Prenatal detection of trisomy 9 mosaicism

Chromosomal mosaicism in amniotic fluid cells poses a serious dilemma in prenatal diagnosis since the observation may represent: (1) pseudomosaicism—an inconsequential tissue culture artefact; or (2) true mosaicism—occurring in approximately 0.0 per cent of amniocenteses with a significant impact on pregnancy outcome. Mosaicism for trisomy 9 was observed in an amniotic fluid specimen obtained for advanced maternal age with two cell lines [46,XX (46 per cent)/47,XX, + 9 (54 per cent)] present in each of four culture flasks. Since more than 75 per cent of newborns with trisomy 9 mosaicism have complex cardiac malformations, a fetal echocardiogram was obtained at 20 weeks' gestation and interpreted as normal. A fetal blood sample (22 weeks' gestation) disclosed only a single trisomy 9 cell among the 100 metaphases analysed. However, a second fetal echocardiogram performed at the time of blood sampling suggested a non-specific cardiac anomaly. Fetal autopsy following elective pregnancy termination revealed several malformations including severe micrognathia, persistence of the left superior vena cava, and skeletal anomalies. Cytogenetic studies of cell cultures derived from several fetal tissues demonstrated trisomy 9 ranging from 12 to 24 per cent.     

Detection of fetal cells in maternal blood

We report the detection of fetal cells in the maternal circulation by enzymatic amplification of a single copy gene sequence that was fetal-specific. Fetal HLA-A2-positive cells were sorted from maternal HLA-A2-negative cells by flow cytometry and confirmed by demonstration of a fetal-specific HLA-DR4 sequence. However, this sequence could not be detected in unenriched maternal DNA prepared at 28 and 32 weeks' gestation. The sensitivity of detection was 1 HLA-DR4-positive cell in 10⁵ HLA-DR4-negative cells. We conclude that prenatal diagnosis of paternally inherited autosomal-dominant genetic defects may be possible by selective gene amplification of maternal peripheral blood. However, preliminary enrichment for fetal cells may be necessary.     

Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Fetal tissue involvement in the late infantile type of neuronal ceroid lipofuscinosis

Prenatal diagnosis in a pregnancy at risk for late infantile neuronal ceroid lipofuscinosis (Batten's disease) was undertaken at 17 weeks' gestation by ultrastructural examination of amniotic fluid cells. The presence of curvilinear profiles indicated an affected fetus and the diagnosis was confirmed, after the pregnancy was terminated, by the finding of many typical curvilinear profiles in multiple tissues which included skin, amnion, umbilical vessels, blood, liver, and brain. Comparison between the involved cells in the amniotic fluid and fetal tissues suggests that these cells are probably derived from the periderm, and possibly also from the amnion. The prominent presence of cytosomes in the periderm and intermediate cells of the fetal epidermis and occasionally also in the endothelial cells of the dermis suggests that fetal skin may be a useful alternative site for assessing fetal involvement. Control specimens of the amniotic fluid, fetal skin, amnion, and liver showed no similar cytosomes. However, some control amniotic fluid samples did contain cells with large collections of irregular trilaminar membranes, and these could be open to misinterpretation. It is important that only typical curvilinear profiles are considered as an indication of an affected pregnancy.     

Incidental detection of premature centromere separation in amniocytes associated with a mild form of Roberts syndrome

Premature centromere separation (PCS) was detected in amniocytes after an amniocentesis was done because of markedly elevated maternal serum alpha-fetoprotein values in a healthy primiparous young woman. PCS has been associated with the Roberts-SC phocomelia syndrome (RS). By 23 weeks' gestation, ultrasonic evaluations did not reveal abnormal fetal development. The pregnancy continued and a male infant was born with mild manifestations of RS. PCS was confirmed in cord blood lymphocytes. This case illustrates that PCS, when detected in amniotic fluid cell cultures, requires a thorough evaluation.     

First-trimester prenatal diagnosis of Sanfilippo C disease

At 10 weeks' gestation, chorionic villus biopsy was obtained from a woman at risk for Sanfilippo type C disease. Acetyl-CoA: α-glucosaminide N-acetyltransferase activity was markedly deficient. The pregnancy was terminated at 12 weeks' gestation and follow-up study on fetal fibroblasts confirmed the diagnosis.     

