Prenatal diagnosis of beta-thalassaemia by coelocentesis

Coelomic fluid and placental tissue were obtained from four women undergoing termination of pregnancy at 7-9 weeks gestation for psychological reasons. All four women and their partners were known carriers of beta-thalassaemia and DNA analysis in their blood identified the mutation carried by each of them. Allelespecific polymerase chain reaction and denaturing gradient gel electrophoresis techniques were used to detect and identify the mutations in the DNA extracted from the coelomic cells and placental tissue. Three fetuses were found to be carriers of either the paternal or maternal mutation, while one was found to be affected by beta-thalassaemia. There was concordance in the results obtained from the chorionic villi and coelomic cells. Amplification of the apolipoprotein B gene variable number tandem repeats (VNTR), in the DNA of the coelomic cells showed normal sagregation of alleles in the fetuses, thus excluding maternal contamination. The results of this study demonstrate that coelocentesis may be a reliable alternative technique for the diagnosis of beta-thalassaemia from as early as 7 weeks gestation.     

Prenatal diagnosis of triploidy and trisomy 21 through fetal erythroblasts isolated from maternal blood

BACKGROUND: A long-sought goal of medical genetics has been the development of prenatal diagnostic procedures that do not endanger the conceptus. The safety of noninvasive methods for prenatal diagnosis would be especially attractive because they could be extended to all pregnant women, regardless of their ages or histories. Noninvasive prenatal diagnosis for the entire population might be possible recovering fetal cells from maternal blood. For this purpose, we have studied fetal erythroblasts. MATERIALS AND METHODS: To evaluate the potential of the method for clinical use, we studied maternal blood samples from 11 women referred to us for prenatal diagnosis between 15 and 20 weeks of gestation. For simple and effective enrichment of fetal nucleated erythrocytes from peripheral maternal blood, we combined a triple density gradient and magnetic-activated cell sorting (MACS) of anti-CD71 transferrin receptor antibody labeled cells. The isolated cells were analysed by using dual-colour interphase fluorescent in situ hybridization (FISH) with X-, Y-, 18- and 21-specific DNA probes. RESULTS: Chromosomal abnormalities detected on enriched fetal cells include trisomy 21 and triploidy. CONCLUSIONS: Based on the current results it is suggested that the technique described here is a simple, fast, efficient and reliable method for non invasive prenatal diagnosis.     

Fetal cells in the maternal blood: a step towards non-invasive prenatal diagnosis? Review of the literature

Prenatal diagnosis of genetic abnormalities requires nucleated fetal cells which are currently obtained by invasive techniques such as amniocentesis, chorionic villus sampling and percutaneous umbilical blood sampling. Each of these entails a risk to the foetus and sometimes to the mother. Nucleated fetal cells have been reported to be present in maternal blood. Recovery of fetal cells from maternal blood would allow a noninvasive prenatal diagnosis. Their rarity (1 fetal cell for 10(6) to 10(8) maternal cells) presents a technical challenge. Due to the small number of fetal cells, sensitive analysis techniques such as PCR and FISH are necessary. Some degree of fetal cells enrichment in the maternal blood sample often precedes the analysis. Different techniques are used for the enrichment: discontinuous density gradient, magnetic activated cell sorting, fluorescence activated cell sorting, micromanipulator.... Several prenatal diagnosis have already been performed from maternal venous blood samples: diagnosis of gender, RhD blood genotype, Duchenne muscular dystrophy and hemoglobinopathy by PCR, diagnosis of gender and chromosome aneuploidy by FISH. Many teams are working on this subject. It is difficult to compare the studies because the techniques of enrichment and analysis vary. We review the different strategies chosen for prenatal diagnosis from maternal blood and discuss the results.     

