Mosaicism and accuracy of prenatal cytogenetic diagnoses after chorionic villus sampling and placental biopsies

Discrepant chromosome findings in placenta and fetus (false negative and false positive) after chorionic villus sampling (CVS) are mainly due to confined mosaicism. Non-mosaic normal or abnormal chromosome counts after direct preparation and culture nearly always correctly reflect the fetal chromosome constitution. False-negative results have almost exclusively been restricted to cytotrophoblast cells not representing a fetal chromosome abnormality. Diagnosis of placental mosaicism definitely requires an adequate follow-up by amniocentesis, fetal blood sampling, or sonography before a pregnancy is terminated. When direct preparations and cultured cells are used for cytogenetic diagnoses and placental mosaicism is not taken as proof for a chromosomal abnormality in the fetus, CVS is an accurate diagnostic tool.     

Prenatal diagnosis of purine nucleoside phosphorylase deficiency in the first and second trimesters of pregnancy

Prenatal diagnosis was performed in two successive pregnancies of a mother with a previous child with purine nucleoside phosphorylase (PNP) deficiency. In one pregnancy, an affected fetus was diagnosed in the 18th week of gestation after the demonstration of PNP deficiency in cultured amniotic fluid cells. Also an abnormal purine nucleoside profile was found in the amniotic fluid. The diagnosis of an affected fetus was confirmed by the analysis of cultured fetal skin fibroblasts and placental villi. The complete deficiency of PNP activity in placental villi confirms that the prenatal diagnosis of this disorder is possible by the direct investigation of chorionic villi. In the subsequent pregnancy, a heterozygous fetus was predicted in the tenth week of pregnancy by using chorionic villi.     

Management of prenatally detected trisomy 8 mosaicism

We report on ten pregnancies with trisomy 8 mosaicism. Nine cases were prenatally detected in chorionic villi (n=6), amniotic fluid (AF) cells (n=2) or fetal blood (FB) lymphocytes (n=1). Follow-up laboratory investigations showed confined placental mosaicism (CPM) or pseudomosaicism in eight cases. In one case with ultrasound abnormalities, trisomy 8 mosaicism was detected in FB cells although cultured AF cells showed normal cells only. Another case of mosaic trisomy 8 was prenatally missed; cytogenetic analysis of short-term cultured villi revealed a normal male karyotype, while postnatally, trisomy 8 mosaicism was detected in peripheral blood lymphocytes and skin fibroblasts of the affected child. These findings indicate the difficulties in the prenatal diagnosis of trisomy 8 mosaicism. When found in chorionic villi, it mostly represented CPM, while in a case of true fetal trisomy 8 mosaicism, the cytotrophoblast cells showed a normal karyotype. So, the cytotrophoblast compartment of chorionic villi is a poor indicator of the presence or absence of fetal trisomy 8 mosaicism. Follow-up investigations including amniocentesis and especially fetal blood sampling are required to come to a definite prenatal diagnosis of trisomy 8 mosaicism. Copyright © 2001 John Wiley & Sons, Ltd.     

Resorbed co-twin as an explanation for discrepant chorionic villus results: Non-mosaic 47,XX, +16 in villi (direct and culture) with normal (46,XX) amniotic fluid and neonatal blood

Non-mosaic trisomy 16 was observed in chorionic villus cytotrophoblasts (direct) as well as cultured mesenchymal core cells derived from the pregnancy of a 38-year-old woman. Chromosome preparations from amniotic fluid and neonatal cultures (cord blood) were 46,XX. Normal fetal growth as determined by serial ultrasound examinations occurred throughout the pregnancy, which resulted in a healthy 2724 g female. Multiple biopsies taken from the umbilical cord, placental cotyledons, and fetal membranes were 46,XX. However, a placental nodule and three of six cultures initiated from membranes (amnion and chorion) showed 46,XX/47,XX, + 16 mosaicism. We propose that the trisomy 16 cells arose from residual villi derived from a trisomic cotwin that never developed. This case further demonstrates that normal fetal growth may presage normal outcome irrespective of cytogenetic findings in cytotrophoblasts (direct) and cultured mesenchymal core cells.     

Apparent non-mosaic trisomy 16 in chorionic villi: Diagnostic dilemma or clinically significant finding?

A case is presented in which apparent non-mosaic trisomy 16 was found in chorionic villi (direct and culture) obtained from a patient undergoing first-trimester prenatal diagnosis. The fetal karyotype subsequently was determined to be 46,XX by follow-up amniocentesis. Serial ultrasonographic examinations revealed placental sonolucencies and intrauterine growth retardation. At 37 weeks, a small-for-gestational-age female was delivered by Caesarean section for fetal distress. Postnatal cytogenetic studies revealed a normal female karyotype in cord blood and mosaic trisomy 16 in plaental tissues. These findings suggest that in cases where aneuploidy is confined to placental tissues, it may have biological significance, as evidenced by the apparent placental dysfunction and poor fetal growth in this case.     

The safety of fetoscopy: (I). Effect of umbilical vessel puncture on the fetal heart rate and cord histology

The fetal heart rate (FHR) was continuously monitored during 42 umbilical vessel punctures performed at the placental insertion of the cord in 24 diagnostic fetoscopies in which pure fetal blood was obtained. In only one patient did a deceleration first appear during puncture and aspiration of fetal blood. In two patients decelerations preceded fetoscopy and in two others they began during the fetoscopy but before puncture of an umbilical vessel. In 19 patients, the FHR did not change at all during the procedure. Fetal haemorrhage after sampling was either absent or minimal. Six pregnancies were terminated because a positive diagnosis had been made and 18 healthy babies were born. Umbilical cords were examined after 7 terminations of pregnancy and after 6 deliveries. In the former group the puncture could just be seen with the naked eye and the needle track was demonstrated histologically in 6. No traces of the puncture or other abnormalities were found in the cords after delivery. Fetal blood sampling from umbilical cord vessels, particularly at the placental insertion of the cord, is the technique of choice since pure fetal blood can be obtained without increasing the risk of fetoscopy.     