Maternal age-specific risks for trisomies at 9—14 weeks' gestation

This study provides data on the incidence of fetal trisomies 21, 18, and 13 at 9–14 weeks' gestation in women aged 35–45 years and estimates of maternal age-specific risks in women aged 20–45 years. Our data from 5814 singleton pregnancies undergoing first-trimester karyotyping for the sole indication of maternal age ⩾ 35 years were combined with those from two previous reports and the incidence of the trisomies was calculated from a total of 15 793 pregnancies. Comparison of incidences at 9–14 weeks' gestation with published data at 15–20 weeks' gestation and in livebirths demonstrated that at birth the maternal age-specific incidence of trisomy 21 is 33 per cent lower than at 15–20 weeks' gestation and 54 per cent lower than at 9–14 weeks' gestation. Furthermore, the relative frequency of trisomies 18 and 13 decreases from 30 per cent at 9–14 weeks to 22 per cent at 15–20 weeks and 14 per cent at birth.     

Umbilical cord haematoma as a complication of intrauterine intravascular blood transfusion

Between October 1985 and February 1989, 49 ultrasound-guided intravascular fetal blood transfusions were performed in 16 patients (14 with rhesus (Rh) isoimmunization, 2 with non-immunologic hydrops fetalis (NIHF)). As an intra-operative complication, perivascular haematoma of the cord occurred in three patients (7 per cent). In two cases, fetal bradycardia necessitated delivery by Caesarean section at 30 and 32 weeks' gestation, respectively. In the third case, fetal bradycardia developed during transfusion, at 31 weeks' gestation, but normalized within 3 min. The baby was delivered as planned at 36 weeks of gestation, after another transfusion at 34 weeks. Dislodgement of the needle tip into perivascular tissue, caused by sudden fetal or maternal movements, is the reason for this complication. The haematoma develops as a result of delayed recognition and continuous transfusion into Wharton' s jelly. Cord haematoma may be diagnosed in time by continuous ultrasound imaging, as illustrated in case 3. To minimize the risk of needle dislodgement during transfusion, sedation of the mother and complete immobilization of the fetus by injecting a short-acting muscle relaxant into the umbilical vessel are recommended.     

Prenatal ultrasonographic diagnosis of fetal cerebral ventriculitis associated with asymptomatic maternal cytomegalovirus infection

Cytomegalovirus is the most common cause of congenital viral infection. In utero infection is usually suspected in patients with growth-retarded fetuses or when maternal illness precipitates serological investigations. A case is presented where routine ultrasound examination at 30 weeks' gestation in an asymptomatic patient demonstrated mild fetal ventriculomegaly. Transvaginal ultrasound enabled the visualization of intraventricular adhesions and small periventricular cysts. The suspected diagnosis of in utero cytomegalovirus infection was confirmed by the presence of IgM antibodies in fetal blood and subsequently by isolation of the virus from the infant's urine. The presence of mild fetal ventriculomegaly should prompt transvaginal brain imaging.     

Practical experience using transabdominal chorionic villus biopsies taken after 16 weeks' gestation for rapid prenatal diagnosis of chromosomal abnormalities

Placental biopsy was performed on 81 patients at greater than 16 weeks' gestation. The major indication for such biopsies was an increased risk of chromosomal abnormality because of either abnormal ultrasound findings or late presentations for advanced maternal age. Six abnormal karyotypes resulting in elective termination were found. The use of rapid karyotyping by this procedure as an alternative to amniocentesis or fetal blood sampling is discussed.     

