Prenatal diagnosis with repetitive in situ hybridization probes.

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY, +21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries.

In utero stem cell therapy.

In utero stem cell transplantation offers the potential for treating a number of genetic disorders. The combination of fetal immunotolerance and fetal marrow space makes the fetus an excellent transplant recipient. Experiments on the mouse, sheep and rhesus monkey have indicated that in utero transplantation is feasible. Human trials are currently beginning.

Supplementing iron intravenously in pregnancy. A way to avoid blood transfusions.

OBJECTIVE: To determine the safety and efficacy of maternal intravenous iron administration to avoid blood transfusion in patients who cannot use oral preparations. METHODS: Patients with persistent iron-deficiency anemia who had one of the following indications were included in this study: severe side effects from oral preparations, lack of improvement despite oral iron intake or history of gastrointestinal operations. The total iron amount needed to regenerate iron stores was calculated according to hemoglobin and the patients' weight. Hemoglobin, hematocrit, mean corpuscular volume, serum iron, transferrin and ferritin were evaluated at the start and conclusion of therapy as well as two weeks afterward. RESULTS: Twenty-six patients were included in the study; four of them delivered during the therapy course. One patient developed mild signs of allergy (urticaria) after the test dose and was excluded from the study. The remaining 21 pregnant patients (mean gestational age 28 weeks) completed the therapy course and received a mean of 1,000 mg of elemental iron. The hemoglobin was increased from 8.4 +/- 1.0 to 10.1 +/- 0.6 g/dL at the start and end of therapy, respectively (P < .01) and continued to rise to 10.9 +/- 0.6 g/dL two weeks later (P < .01). The serum iron was increased from 3.9 +/- 2.0 mumol/L at the start of therapy to 15.5 +/- 7.2 at the end (P < .01). The transferrin was decreased from 47.0 +/- 7.8 to 41.4 +/- 5.3 to 37.1 +/- 11.8 mumol/L at the start of, end of and two weeks after therapy, respectively (P < .01). Ferritin levels were increased from 2.9 +/- 2.7 ng/mL at the start to 122.8 +/- 87.1 at the end of therapy (P < .01) and decreased to 109.4 +/- 90.7 ng/mL two weeks after treatment (not significant). Only mild and transient side effects were occasionally reported. CONCLUSION: Intravenous iron administration during pregnancy is an effective method of regenerating hemoglobin and iron stores. It should be considered for patients with severe iron-deficiency anemia who cannot use oral preparations.

Prenatal diagnosis of inherited metabolic diseases.

Advances in the prenatal diagnosis of inherited metabolic disease have provided new reproductive options to at-risk couples. These advances have occurred in both sampling techniques and methods of analysis. In this review we present an overview of the currently available prenatal diagnostic approaches for the diagnosis of metabolic disease in a fetus.

The distinction between arylsulphatases in chorionic villi.

The relatively high activity of arylsulphatase C (ASC) in the placenta is a potential risk for the misdiagnosis of arylsulphatase A (ASA) or arylsulphatase B (ASB) deficiency in chorionic villus sampling when assayed by synthetic substrates. A clear distinction between these enzymes can be achieved in either the direct villi or the cultured villi cells. Interestingly, the activity of ASC differed significantly in cultured villi cells when prepared by two different methods, namely, minced villi versus treatment with trypsin and collagenase, while ASA and ASB were not affected by these treatments. Whether ASC was directly affected by one of these treatments or whether a selection of cells with different ASC levels was achieved is not yet clear, but this phenomenon clearly indicates the importance of precise definition of CVS preparations to correlate with the enzyme activity data.

Fetal acrania: five new cases and review of the literature.

Acrania is a lethal malformation in which there is an absence of the flat skull bones covering the brain. Five new cases are described, and a review of the English-language medical literature is presented. The sonographic differential diagnosis of acrania includes anencephaly, large cephalocele, osteogenesis imperfecta, and hypophosphatasia. The diagnosis of acrania can be established sonographically even in the first trimester if a large mass of disorganized brain tissue covered only by a thin membrane is detected.

Prenatal diagnosis of Chediak-Higashi syndrome.

We report the first prenatal diagnosis of an affected fetus with Chediak-Higashi syndrome (CHS). Diagnosis was accomplished via fetal blood sampling at 17 menstrual weeks and was confirmed after birth. Retrospective measurement of the largest acid phosphatase-positive lysosomes in cultured amniotic fluid cells and chorionic villus cells showed that in CHS these lysosomes are significantly larger than those in normal cells. This method may be used for prenatal diagnosis of CHS by amniocentesis and chorionic villus sampling (CVS).

Replication asynchrony increases in women at risk for aneuploid offspring.

