Bernd Eiben

Rapid Prenatal Diagnosis of Aneuploidies in Uncultured Amniocytes by Fluorescence in situ Hybridization

Objective: A new method in prenatal diagnostics allows to demonstrate certain numeric chromosomal aneuploidies in amniotic cells within 24 h in contrast to conventional methods which take 1–3 weeks. Materials: The experience with this rapid fluorescence in situ hybridization (FISH) method is compared to standard karyotyping and its clinical relevance is described in a large clinical pilot study. FISH on uncultured amniocytes has been performed from 12 weeks of gestation to the third trimester using commercially available chromosome-specific DNA probes for chromosomes 13, 18, 21, X and Y. Results: FISH was performed successfully in 3,150 prenatal cases. All trisomies 13, 18 and 21 and all cases with gonosomal aberrations were detected by FISH analysis. Neither false-positive nor false-negative results were obtained using FISH. For all analyzable disorders the FISH results were in complete agreement with standard cytogenetics. Conclusions: In our experience, FISH is a valuable and reliable method for rapid diagnosis of numeric chromosomal aneuploidies.

A prospective comparative study on fluorescence in situ hybridization (FISH) of uncultured amniocytes and standard karyotype analysis

Fluorescence in situ hybridization (FISH) on uncultured amniocytes and standard cytogenetic analysis after amniocentesis have been performed for 904 samples. The experience with the FISH method and its clinical relevance is described in a large clinical pilot study. Commercially available chromosome‐specific DNA probes for chromosomes 13, 18, 21, X and Y were used. FISH assays were performed from 12 weeks of gestation to the third trimester. In 96 per cent of the cases, hybridization was performed successfully. At least 50 nuclei for all probes could be counted in 88 per cent of the cases and in 8 per cent between 10 and 49 nuclei were scored. All trisomies 13, 18 and 21 and all cases with gonosomal aberrations were detected by FISH analysis with the exception of one case of trisomy 21 in which hybridization failed due to technical problems. Neither false‐positive nor false‐negative results were obtained with the DNA probes, in complete agreement with standard cytogenetics. In our experience, FISH is a valuable and reliable method for rapid diagnosis. Consequences of FISH diagnosis are discussed.

The predictive value of chorionic villus histology for identifying chromosomally normal and abnormal spontaneous abortions

SummaryTo evaluate the significance of placental histology, a collaborative histological and cytogenetic study on the products of 123 spontaneous abortions of 6–19 week pregnancies was performed. From each sample, 2–3 g was dissected randomly and analysed histologically with no prior knowledge of the karyotype. Chromosomes were prepared from the remaining chorionic villi after an overnight incubation in culture medium; they were analysed by Q- or G-banding. The frequency and type of chromosome anomalies detected are comparable to those seen in other studies. Altogether, 49.6% were chromosomally abnormal, trisomies predominating (49.2%), followed by polyploidy (27.9%) and monosomy X (14.5%). The histological classification failed for technical reasons in 11 cases (8.9%). To classify the remaining cases, 18 different histological criteria were considered independently by two pathologists. A definite diagnosis was not possible for 27 abortions (22.0%), and 50 were classified as being chromosomally abnormal. This proved to be correct in 36 (72.0%), but incorrect in as many as 14 (28.0%). No evidence for an abnormal karyotype was found histologically in samples from 35 abortuses. Nevertheless, 9 of them (25.7%) had an abnormal karyotype. The predictive value of chorionic villus histology seems to be inadequate, as only 62 of 112 samples were correctly classified by histology (55.4%). The value differs little from the a priori probability of an abnormal or normal karyotype in abortuses of corresponding gestational ages.

A cytogenetic study directly from chorionic villi of 140 spontaneous abortions

SummarySpontaneous abortions were studied by analyzing chromosomes directly from chorionic villi. The frequency and the type of anomalies detected among 140 abortuses are in good agreement with those observed by others using conventional tissue cultures. Abnormal karyotypes were found in 48.6% of the cases. Trisomy predominated (66.2%), followed by polyploidy (22.1%), monosomy X (7.4%), and structural anomalies (4.4%). Among the trisomies, the most prevalent were of chromosome 22 (22.2%), 16(22.2%), and 13 (9.5%). The relative frequencies of trisomies, monosomy X, and the different chromosomes involved in trisomies seem to differ between our study and those in which tissue cultures were analyzed. Our low frequency of 45,XO karyotypes and the shift to trisomies of chromosomes whose involvement increases steeply with maternal age are considered due to the approximately 3 year higher mean maternal age in our sample. The sex ratio (male to female) in chromosomally abnormal abortuses was 1.28, which is nearly identical to the 1.2 found in earlier studies. Surprisingly, in chromosomally normal abortions males were significantly outnumbered by females (sex ratio 0.76). Since maternal cell contamination cannot have influenced the sex ratio in our study, we consider it worthwhile to investigate whether failures associated with X inactivation are responsible for pregnancy wastage of some euploid female conceptuses. Knowledge of the karyotypes may serve as a prerequisite for the investigation of non-chromosomal genetic causes of pregnancy wastage.

