Microsatellite analysis provides efficient confirmation of fetal trophoblast isolation from maternal circulation

Fetal trophoblasts can be found in maternal circulation from an early stage of pregnancy and thus provide a potential source of DNA for non-invasive prenatal diagnosis. We have developed a two-step method for trophoblast isolation between the 8th and 12th week of pregnancy. Blood was sampled from 14 women undergoing termination of pregnancy or spontaneous abortion. Immunomagnetic beads precoated with HLA class I and II, and with anti-cytokeratin-18 monoclonal antibodies, were used to remove CD8+ and other maternal cells, and to select for fetal trophoblasts, respectively. Microsatellite analysis was performed on DNA extracted from the isolated, maternal, paternal and placental cells after PCR amplification. Recovery of the trophoblasts was confirmed in 13/14 cases (93%) by the identification of an identical microsatellite pattern for fetal and placental cells. Further evidence was the presence of heterozygous alleles of both maternal and paternal origin. The correct prediction of gender in all five male fetuses was an additional confirmation of trophoblast recovery. We conclude that trophoblasts can be effectively isolated from maternal blood in the first trimester, and by using polymorphic microsatellite markers to confirm sample purity, this method has potential future application in prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

Normal and abnormal fetal cardiac anatomy

The heart is often perceived as a difficult organ to understand by ultrasound during fetal life. This is undoubtedly reflected in the low detection rate of cardiac abnormalities as compared to those of most other organ systems in the fetus. In this article we start by updating classical concepts of cardiac embryology, many of which were previously difficult to understand since they were overly simplistic or purely observational. We then lead on to the structure and growth of the fully formed fetal heart where we review the anatomy and ultrasound appearances in detail and provide comparisons with major abnormalities. We emphasise the fact that a solid understanding of cardiac anatomy can enable those involved in fetal medicine to make full use of the views of the heart that are obtained by ultrasound and which are often only transient. Copyright © 2004 John Wiley & Sons, Ltd.     

The predictive value of cytogenetic diagnosis after CVS based on 4860 cases with both direct and culture methods

Cytogenetic analysis was performed in 4860 chorionic villus samples by means of both direct preparation and long-term culture. The results of the analysis were compared with a classification including all theoretical types of combinations between the chromosomal constitution of the cytotrophoblast, extraembryonal mesoderm, and fetus, with the aim of evaluating the cytogenetic variability along the trophoblast—embryo axis. Eighteen of 29 possible combinations were found demonstrating a considerable heterogeneity. A mosaic conceptus was found in 1·5 per cent of cases, with generalized mosaicisms and confined mosaicisms in 0·2 and 1·3 per cent, respectively. Cytogenetic variability along the trophoblast—embryo axis was found in 1·42 per cent of cases. Results possibly leading to diagnostic errors (false-positive and false-negative results) were found in only 1·38 per cent. False-positive results of direct preparation were the most commonly observed discrepancy (0·8 per cent), while the incidence of false-positive results of the culture method and of both methods was 0·31 and 0·16 per cent respectively. The incidence of false-negative results was 0·1 per cent, with false-negative results of direct preparation 0·08 per cent and false-negative results of both methods 0·02 per cent. False-negative results of the culture method were not found. Our data confirm the high diagnostic accuracy of chorionic villus sampling and the utility of the combined use of the two methods in minimizing diagnostic errors and in reducing the need for follow-up amniocentesis.     

Astomia-agnathia-holoprosencephaly association. Prenatal diagnosis of a new case

A case of agnathia-astomia-holoprosencephaly with prenatal ultrasound diagnosis at 23 weeks is reported and discussed. This lethal neurocristopathy, well known in mammalians, is rarely observed in humans. Prenatal diagnosis features are intrauterine growth retardation, mandible absence or major hypoplasia, holoprosencephaly, cyclopia or hypotelorism, and in some instances frontal proboscis. This malformation is usually sporadic, but may be genetically determined as an autosomal recessive trait, since two cases in the same sibship have been reported.     

Novel mutation and prenatal sonographic findings of glutaric aciduria (type I) in two Taiwanese families

Glutaric aciduria type I (GA I) is an autosomal recessively inherited inborn error with a defect of the enzyme glutaryl-CoA dehydrogenase (GCDH), which has never been diagnosed prenatally in Taiwanese patients. We present the prenatal sonographic findings and mutational analysis data of three children in two Taiwanese families. One patient from each family was diagnosed postnatally due to macrocephaly and neurological deterioration at 4 months and 10 months, respectively. The third child, sister of the first patient, was diagnosed prenatally at 11 weeks' gestation through chorionic villus sampling (CVS). Molecular analysis revealed that the fetus and child in Family 1 were homozygous for a common mutation, IVS10 -2A>C, which has not been reported in the Caucasian population. The patient in Family 2 was a compound heterozygote for IVS10 -2A>C and a novel mutation 749T>C (L238P). After genetic counseling, the couple decided to continue the second pregnancy. However, dilatation of quadrigeminal cistern (QC) and suspicious macrocephaly were noted at 30 weeks. Progressive dilatation of the QC associated with macrocephaly, fronto-temporal atrophy and wide space of perisylvian fissure were found in the follow-up scans. The affected girl was delivered at 37 weeks' gestation by cesarean section. Postnatal magnetic resonance imaging (MRI) studies confirmed the prenatal sonographic findings. With prenatal sonographic findings and mutational analysis presented in the present cases, the feasibility of prenatal diagnosis of GA I in high-risk pregnancy can not be overlooked. Copyright © 2002 John Wiley & Sons, Ltd.     

Birth of healthy children after preimplantation diagnosis of β-thalassemia by whole-genome amplification

Preimplantation genetic diagnosis (PGD) offers couples at risk for transmitting an inherited disorder the possibility to avoid the need to terminate affected pregnancies. PGD for monogenic diseases is most commonly accomplished by blastomere biopsy from cleavage-stage embryos, followed by PCR-based DNA analysis. However, the molecular heterogeneity of many monogenic diseases requires a diagnostic strategy capable of detecting a range of mutations and compound genotypes. With the above considerations, we developed an accurate and reliable strategy for analysis of β-globin gene mutations, applicable for PGD for the wide spectrum of β-thalassemia major mutations in the Chinese population. The strategy involves primer-extension preamplification (PEP), followed by nested PCR and reverse dot blot (RDB) for mutation detection since it facilitates simultaneous analysis of more than one mutation in a single cell. This report describes the application of the strategy in two clinical IVF/PGD cycles at risk for transmitting β-thalassemia major, which resulted in the first thalassemia-free children born after PGD in China. Copyright © 2003 John Wiley & Sons, Ltd.