Fetal heart rate following coelocentesis

For psychological reasons, coelocentesis was performed in 20 women prior to termination of pregnancy, at 6-11 weeks of gestation. The fetal heart rate (FHR) was measured immediately before the procedure and at 1, 5, and 10 min afterward. There was no significant difference between FHR before coelocentesis compared to the values at 1 min (mean = 158, range 114-178; z = -0.629, P = 0.529), 5 min (mean = 160, range 121-179; z = -0.191, P = 0.848), or 10 min (mean 159, range 117-183; z = -0.214, P = 0.83) after the procedure. These findings suggest that coelocentesis does not have a major effect on the fetal cardiovascular system.     

Rescue therapy for the acute respiratory distress syndrome (ARDS)

The potential risk of feto-maternal haemorrhage following coelocentesis was examined in 17 singleton pregnancies at 6-11 weeks of gestation by measuring maternal serum concentration of alpha fetoprotein (AFP) before and 1 and 10 min after the procedure. There was no significant difference between the maternal serum AFP concentration before coelocentesis (median 7.5, range 4.5-21.5 IU) compared to the values at 1 min (median 8.6, range 3.9-17.8; Z = -0.504, P = 0.614), and 10 min (median 7.5, range 5.7-20.6; Z = -0.432, P = 0.666) after the procedure. These findings demonstrate that coelocentesis is not associated with significant feto-maternal haemorrhage.     

Rapid trisomy diagnosis (21, 18, and 13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis

PURPOSE AND METHODS: Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. This requires lengthy laboratory procedures and high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid and inexpensive and may potentially be suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction (F-PCR) using polymorphic small tandem repeats (STRs). RESULTS: In this paper we present data demonstrating that fluorescent PCR amplification of STRs can be used for rapid diagnosis of trisomy 21, trisomy 18, and trisomy 13 and can be successfully applied to both prenatal diagnosis and diagnosis of single cells. This study also reports significant numbers of prenatal diagnoses using quantitative fluorescent PCR. CONCLUSIONS: We believe that further studies of greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for trisomy diagnosis from single cells using multiple STR markers for either preimplantation genetic diagnosis or, potentially, diagnosis from fetal cells isolated from maternal blood.     

Potential role of transforming growth factor-beta 1 in terminating the immune response in patients with Guillain-Barré syndrome

Currently, amniocentesis, chorionic villus sampling (CVS) and fetal blood sampling are used to obtain fetal cells for genetic diagnosis. These invasive procedures pose a small but not negligible risk for the fetus. Efforts have been directed towards the enrichment of fetal cells, such as erythroblasts, from maternal blood and progress has been made in the diagnosis of some chromosomal disorders and in sex determinations. We now report the detection of point mutations in single gene disorders using this method of prenatal diagnosis by enriching fetal cells from maternal blood by magnetic cell sorting followed by isolation of pure fetal cells by microdissection. In two pregnancies at risk for sickle cell anaemia and beta-thalassaemia, we successfully identified the fetal genotypes. Thus, prenatal diagnosis of single gene disorders by recovering fetal cells from maternal circulation appears to be a feasible approach.     

First-trimester fetal chromosomal diagnosis using endocervical lavage: a negative evaluation

A report in the literature indicated that fetal cells could be obtained by endocervical lavage during the first trimester of pregnancy and successfully cultured. This would allow prenatal diagnosis earlier in pregnancy than is currently possible with second-trimester amniocentesis. Therefore, we attempted to confirm these findings. Our results indicated that the cultural cells were maternal in origin. We disagree with interpretations of the data given in the initial report and conclude that first-trimester prenatal diagnosis is not feasible at this time.     

Prenatal diagnosis

The enormous progress witnessed in the field of prenatal diagnosis during the past two decades is likely to continue into the future. Improved imaging techniques are likely to enhance the resolution of noninvasively obtained fetal images considerably over their current excellent quality. Although this undoubtedly will be true for ultrasonography, the increased speed of magnetic resonance equipment may offer a new realm of imaging possibilities. Computerized image processing, analysis, and three-dimensional reconstructions all should make interpretation of fetal images easier and more understandable to the nonspecialist. Advances in molecular genetics will continue to accelerate, greatly expanding the range and accuracy of prenatal diagnosis. The alert pediatrician who is sensitive to genetic issues may, by early detection of pediatric disorders and careful family history assessment, be in a position to identify families at risk for serious genetic conditions and provide the opportunity to make informed decisions on reproductive options that avert a major tragedy. The pediatrician, working with obstetric colleagues, should be part of a team effort to support families going through prenatal testing. Familiarity with these rapidly changing technologies will make it far easier to support the family needing additional explanation about prenatal diagnosis issues.     

