Amniotic Fluid Cell Cultures Research Articles

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 19,432 cells analysed showed some form of chromosome anomaly, terminal deletions (57 per cent) 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 X 10(-3)) before culture was initiated. At harvest, the frequency of abnormal cells was much higher (3 X 10(-2)) corresponding to 3 X 10(-3) mutations per cell per generation accumulating over approximately ten generations in vitro.

Hormone release by primary amniotic fluid cell cultures.

Amniotic fluid cells have been widely used in prenatal diagnosis; however, there is great heterogeneity of the cells and their origin. In this study we analyze the karyotype and release of human chorionic gonadotropin (hCG), human chorionic somatomammotropin (hCS), free estriol (E 3), prolactin (PRL) and progesterone (P) of amniotic fluid cells from primary cultures of six normal and two anencephalic fetuses. In all the amniotic fluid samples there was release of hCG; in one amniotic fluid, in which several tetraploid colonies were found. PRL and P were also released. The heterogeneity of amniotic fluid cell morphology and their hormone release in culture was confirmed. The presence of hormones like hCG supports the trophoblastic origin of some amniotic fluid cells from normal and anencephalic fetuses. Other hormones, such as PRL and P could be used in the differential diagnosis between the karyotype of fetal membranes and the true fetal karyotype. Amniotic fluid cell cultures used in prenatal diagnosis yielded second trimester placental cells without any elaborate methods that could be used as cell models for hormone studies.

3 Chromosome analysis on chorionic villi

Chromosome abnormalities are a major health problem. About 25% of mentally retarded individuals have a chromosome aberration. Very little is known about the causes of non-disjunction giving rise to trisomies, the largest group of chromosomally abnormal individuals. As primary prevention is not possible methods have been developed to study fetal chromosomes with the aim of aborting affected fetuses. Most of the studies have involved screening of women 35 years and older--they have also allowed couples with translocations and other aberrations, who would otherwise fear to reproduce, to achieve a normal family. Hitherto, one of the greatest problems of fetal diagnosis has been the timing of the test in the second trimester of pregnancy with the risk of having to terminate a wanted pregnancy around the twentieth week of gestation. The first trimester approach would overcome this problem. PREVALENCE OF CHROMOSOME ABERRATIONS Chromosome aberrations can be diagnosed by cytogenetic analysis of metaphases from lymphocyte cultures or fibroblast cultures from skin or other tissues, amniotic fluid cell cultures or recently by a direct method or cultures from chorionic villi. Chromosome abnormalities are found with an average incidence of 6 per 1000 live births. The majority of chromosome aberrations at birth are aneuploidies due to a non-disjunctional event. The birth prevalence of viable trisomies rises with advanced maternal age. About 0.3% are chromosomal rearrangements, most of them balanced without phenotypical effect. Only a minority have unbalanced rearrangements with more or less severe developmental abnormalities. Inherited translocations have a high risk of unbalanced offspring depending on the type of rearrangement and in certain cases on the sex of the carrier parent. In sporadic aberrations the recurrence risk is about 1% (Stene and Mikkelsen, 1984). A much higher prevalence of chromosome aberrations was observed in spontaneous abortions (Hassold et al, 1980). Also, studies of perinatal deaths show a higher prevalence of chromosome aberrations compared to live birth figures (Kuleshov, 1976).

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 to 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 Fabry's disease by direct analysis of chorionic villi.

Six pregnancies of three carriers for X-linked Fabry's disease, were monitored by chromosome and enzyme analysis. Two affected male fetuses were detected by the demonstration of alpha-galactosidase deficiency in amniotic fluid cells and chorionic villi respectively. The use of chorionic villi enabled a diagnosis within a few hours after sampling in the ninth week of pregnancy whereas the use of amniotic fluid cells in the earlier case required two weeks of culturing after amniocentesis in the 16th week. Four female fetuses were found; heterozygosity was demonstrated in one by analysis of clones in the primary amniotic fluid cell culture.

Technical and biological aspects of pseudomosaicism and mosaicism.

In 471 amniotic fluid cell cultures, single abnormal cells were found to be randomly distributed. The expected number of pseudomosaicisms for aneuploidy due to randomly distributed cells was 3.9, and the observed number was 4. Expected number of mosaicism for aneuploidy was 0.6, and none was observed. The findings indicated that pseudomosaicisms as well as mosaicisms may be diagnosed from randomly distributed single abnormal cells. The probability of erroneously confirming mosaicism increased with the number of cells analysed in the second culture.