Prenatal diagnosis of mosaicism for triploidy and trisomy 13

Mosaicism for trisomy 13 and triploidy was detected by amniocentesis performed at 18 weeks' gestation because of fetal anomalies. Pregnancy continued and a live-born male was delivered vaginally at 37 weeks. The infant had features common to both trisomy 13 and triploidy: intrauterine growth retardation (IUGR), small abnormal ears, cleft palate, and a small jaw. In addition, he had complete cutaneous syndactyly of fingers 3 and 4 and partial syndactyly of the toes, as seen in triploidy. Mixoploidy for trisomy 13 and triploidy was confirmed postnatally in blood, skin, and placenta. Examination of chromosome heteromorphisms and DNA markers suggested the presence of two maternal contributions in the triploid cell line. In addition, the extra chromosome 13 in the trisomic cell line was derived from the mother. Copyright © 2001 John Wiley & Sons, Ltd.     

Tumour-associated antigens in maternal and fetal blood

Normal levels of cancer-associated antigen (CA) 19-9, neurone-specific enolase (NSE), cancer-associated antigen (CA) 125, and mucin-like carcinoma-associated antigen (MCA) during pregnancy were determined in 87 mothers and fetuses, using a solid-phase sandwich enzyme immunoassay. CA 19-9 concentrations were higher in the fetuses, whereas the other three tumour-associated antigen levels were higher in the mothers. Only fetal NSE and MCA levels were positively correlated with those in maternal serum. Contrary to adult samples, no difference was demonstrated between male and female fetal levels of CA 125. MCA was the only maternal marker that increased significantly with gestational age between 20 and 34 weeks' pregnancy.     

Maternal platelet size as a marker for fetal trisomies 18 and 13

Maternal and fetal platelet size and glycoprotein expression were measured in 14 pregnancies complicated by fetal aneuploidy between 20 and 24 weeks' gestation. Flow cytometry was used to determine platelet size and surface glycoprotein Ib (GPIb) and GPIIIa expression both before and after stimulation with adenosine diphosphate (ADP). The data were compared with results obtained from 35 normal paired maternal and fetal controls. In fetuses affected with trisomies 18 and 13, but not trisomy 21, the maternal and fetal platelet sizes were significantly higher than those of the normal controls. Furthermore, the increase in fetal platelet size was significantly associated with the increase in maternal platelet size. Increase in maternal platelet size may be of potential value as a marker for fetal trisomies 18 and 13.     

Early amniocentesis and fetal nuchal translucency in women requesting karyotyping for advanced maternal age

Fetal nuchal translucency was measured at 11–14 weeks' gestation in 97 pregnancies referred for early amniocentesis for advanced maternal age. The nuchal translucency was abnormal in 11 fetuses and the fetal karyotype was abnormal in five of these 11 cases. The karyotype was normal in 86 cases with normal nuchal translucency. The culture failure and miscarriage rates associated with early amniocentesis were 3·3 per cent and 2·2 per cent respectively. Amniotic fluid leakage occurred in 6 per cent of cases. In women requesting fetal karyotyping for advanced maternal age without additional biochemical screening, fetal nuchal translucency should be measured at 11–14 weeks. If the nuchal thickness is ≥ 3 mm, a first-trimester diagnostic procedure is indicated; however, if it is <3 mm, amniocentesis should be delayed until 16 weeks' gestation.     

Detection and isolation of fetal cells from maternal blood using the flourescence-activated cell sorter (FACS)

The presence of fetal cells in the maternal circulation during pregnancy has been suggested by repeated observations of small numbers of cells containing Y chromatin or a Y chromosome in the blood of pregnant women. With the fluorescence-activitated cell sorter (FACS), we have used antibodies to a paternal cell surface (HLA) antigen, not present in the mother, to select fetal cells from the lymphocyte fractions of a series of maternal blood samples, collected as early as 15 weeks of gestation. These sorted cells have been examined for a second paternal genetic marker, Y chromatin. Y chromatin-containing cells were found among the sorted cells from prenatal maternal blood specimens in 8 pregnancies subsequently producing male infants whose lymphocytes reacted with the same antibodies to paternal antigen used for sorting with the FACS. In each of 17 pregnancies resulting in male infants who failed to inherit the antigen detected by the antibodies used for cell sorting, Y chromatin-containing cells were not found prenatally. The use of two paternal genetic markers, a cell surface antigen and nuclear Y chromatin, to identify fetal cells in maternal blood permits us to conclude that these cells are present in the mother's circulation, as early as 15 weeks gestation. Further development of the techniques reported here could lead to widespread screening of maternal blood samples during pregnancy for detection of fetal genetic abnormalities.     