Roe v. Wade and the euthanasia debate

Epidermolysis bullosa (EB) is a group of heritable diseases which manifest with blistering and erosions of the skin and mucous membranes. Due of life-threatening complications and significant long-term morbidity associated with the severe, neonatal lethal (Herlitz) form of junctional EB (H-JEB), there has been a demand for prenatal diagnosis from families at risk for recurrence. Previously, the only reliable method of prenatal diagnosis of EB was a fetal skin biopsy performed at 16-20 weeks' gestation and analysed by electron microscopy. Recently, the genes LAMA3, LAMB3, and LAMC2, encoding the polypeptide subunits of laminin 5, an anchoring filament protein, have been shown to contain mutations in H-JEB. In this study, direct detection of pathogenetic mutations in the laminin 5 genes was used to perform polymerase chain reaction (PCR)-based prenatal testing. DNA was obtained by chorionic villus sampling (CVS) at 10-15 weeks or amniocentesis at 12-19 weeks' gestation in 15 families at risk for recurrence of JEB. In 13 cases, the fetus was predicted to be either genetically normal or a clinically unaffected carrier of a mutation in one allele. These predictions have been validated in all cases by the birth of a healthy child. In two cases, an affected fetus was predicted, and the diagnosis was confirmed by subsequent fetal skin biopsy. These results demonstrate that DNA-based prenatal testing offers an early, expedient, and accurate method of prenatal diagnosis or an exclusion of Herlitz JEB.     

Prenatal diagnosis of vein of Galen aneurysmal malformation: report of two cases with proposal for prognostic indices

Vein of Galen aneurysmal malformation (VGAM) is a rare, intracranial vascular anomaly that, until recently, has usually been diagnosed postnatally. Today, however, with the advances in high-resolution ultrasonography and colour Doppler, prenatal diagnosis is relatively easy. Due to novel intravascular embolization techniques, the prognosis of neonates with VGAM has markedly improved. A healthy infant with normal neurodevelopmental and cardiovascular status is now a reality. For the best outcome, however, careful planning of the appropriate time, mode, and place of delivery should be undertaken. To achieve this goal, in utero prognostic factors should be determined. This report illustrates, for the first time, prenatal ultrasonographic indices that may predict the outcome in two cases with a prenatal diagnosis of VGAM. The indices included mapping of intracranial feeding arteries, assessment of the width of the straight sinus, assessment and measurement of flow in the straight sinus, existence of 'steal' retrograde aortic flow, and the appearance of high-output cardiac state. By using these prenatal ultrasonographic parameters, fetal outcome may be predicted and appropriate management decided upon.     

Prenatal diagnosis: choices women make about pursuing testing and acting on abnormal results

Liberalization of abortion laws in several US states (e.g., New York and California) coincided with the development of prenatal techniques, which diagnose chromosomal abnormalities and biochemical disorders. Increased use of prenatal diagnostic services has not been accompanied by adequate examination of the decision making process women undergo when contemplating prenatal diagnosis, pregnancy termination, or experimental fetal therapy. The limited literature exploring these issues indicates that many women do not know as much as possible about the health of their fetus. Women who are at risk of abnormal pregnancy tend to become distressed and willing to accept invasive testing, even when they know the significant, albeit low, risks of such testing. Women's perceptions of risk, which stem from complex psychologic-phenomena, are likely to be very inconsistent with objective reality. Neither counseling nor education can easily change these misperceptions. Nevertheless, counseling can at least alter misperceptions enough so they move closer to objective reality. On the other hand, counseling can sway perceptions and choices made based on these perceptions. Decision making is even more complex and emotional when women encounter abnormalities. Considerable social, moral, and psychologic factors influence this process, making this a very problematic area to study. Almost all women who carry an abnormal fetus with a very serious prognosis and a high degree of diagnostic certainty chose to terminate the pregnancy. The decision is much more difficult for women carrying a fetus with less diagnostic or prognostic certainty. Insufficient data exists to determine how they handle these management decisions. Women tend to opt for abortion in cases of chromosomal abnormalities, regardless of the severity or certainty of the outcome. Women carrying a fetus with anatomic disorders with prognostic uncertainty or less severity choose to abort at lower rates. More research is needed to understand decision making processes.     