Lack of syringe toxicity to amniotic fluid cells in culture

Toxicity to amniotic fluid from many brands of syringes has been reported in the past few years. Thirty amniocentesis samples were exposed to Becton-Dickinson syringes for varying lengths of time and evaluated for growth rate. Six amniocentesis samples were exposed to the syringes for prolonged periods at varying temperatures and also evaluated for growth rate. No evidence of syringe toxicity was found for exposure periods up to 24 h, regardless of storage temperature.

Chromosomal mosaicism and maternal cell contamination in chorionic villi cultures.

In a series of 110 chromosome analyses, after chorionic villus sampling, mosaics were found in 3 out of 106 long term cultures. In all 3 cases, subsequent amniotic fluid cell cultures showed normal karyotypes. Maternal cell contamination was encountered in one long term culture derived from carefully dissected chorionic villi.

Low frequencies of apparently fragile X chromosomes in normal control cultures: a possible explanation.

Low frequencies of apparently fragile X [fra(X)] chromosomes have been reported in normal control, short-term, whole blood cultures, and they have been noted in both amniocyte and fetal blood cultures. However, there is currently no universal agreement on the lowest frequency for fra(X)(q27) that is diagnostic for the fragile X syndrome. Here, we present our observations on low levels of apparently fra(X) chromosomes in normal samples. We observed frequencies of 0.5% in short-term whole blood cultures and 0.9% in amniotic fluid cell cultures. In 1982, Steinbach et al. described nonspecific telomeric structural changes (TSC) and suggested that such low frequencies of apparently fra(X) chromosomes in normal material may be occurring by the same mechanism that is responsible for TSC formation. To determine if TSC formation can explain the significant baseline frequencies of fra(X) in normal controls, 10,457 cells were screened from 178 individuals referred for fra(X) analysis. Our findings indicated that TSC are not randomly distributed across chromosomes but tend to occur at specific sites. Based on our observations, we offer the hypothesis that the low frequency of apparent fra(X) in normal individuals may be due to nonrandom TSC distribution.

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.

Duration of nocturnal tooth contacts during bruxing

11. Ogus, HD, and Toiler, PA: Common disorders of the temporomandibular joint. Dental Practitioner Handbook No. 26, 1981. Oberg, T, Carlsson, GE, and Fajers, CM: The temporomandibular joint--A morphological study on human autopsy material. Acta Odontol &and 29:349, 1971. Griffin, CJ. Hawthorn R, and Harris, R: Anatomy and histology of the human temporomandibular joint. Monogr Oral Sci 4~1, 1975. Smith, RJ: Mandibular biomechanics and temporomandibular function in primates. Am J Phys Anthropol 49:341, 1978. Boering, G Anatomical and physiological considerations regarding the temporomandibular joint. Int Dent J 29:245, 1979. Kivirikko, KI: Urinary excretion of hydroxyproline in health and disease. Int Rev Connect Tissue Res 5:93, 1970. Bergman, I, and Loxley, R: The determination of hydroxyproline in urine hydroxysates. Clin Chim Acta 27:347, 1970. Prockop, DJ, Kivirikko, KI, Tuderman, L, and Guzman, NA: The biosynthesis of collagen and its disorders. N Engl J Med 30:13, 1979. Munksgaard, EC, Rhodes, M, Mayne, R, and Butler, WT: Collagen synthesis and secretion by rat incisor odontoblasts in organ culture. Eur J Biochem 82:609, 1978. Crouch, E, and Bornstein, P: Collagen synthesis by human amniotic fluid cells in culture: Characterization of procollagen with three identical proalpha 1 (I) chains. Biochemistry 17~5499, 1978 Narayanan, AS, and Page, RC: Biochemical characterisation of collagens synthesised by hbroblasts derived from normal and diseased human gingiva. J Biol Chem 251:5464, 1976. 12.

The prenatal diagnosis of the cerebro-hepato-renal syndrome of Zellweger.

The prenatal diagnosis of the cerebro-hepato-renal syndrome of Zellweger (CHRS) was made by assaying the levels of very long chain fatty acids (VLCFAs) in amniotic fluid cell cultures, obtained by amniocentesis at 16 1/2 weeks of pregnancy. The family-at-risk, because they had previously borne a child with CHRS, accepted these results as indications of an affected fetus, and chose to terminate the pregnancy at 20 1/2 weeks of gestation. The diagnosis was confirmed by the phenotype of the aborted fetus and the presence of markedly elevated levels of VLCFAs in fetal liver homogenates. The prenatal diagnosis of CHRS, which can now readily be determined from amniotic fluid cell cultures, is an important step in genetic counselling of families-at-risk for this disease.