Trisomy 16 fetus surviving into the second trimester

A 27-year-old gravida 4, para 3 was found to have anhydramnios at 14 weeks' gestation following a size/date discrepancy noted at her routine prenatal visit. A detailed ultrasound revealed multiple fetal anomalies including congenital heart defect, chest hypoplasia, and bilateral dysplastic kidneys. Karyotype revealed trisomy 16 in 15/15 cells from a tissue specimen obtained from the fetal cord insertion site following elective pregnancy termination.     

Prenatal diagnosis of a familial complex chromosomal rearrangement involving chromosomes 5, 10, 16 and 18

We report one case of a familial complex chromosomal rearrangement (CCR) involving four different chromosomes 5, 10, 16 and 18. The CCR was detected prenatally at 20 weeks' gestation because of advanced maternal age and history of recurrent miscarriages. Cytogenetic analysis of cultured amniotic fluid cells with GTG banding showed a 46,XX,t(5;16;10;18)(q13;q22;q11.2;q21) karyotype. Parental cytogenetic study revealed that the mother has the same CCR. RBG banding, high-resolution banding and fluorescence in situ hybridization (FISH) were used to characterize further and confirm the conventional banding data. No physical abnormalities were shown in the targeted fetal ultrasonography examination. The parents decided to continue the pregnancy. The child is now 2 years old and has neither congenital anomalies nor evidence of delayed psychomotor development. The fetal targeted ultrasound and FISH analysis helped us reassure fetal status. Copyright © 2002 John Wiley & Sons, Ltd.     

Prenatal diagnosis of autosomal dominant microcephaly and postnatal evaluation with magnetic resonance imaging

A case of fetal autosomal dominant microcephaly was prenatally diagnosed with ultrasonography in a woman with previously undiagnosed microcephaly. At the time of initial ultrasonographic assessment, the mother was identified to have a markedly small cranium, consistent with maternal microcephaly. The ultrasonographic examination showed the fetal head size to be four standard deviations below the mean for gestational age. Gesta-tional dating from the other biometric parameters and from the last menstrual period was consistent with 31 weeks' gestation. Neurosonographic evaluation of the fetus revealed no obvious structural abnormalities. Serial ultrasonographic examinations at 35 and 38 weeks' gestation showed no changes in the fetal head size. A 2·64 kg male fetus was delivered at term. Neonatal assessment showed the fetal head circumference to be less than the second percentile for gestational age. Neurologic assessment of the neonate with magnetic resonance imaging showed abnormal development of the brain, with small cerebellar and cerebral hemispheres, and pachygyria. These images are compared with the magnetic resonance images of the mother. Our findings of maternal and fetal microcephaly are consistent with autosomal dominant microcephaly. To our knowledge, this is the first report of the prenatal diagnosis of autosomal dominant microcephaly.     

Severe early-onset fetal growth restriction: What do we tell the prospective parents?

Fetal growth restriction (FGR) is a common complication of pregnancy, associated with higher risk of perinatal mortality and adverse health and developmental outcomes for surviving infants. True FGR relates to a pathological restriction of fetal growth resulting from complex interactions between maternal, placental, fetal, and environmental factors. Early-onset FGR (onset <32 weeks' gestation) is often first suspected at routine mid-trimester sonographic assessment of fetal morphology, or identified as part of the placental syndrome, commonly maternal pre-eclampsia. Prenatal investigations may identify the cause of FGR. Timing of delivery is guided by serial sonographic surveillance of fetal growth and well-being and maternal condition, balancing the risk of stillbirth with the benefits of advancing gestation. This is particularly pertinent to severe early-onset FGR, a leading iatrogenic cause of very preterm birth. Prognosis is largely determined by the severity of FGR and its causes, gestation at birth, and birthweight. Pregnancy termination may be considered. Antenatal care and delivery in a tertiary center, provided by a multi-disciplinary team with expertise in managing high-risk pregnancies, are imperative to optimizing outcomes.