Prenatal diagnosis by minimally invasive first-trimester transcervical sampling is unreliable

OBJECTIVE: We investigated whether reliable prenatal diagnosis is possible from fetal cells harvested transcervically in first-trimester pregnancies. STUDY DESIGN: Fetal cells were obtained transcervically from 87 women undergoing pregnancy termination. Fetal gender was determined in 51 pregnancies with three different polymerase chain reaction techniques and in 36 pregnancies with fluorescent in situ hybridization. In known male pregnancies the number of male fetal cells present was also determined. RESULTS: Polymerase chain reaction detected male deoxyribonucleic acid in up to 79% of cases in male pregnancies and up to 45% of cases in female pregnancies. Fetal gender was correctly predicted in up to 72% of cases with fluorescent in situ hybridization. However, fetal cells were identified in < 40% of informative male pregnancies and were present in low numbers-0.7% to 3.4% in swabs and 4.4% to 24.8% in flushes. CONCLUSION: The use of fetal cells obtained by minimally invasive first-trimester transcervical sampling is unreliable for prenatal diagnosis.     

The initial Australian experience of technetium-99M sestamibi scintimammography: a complementary test in the management of breast cancer

Current prenatal diagnosis relies on invasive methods such as amniocentesis and chorionic villus sampling. Because these methods carry a low, but finite risk of pregnancy loss, noninvasive genetic screening techniques are the focus of intense research. Isolating fetal cells from maternal blood for genetic analysis is the least invasive method currently being investigated. We discuss the various methods that have been used to isolate these cells. Nucleated red blood cells have emerged as the ideal fetal cell type. This is because they have the DNA material necessary for genetic analysis, they are consistently present in maternal blood, they can be easily identified based on their morphology, and they have a definite gestational life span.     

Molecular, cytogenetic, and clinical characterisation of six XX males including one prenatal diagnosis

Cytogenetic analysis, fluorescent in situ hybridisation (FISH), and molecular amplification have been used to characterise the transfer of Yp fragments to Xp22.3 in six XX males. PCR amplification of the genes SRY, RPS4Y, ZFY, AMELY, KALY, and DAZ and of several other markers along the Y chromosome short and long arms indicated the presence of two different breakpoints in the Y fragment. However, the clinical features were very similar in five of the cases, showing a male phenotype with small testes, testicular atrophy, and azoospermia. All these patients have normal intelligence and a stature within the normal male range. In the remaining case, the diagnosis was made prenatally in a fetus with male genitalia detected by ultrasound and a 46,XX karyotype in amniocytes and fetal blood. Molecular analysis of fetal DNA showed the presence of the SRY gene. FISH techniques also showed Y chromosomal DNA on Xp22.3 in metaphases of placental cells. To our knowledge, this is the second molecular prenatal diagnosis reported of an XX male.     

Prenatal diagnosis and management of fetal hydrocephaly and lissencephaly

Two cases of prenatal diagnosis of lissencephaly are presented in the context of a series of 118 cases of prenatally diagnosed hydrocephalus. Within this series there was one case of Walker-Warburg syndrome and another of Miller-Dieker syndrome. It is stressed that the cases reported here of ventriculomegaly diagnosed in utero show a very different outcome from those in published studies of fetal hydrocephalus which only deal with patients in whom the diagnosis was determined after birth. In those postnatal series there is a considerable selection bias, and the fate of the fetuses reported here was much worse than in postnatal series. Of the 118 fetuses 6 had fetal infections, 6 had chromosomal abnormalities, 26 had associated spina bifida, 64 fetuses had associated other anomalies, and only 28 had isolated hydrocephalus. Although it is difficult to determine the prognosis individually after prenatal diagnosis of ventriculomegaly, the data presented here may be helpful in counseling parents prenatally. The counseling should be performed with the collaboration of obstetricians, pediatricians, surgeons, and geneticists.     