European collaborative study on prenatal diagnosis: Mosaicism, pseudomosaicism and single abnormal cells in amniotic fluid cell cultures

A report is given of the results of a European collaborative study on mosaicism, pseudomosaicism and single abnormal cells in amniotic fluid cell cultures. The mean frequency of cases with mosaicism was 0.10 per cent, with pseudomosaicism 0.64 per cent and with single abnormal cells 2.83 per cent in a series of 44 170 amniotic fluid samples. There was no significant difference between the colony (in situ) and the flask method with regard to the frequency of mosaicism. Pseudomosaicism and single abnormal cells were more frequent in cases studied with the flask method probably due to other factors than the method of cultivation of the cells. The frequency of maternal cell contamination was 0.17 per cent and the frequency of wrong sex assignment was 0.11 per cent. A more correct estimation is obtained if these frequencies are doubled. There was a considerable variation between laboratories with regard to the frequencies given above. One reason for this variation is that there are no sharp limits between mosaicism, pseudomosaicism and single abnormal cells. Thus the material contained cases diagnosed as having pseudomosaicism which turned out to be mosaics at birth and to have an abnormal phenotype. These cases were very rare but pose a definite problem in prenatal cytogenetic diagnosis.

Mosaicism or Pseudomosaicism

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.

A culture vessel for amniotic fluid cells allowing faster preparation of chromosome slides.

A new culture vessel for amniotic fluid culture is presented (flaskette). It consists of a microscope slide, on top of which a culture chamber is mounted. Amniotic fluid cell cultures using in situ technique in the flaskette were compared to subcultured samples in ordinary (Falcon) tissue culture bottles. Working time was reduced by using this new culture vessel because of a very simple harvest procedure, allowing simultaneous harvest of 15 samples. The interval between amniocentesis and harvest was shorter for the in situ technique than for the subcultivation technique. The frequency of aneuploidy in individual metaphases was higher with the subcultivation technique, while there was no difference in the frequency of structural anomalies.

A Canadian collaborative study of mosaicism in amniotic fluid cell cultures.

Data on chromosomal mosaicism was collected retrospectively on 12 386 amniotic fluid samples cultured over a 10 year period in 14 Canadian centres. Level I mosaicism (a single abnormal cell--excluding single cell monosomy) was encountered in 863 cases (7.1 per cent). Level II mosaicism (multiple cells with the same abnormality in a single flask or colony) was encountered in 138 cases (1.1 per cent). Level III mosaicism (multiple cells distributed over multiple flasks or colonies) was encountered in 34 cases (0.3 per cent). Analysis of the details of these cases allowed five major conclusions to be drawn: (1) Single cell abnormalities should not be taken as an indication of true fetal mosaicism. Only rarely will this interpretation prove to be incorrect. (2) Mosaicism involving multiple cells confined to a single flask should not be regarded as an indication of true fetal mosaicism. Only occasionally will this interpretation prove to be incorrect. (3) Mosaicism involving multiple cells distributed over more than one flask should be regarded as a strong indication of true fetal mosaicism. Sixty per cent will be confirmed by karyotype analysis of the fetus or infant. (4) Mosaicism of the XX/XY type is usually due to maternal cell contamination. Occasionally it can be a female fetus with XY cells from an unknown source. (5) The in situ or colony method of chromosome analysis has no clear advantage over the flask method for either the detection of true fetal mosaicism or for the ability to distinguish true mosaics from false positives.