Trisomy of human chromosome 18: Molecular studies on parental origin and cell stage of nondisjunction

We investigated the parent and cell division of origin of the extra chromosome 18 in 62 aneuploids with a free trisomy 18 by using chromosome-18-specific pericentromeric short-sequence repeats. In 46 cases, DNA of patients was recovered from archival specimens, such as paraffin-embedded tissues and fixed chromosomal spreads. In 56 families, the supernumerary chromosome was maternal in origin; in six families, it was paternal. Among the 56 maternally derived aneuploids, we could exclude a postzygotic mitotic error in 52 cases. Among those in which the nondisjunction was attributable to an error at meiosis, 11 were the result of a meiosis I nondisjunction and 17 were caused by a meiosis II error. This result differs markedly from findings in acrocentric chromosomes where nondisjunction at maternal meiosis I predominates. Among the six paternally derived cases, two originated from a meiotic error, indicating that a nondisjunction in paternal meiosis is not as rare as previously suggested.

On the complication risk of early amniocentesis versus standard amniocentesis.

In the last 6 years early amniocentesis for the prenatal diagnosis of chromosome aberrations has been established in many centers worldwide, but knowledge about the gynecological safety of the procedure is sparse. From 1990 to 1995 at the Evangelisches Krankenhaus Oberhausen (Germany) 3,277 early amniocenteses (between weeks 11 and 14) and 1,808 standard amniocenteses were performed in low-risk indication groups (advanced maternal age and anxiety). A complete follow-up including reports of fetal outcome was obtained in 4,444 cases (87.5%). A pregnancy age-related abortion rate was determined with a slightly higher rate of abortions up to week 28 of gestation in early amniocentesis. The total abortion rate up to week 28 after the procedure for cases with complete follow-up was 2% in early amniocentesis. Compared to standard amniocentesis performed under the same clinical conditions with an abortion rate of 1.3%, there is no statistical difference between early and standard amniocentesis (p = 0.0971). Hip and foot dislocations (22 cases) and pulmonary distress syndromes (8 newborns) showed no significant correlation with the gestational week. Given the high normal background rate of spontaneous abortions in the early period of pregnancy without an invasive procedure, early amniocentesis can be considered as a safe alternative to chorionic villus sampling and standard amniocentesis.

Partial deletion of 4p in fetal cells not present in chorionic villi

A case of a prenatal diagnosis at the second trimester is presented showing a normal karyotype in 12 metaphases from chorionic villi. In all cultured amniotic cells, however, and also in all fetal fibroblasts analyzed after abortion a structural anomaly (46,XY;del 4(pter→p15.2) was detected. Prenatal diagnosis was performed because of intrauterine growth retardation, cleft lip and esophagus atresia by ultrasound. The fetal stigmata are compatible with the Wolf Hirschhorn syndrome. We conclude that amniocentesis may be indicated notwithstanding a normal CV‐diagnosis in those rare pregnancies with a characteristically abnormal ultrasound.

Retrospective study of the parental origin of the extra chromosome in trisomy 18 (Edwards syndrome)

The parental origin of the extra chromosome in trisomy 18 was traced in 30 informative families using highly polymorphic (CA) repeats mapped on the long arm of chromosome 18. Proband DNA was recovered from slides of chromosome preparations in 28 cases and from paraffin-embedded tissues in two cases. The extra chromosome was found to be of maternal origin in 26 cases (86.7%), and paternal origin in 4 cases (13.3%).

Tissue-specific 45,X0/47,XY,+13 mosaicism in an 18-year-old woman.

SummaryThe case of an 18-year-old woman with a 47,XY,+13 karyotype in lymphocytes and a 45,X0 karyotype in all other tissue analyzed is presented. The proposita shows no stigmata of the Patau syndrome and no masculinization.

A Retrospective Evaluation of Second-trimester Serum Screening for Fetal Trisomy 18: Experience of Two Laboratories

A retrospective study on screening methods for fetal trisomy 18 has been carried out in two different laboratories using the serum parameters: total human chorionic gonadotropin (hCG), unconjugated oestriol (uE3), and α‐fetoprotein (AFP) in different combinations and in single marker protocols. Laboratory A (LA) utilized a radio‐immunoassay to examine 38 fetal trisomy 18 cases and laboratory B (LB) utilized an enzyme‐immunoassay to examine 33 trisomy 18 cases. As unaffected references the whole routine cohorts of each laboratory were used (LA: 29 043; LB: 4264). In both trisomy 18 study groups the median hCG and uE3 multiples of the median (MoM) values were markedly declined (LA: 0.21 MoM, 0.37 MoM; LB: 0.31 MoM, 0.44 MoM). Even after exclusion of trisomy 18 cases with combined neural tube or ventral wall defects the medians of AFP MoM values were only moderately declined (LA: 0.73 MoM; LB: 0.8 MoM). Receiver–operator characteristic (ROC) curves after multivariate discriminance analysis and single marker evaluation demonstrated that the difference of efficiency between a combination of hCG, uE3 and AFP, and a combination of hCG and uE3 is small but that any of these combinations are more efficient than a combination of hCG and AFP or single marker protocols, respectively. At a risk cut‐off generating a false‐positive rate of one per cent the most effective marker combination detected 31 of 38 (81.6 per cent) affected pregnancies in LA and 25 of 33 (75.8 per cent) in LB. The differences in sensitivity and specificity seem to be due to the different analytical systems being utilized by the two laboratories. Copyright