Prenatal diagnosis of congenital cardiomyopathies. Current reality in the south of the country

In order to make an actual perspective about prenatal diagnosis of congenital heart disease in the area of influence of our department, a prospective study including 948 fetus and 185 newborn was done, 348 fetus and 20 newborn evaluated during 1993 (group I) and the remaining during 1994 (group II). In both groups indications for fetal echocardiography were mainly maternal (18%) and familiar (14%) factors, but occurrence of CHD were respectively 2% and 0% for them. Fetal factors for echocardiography account for 7%, namely arrhythmias (7%) and obstetric suspicion of CHD (6%), but occurrence of CHD was respectively 13% and 32% for group I and 36% and 48% for group II. In the newborn with serious CHD, risk factors could be identified in 30% in group I and 36% in group II, being respectively 15% and 7% referred for fetal echocardiography. It is concluded that although a rise in the number of fetus evaluated and a better obstetric accuracy have occurred, the rate of prenatal diagnosis of CHD is still very low, pointing to necessity of continuing our actual policy of teaching and spreading this area, specially in the primary health care units.     

Revascularization of internal iliac arteries during aortoiliac surgery: a multicenter study

Prenatal screening for fetal abnormalities in an accepted part of modern obstetric management. Improvements on current screening procedures need to address increased diagnostic efficacy and earlier diagnosis. This study evaluates diagnostic efficacy of PAPP-A and F beta-hCG in the detection of first trimester pregnancy abnormalities, including Down syndrome (DS). Of 731 pregnant volunteers, obtained from a mature age population undergoing chorionic villus sampling (CVS), 17 DS and 11 compromised (six numerical (excluding sex chromosome) aneuploidies, five spontaneously failed) pregnancies were detected. Application of an algorithm, which combines PAPP-A and F beta-hCG levels with material age, detected 66.6 per cent of DS pregnancies for a five per cent false positive rate. Similarly, for a 1-2 per cent recall rate, 72.2 per cent of compromised pregnancies were detected. This report supports the notion that prenatal screening at 9-12 weeks of pregnancy is achievable with PAPP-A and F beta hCG quantitation. Whereas mid-gestational screening targetted the detection of fetal abnormalities, screening earlier in pregnancy will detect other pregnancy-related abnormalities, in addition to aneuploidy.     

Severe obesity as an explanatory factor for the black/white difference in stage at diagnosis of breast cancer

Black women with breast cancer are less likely than white women to be diagnosed while their disease is still at a localized stage. Racial differences in the prevalence of obesity in the United States have also been documented. This study was undertaken to determine the extent to which the observed racial difference in stage at diagnosis of breast cancer could be explained by racial differences in obesity, specifically severe obesity. This was a population-based, retrospective study of 145 black women and 177 white women in Connecticut who were diagnosed with breast cancer between January 1987 and March 1989. Severe obesity was associated with both race and stage at diagnosis: Black women were significantly more likely than white women to be severely obese (26% vs. 7%, respectively), and severe obesity was significantly associated with diagnosis at TNM stage II or greater (multivariate-adjusted odds ratio = 3.10, 95% confidence interval (CI) 1.28-7.52). Adjustment for severe obesity in a logistic regression model reduced the risk of later stage at diagnosis in blacks relative to whites by 33%, from an odds ratio of 1.98 (95% CI 1.22-3.19) to one of 1.66 (95% CI 1.01-2.73). The higher prevalence of severe obesity among black women may play an important role in explaining their relative disadvantage in stage at diagnosis of breast cancer.