several key steps including: (1) reduced colcemid concentration, (2) reduced exposure to trypsin-EDTA, and (3) maintaining cells in single suspension by adjusting cell concentration appropriately. Chromosomes with banding resolution up ot 800 bands per haploid set can be routinely produced. The described methodology is particularly useful in defining and establishing the clinical significance of subtle structural aberrations.     

Prenatal diagnosis of inherited metabolic disorders by quantitation of characteristic metabolites in amniotic fluid: Facts and future

An attempt is made to summarize as completely as possible what is known about the prenatal diagnosis of amino– and organic acidurias by direct measurement of characteristic metabolites in amniotic fluid, and to indicate which disorders can potentially be diagnosed prenatally by direct quantitation of metabolites. Furthermore, the disorders are mentioned in which the prenatal diagnosis was proven to be unsuccessful by this approach. The prenatal diagnoses of a case of propionic acidemia and a case of tyrosinemia type I in the 11th and 12th week of gestational age, respectively, are reported and the prospects of performing amniocentesis in the first trimester for prenatal diagnosis are discussed.     

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.     

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.     

The effect of oxygen tension on colony formation and cell proliferation of amniotic fluid cellsin vitro

The effects of reduced oxygen concentrations in the gas phase over the culture medium on colony formation and cell proliferation were investigated in high and low cell density primary and secondary cultures of amniotic fluid cells. Using two standard culture methods (25 cm² plastic flasks and Leighton type tubes) a significantly reduced culture time was observed at high cell density for mass cultures by incubation within a low oxygen tension gas phase (2.5 per cent to 7.5 per cent O₂) instead of conventional air (18 per cent O₂). At low cell density colony formation was significantly enhanced in cultures grown at reduced oxygen tension. Using gas permeable membranes as support, lowering the oxygen tension from 7.5 per cent to 2.5 per cent yielded an increase in plating efficiency of cells from approximately 5 per cent to 25 per cent, whereas plating efficiency was less than 2 per cent for cells grown at ambient 18 per cent O₂. It is suggested that low oxygen tension in the gas phase is an effective means of enhancing clonal growth in amniotic fluid cell cultures, thereby reducing both culture time and risk of culture failure.     

Pseudomosaicism,true mosaicism,and maternal cell contamination in amniotic fluid processed with in situ culture and robotic harvesting

This study was designed to test the usefulness of the common definitions for maternal cell contamination, true mosaicism, and pseudomosaicism for amniotic fluid specimens processed by in situ culture and robotic harvesting. We prospectively studied 4309 consecutive amniotic fluid specimens processed with these methods and found that 0.84 per cent had maternal cell contamination, 0.28 per cent had true mosaicism, and 5.4 per cent had pseudomosaicism. Although the frequencies of maternal cell contamination and true mosaicism were comparable to those in similar published studies, the frequency of pseudomosaicism was more than twice as high as that in previous reports. This finding is most likely not due to the method, but rather to a more accurate estimate of the actual frequency of pseudomosaicism in amniotic fluid cultures than reported heretofore. Follow-up clinical information was available on 72 per cent of the cases. In three cases of true mosaicism involving structural anomalies, the results of cytogenetic follow-up studies on the neonates were normal. None of the pseudomosaic cases involving trisomy 8, 13, 18, or 21; triple X; or monosomy X were associated with newborns who had birth defects.     

The amniotic band syndrome: A possible complication of amniocentesis

Malformations of the upper distal extremities were noted in an otherwise healthy infant whose mother underwent diagnostic amniocentesis. A causal relationship is postulated.     

Genetic amniocentesis: 505 cases performed before the sixteenth week of gestation

Between May 1987 and November 1988, 505 early amniocentesis within the 15th week of gestation were performed at the First Department of Obstetrics and Gynaecology,‘L. Mangiagalli’ of the University of Milan and at the Department of Obstetrics and Gynaecology of ‘Gaslini’ hospital in Genoa. A total number often abnormal fetal karyotypes were diagnosed. In addition, one case of pseudomosaicism (not confirmed on fetal blood) and one case of osteogenesis imperfecta type II (observed at ultrasound examination) were also detected. Eleven pregnancies were therefore terminated because of an abnormal fetus. Out of 494 pregnancies (excluding terminated pregnancies) there were 16 fetal losses within the 28th week; ten of these occurred in the 2 weeks following the procedure. There were 475 live-births, of which 447 were term deliveries and the other 28 deliveries occurred before the 37th week of gestation.     

Early amniocentesis: Experience of 222 consecutive patients, 1987–1988

Early amniocentesis from 9 to 14 weeks' gestation provides a safe and accurate method of prenatal diagnosis of cytogenetic and biochemical disorders. There was a 100 per cent success rate in culturing the amniotic cells from 222 samples obtained between 9 and 14 weeks' gestation. Follow-up of the patients to delivery revealed an abortion rate of 1·4 per cent. Among the 207 live- and stillborn infants, only one had a congenital abnormality (bilateral talipes equino-varus) and no infant had respiratory distress syndrome or pneumonia. Eleven pregnancies were terminated following the detection of a chromosomal, biochemical, or congenital abnormality (5·0 per cent). However, before the procedure of early amniocentesis becomes routine clinical practice, it requires appraisal by a randomized clinical trial.     

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.     

False-negative amniotic fluid acetylcholinesterase in a case of meningo-encephalocele

We describe a patient with a significantly elevated serum alphafetoprotein (AFP) concentration at 17 weeks of gestation, who showed only a marginally increased amniotic fluid AFP and lacked the second rapidly migrating band of acetylcholinesterase electrophoresis. Ultrasound examination revealed an encephalocele and ventriculomegaly. Autopsy showed that the encephalocele was not covered by skin.