Advances in the Prenatal Diagnosis of Hematologic Diseases

Abstract Prenatal diagnosis of hematologic diseases can now be performed with fetal blood, fetal amniotic fluid cell DNA, and fetal chorionic villi DNA. Some hemoglobinopathies can be detected by all three methods, and the choice will depend on the available obstetric and laboratory techniques, as well as the time of presentation of the pregnancy. Hopefully, further development of molecular probes and techniques will soon expand these options to all of the globin disorders. Detection of coagulation disorders in utero currently requires samples of pure fetal blood. Gene cloning is accomplished for some (factor IX and antithrombin III) and is underway for others (factor VIII), and further investigation is necessary to determine whether deficiencies in these gene products are due to gene deletion or to mutant genes linked to polymorphic restriction enzyme sites of diagnostic use. Thus, molecular biology may be applied to prenatal diagnosis of the clotting problems, but this has not yet been accomplished. Disorders affecting the number and/or function of erythrocytes, leukocytes, and platelets can be diagnosed by analysis of fetal blood. Blood samples will continue to be required until more is known about the molecular biology of hematopoiesis. Syndromes that can be diagnosed by chromosome studies should be revealed in cultures of amniotic fluid cells, fetal blood lymphocytes, and chorionic villi cells. Cultured cells can be examined for karyotypes, Y-chromatin, spontaneous or induced chromosome breakage, DNA repair, SCEs, and translocations. The techniques for culturing amniotic cells and fetal blood white cells are established, and those for growing cells from chorionic villi are improving rapidly. Direct preparations of cells from villi only may suffice for some of the above analyses. The study of hematologic disease in utero has thus come full circle, from the use of amniotic cells to determine the sex in X-linked disorders, to fetal blood sampling for the analysis of gene products, then back to amniocentesis for DNA, and now earlier in gestation to chorionic villi. All of this has occurred in less than ten years, and it is anticipated that developments in the next ten years will be equally dramatic. The future should bring all prenatal testing into the first trimester, use molecular probes, and provide for both early diagnosis and early treatment of genetic hematologic disease.

Advances in the prenatal diagnosis of hematologic diseases

Prenatal diagnosis of hematologic diseases can now be performed with fetal blood, fetal amniotic fluid cell DNA, and fetal chorionic villi DNA. Some hemoglobinopathies can be detected by all three methods, and the choice will depend on the available obstetric and laboratory techniques, as well as the time of presentation of the pregnancy. Hopefully, further development of molecular probes and techniques will soon expand these options to all of the globin disorders. Detection of coagulation disorders in utero currently requires samples of pure fetal blood. Gene cloning is accomplished for some (factor IX and antithrombin III) and is underway for others (factor VIII), and further investigation is necessary to determine whether deficiencies in these gene products are due to gene deletion or to mutant genes linked to polymorphic restriction enzyme sites of diagnostic use. Thus, molecular biology may be applied to prenatal diagnosis of the clotting problems, but this has not yet been accomplished. Disorders affecting the number and/or function of erythrocytes, leukocytes, and platelets can be diagnosed by analysis of fetal blood. Blood samples will continue to be required until more is known about the molecular biology of hematopoiesis. Syndromes that can be diagnosed by chromosome studies should be revealed in cultures of amniotic fluid cells, fetal blood lymphocytes, and chorionic villi cells. Cultured cells can be examined for karyotypes, Y-chromatin, spontaneous or induced chromosome breakage, DNA repair, SCEs, and translocations. The techniques for culturing amniotic cells and fetal blood white cells are established, and those for growing cells from chorionic villi are improving rapidly. Direct preparations of cells from villi only may suffice for some of the above analyses. The study of hematologic disease in utero has thus come full circle, from the use of amniotic cells to determine the sex in X-linked disorders, to fetal blood sampling for the analysis of gene products, then back to amniocentesis for DNA, and now earlier in gestation to chorionic villi. All of this has occurred in less than ten years, and it is anticipated that developments in the next ten years will be equally dramatic. The future should bring all prenatal testing into the first trimester, use molecular probes, and provide for both early diagnosis and early treatment of genetic hematologic disease.

Fragments of Myelin Basic Protein Derived from Human Brain are not Mitogenic in Cultures of Human Amniotic Fluid Cells and Other Cells

The possible mitogenic effects of a number of preparations of the myelin basic protein (MBP) of human brain have been investigated in various cell types in culture, including human amniotic fluid cells. Intact human MBP, as well as fragments derived by BNPS-skatole and cathepsin D treatment, and isoelectric focusing fractions of human MBP, showed no mitogenic activity. These results are consistent with recent findings that the fibroblast growth factor (FGF) activity of bovine brain does not originate from MBP.

Inability of second trimester human amniotic fluid cell cultures to aromatize C19-steroids.

Amniotic fluid (AF) and fibroblast (F) cell types, derived from human amniotic fluid by amniocentesis in the second trimester, were cultured using media of varying composition and their ability to transform C19-steroids to oestrogens was investigated by fluorimetry, radioimmunoassay, tritium release and measurement of cytochrome P450. Although the cells were shown to be metabolically active by the presence of mitotic figures, the incorporation of tritiated leucine into protein and the production of the beta-subunit of hCG, no evidence of aromatization was obtained despite the inclusion in the medium of a number of steroids known to be transformed to oestrogens in several in vivo and in vitro preparations. The addition of reported stimulants of aromatase activity, viz. hCG, FSH, diethylstilbestrol, prostaglandins E2 and F2 alpha and dibutyryl cyclic AMP, was without effect.