We attempted to demonstrate a relation between a loss of replication control, centromere dysfunction, and predisposition to non-disjunction. Couples with a Down syndrome offspring were the high-risk probands. One-color FISH (fluorescent in-situ hybridization) was applied to interphase nuclei (lymphocytes). Replication pattern of two pairs of alleles, RB-1 and 21q22, were studied, and the rate of aneuploidy was estimated using two alpha-satellite probes of chromosomes 8 and 18. Our results suggest the existence of an association between replication timing and the rate of non-disjunction. A higher rate of allele asynchrony and aneuploidy was found in older women and in mothers of a Down syndrome offspring. These findings may reflect a predisposition for meiotic non-disjunction in these women.

Johanson-Blizzard syndrome: a prenatal ultrasonographic diagnosis.

Johanson-Blizzard syndrome is a rare autosomal recessive disorder characterized by aplasia of alae nasi, pancreatic insufficiency, aplasia cutis, anorectal anomalies and postnatal growth restriction. In this case report, we describe the prenatal sonographic findings of Johanson-Blizzard syndrome in a 21-week pregnancy of a consanguineous couple. Sonographic findings of aplastic alae nasi (beak-like nose) and dilated sigmoid colon led to the prenatal diagnosis. This is the first report of the prenatal sonographic diagnosis of Johanson-Blizzard syndrome.

Interchromosomal effect leading to an increase in aneuploidy in sperm nuclei in a man heterozygous for pericentric inversion (inv 9) and C-heterochromatin.

We describe a man with pericentric inversion 9 and constitutive heterochromatin, and a high disomy rate in his sperm cells (with all probes analyzed). The disomy rate was estimated with the following probes: 8, 9, 18, X, and Y, and was significantly higher than that in control sperm cells, while chromosome 9 showed the highest disomy frequency. The probes of X and Y together showed the same disomy frequency as X and Y alone, which indicates the same nondisjunction rate in the first meiotic division. We suggest that the interchromosomal effect found in this man differed from other findings in sperm cells of men carrying an inversion in terms of the difference in the length of the heterochromatin between the two chromosomes 9. Also, it is well known that the effect of inversion 9 with increased heterochromatin is highly variable and may even vary in members of the same family.

Fetal cells in the uterine cervix: a source for early non-invasive prenatal diagnosis.

Various non-invasive techniques for prenatal diagnosis have been under investigation. We evaluated the success of fetal sexing using a non-invasive technique for obtaining fetal cells, uterine cervix brushing, in combination with FISH. Thirty pregnant women who completed between 6 and 10 weeks of gestation and who were scheduled to undergo pregnancy termination were included in the study. A Pap smear cytobrush was inserted through the external os to a maximum depth of 2 cm and removed while rotating it a full turn. The material that was caught on the brush was spread on four microscope slides. Two-color FISH was used for fetal sexing. Following pregnancy termination, a placental sample was used for full karyotyping. In 29/30 cases FISH diagnosis was confirmed by chromosomal analysis. The only male case in which a Y chromosome was not seen was from a pregnancy of 6 weeks 6 days gestational age. One case was mosaic of 46,XY/47,XXY (25%). In most cases (7/13) the Y chromosome was already identified in the first analyzed slide. With the use of a cytobrush fetal cells can be easily obtained for the purpose of prenatal diagnosis of chromosomal disorders.

Uterine cavity lavage: adding FISH to conventional cytogenetics for embryonic sexing and diagnosing common chromosomal aberrations.

This study was undertaken to examine the efficacy for early prenatal diagnosis of uterine cavity lavage at the level of the internal os and to assess the rate of maternal contamination. In phase I, uterine cavity lavage was performed in 38 women scheduled for pregnancy termination between 6 and 12 weeks. In addition to short- and long-term cultures, one-colour FISH (fluorescence in situ hybridization) with Y and X probes was used for fetal sexing. Two-colour FISH was used in all known male fetuses for the assessment of maternal contamination. In phase II, lavage was performed on 16 women. Fetal sex was diagnosed with direct labelled X and Y probes and common numerical chromosomal aberration was attempted with 18 and 21 direct labelled probes. Fetal sexing was successful in all cases in phases I and II. Out of 34 patients in which tissue was obtained, only FISH was done in six. Long-term cell cultures were successful in the other 28 cases, but complete karyotyping in 19 (56 per cent). No chromosomal aberration was found with the direct labelled probes 18 and 21 in FISH. Maternal contamination was assessed to be 5-10 per cent. This simple and easy-to-master technique is very effective in obtaining fetal cells early in pregnancy for genetic diagnosis, especially by FISH. However, the safety of the procedure must be tested in ongoing pregnancies.