Pure fetal blood samples obtained by cordocentesis: Technical aspects of 322 cases

Three hundred and twenty-two percutaneous umbilical blood samplings were performed over 4 years in our prenatal diagnostic centre. A 3·5 MHz sector ultrasound transducer was used to guide a 22·5-gauge needle under local anaesthesia. Sampling was performed fcir rapid fetal karyotyping (within 72 h) in 120 cases, for diagnosis of fetal toxoplasmosis in 133 cases, for determination of the severity of Rh immunization in 15 cases, and for diagnosis of congenital rubella in 4 cases. Pure fetal blood was obtained in 98·7 per cent of the cases after two attempts. The approach to the cord was either transamniotic or trans-piacental. Puncturing was preferentially done at the placental insertion of the cord (72·2 per cent of the cases) and the mean blood sample volume was 3·5 ml. The rate of fetal death in utero was 1 9 per cent, including two cases of amnionitis, one trisomy 18, and one severe bradycardia. The failures were due to sampling at an early stage of pregnancy (before gestation week 18), to maternal obesity, oligohydramnios, and the inexperience of the operator.     

Identification of a case of maternal uniparental disomy of chromosome 10 associated with confined placental mosaicism

We report a case of maternal uniparental disomy of chromosome 10 discovered after chorionic villus sampling (CVS). Direct preparations revealed mosaic trisomy 10, while cultured CVS cells, as well as amniotic fluid cells, showed only a normal 46,XY complement. DNA analysis using microsatellite markers showed both chromosomes 10 to have been inherited from the mother. The pregnancy was complicated by polyhydramnios. A phenotypically normal male infant of appropriate size was delivered by Caesarean section at 41 weeks' gestation. Since only the direct preparations showed trisomy 10, this case illustrates the importance of CVS direct preparations in the detection of pregnancies at risk of uniparental disomy (UPD). Although the increased frequency of confined placental mosaicism (CPM) diagnosed when direct preparations are performed has been viewed negatively, identification of both CPM and UPD may have biological and clinical significance for a pregnancy. Even though only a single case of maternal disomy 10 is reported here, the apparently normal phenotype provides evidence that there are no major imprinted loci on chromosome 10 that affect in utero growth and development. However, other potential effects such as mental retardation will require long-term follow-up of this as well as additional cases.     

Transabdominal fine needle biopsy from chorionic villi in the first trimester

A technique for sampling first trimester chorionic villi for prenatal diagnosis by transabdominal fine needle biopsy is described. Specimens of chorionic villi were obtained from 49 out of 58 women, a success rate of 84.5 per cent. No fetal or maternal complications were demonstrated in the period before abortion. The procedure is useful for obtaining fetal tissue for culturing, DNA analysis and direct chromosome analysis.     

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. Copyright © 2001 John Wiley & Sons, Ltd.     

Fetoscopy and fetal blood sampling in the management of a twin pregnancy with 45,X/46,XX amniotic fluid cell mosaicism and a suspected fluid sampling error

A 37 year-old woman with a twin pregnancy underwent amniocentesis to exclude fetal chromosome abnormality. The results indicated that both fetuses were mosaics, with 45,X and 46,XX, cell lines. Since it was suspected from the ultrasound scan that the twins were dizygotic, the result was questioned. Fetoscopy and fetal blood sampling were performed and karyotyping the fetal lymphocytes confirmed that one twin was indeed a mosaic, 45,X/ 46,XX, but the other had a normal male chromosome complement. The pregnancy resulted in the birth of a phenotypically normal girl, in whom the 45,X/46,XX mosaicism was confirmed, and a normal boy.     

A fetus with a chromosome 13 ring and placenta with chromosome 13 rod/ring mosaicism

A fetus was identified by prenatal cytogenetic diagnosis as having a karyotype 46,XY,r(13) (p11q13). Termination of the pregnancy yielded a severely malformed fetus. Fetal abnormalities included anencephaly, imperforate anus and urethral meatus, severe talipes, syndactyly, cardiac defects and other anomalies. Confirmatory studies on cultured placental villi cells indicated a second cell line, 46,XY, −13,+ 13qter→cen::13ql3→qter. This cell line was not detectable in cells derived from the fetus despite extensive studies. It seems likely that the two cell lines arose simultaneously with selection favouring the 46,XY,r(13) line. How the chromosome rearrangements may have arisen is discussed. We are unaware of other cases where a cell line identifiable by a chromosome abnormality appeared to be confined to placental tissue. However, studies on placental tissue may be helpful in understanding the origin of other unbalanced de novo rearrangements.     

Transabdominal chorionic villi sampling for first trimester fetal diagnosis. First 26 pregnancies followed to term

A consecutive series of 26 women followed to term after first trimester transabdominal chorionic villi sampling is presented. The clinical application of transabdominal chorionic villi sampling (TA-CVS) seems to have certain advantages, especially from the patients' point of view, but also in regard to successful sampling and to the complication ratio. The results in this clinical trial revealed no cases of abortions, no signs of placental damage and no cases of vaginal bleeding or infections.     

Mosaicism for trisomy 12: Four cases with varying outcomes

Trisomy 12 observed in chorionic villus sampling (CVS) may reflect generalized mosaicism or indicate mosaicism confined to only the placenta. In this report, four cases of trisomy 12 observed in CVS or cultured placental biopsies with varying outcomes are presented. Seven dinucleotide repeat polymorphisms for chromosome 12 were used to determine the chromosome 12 origins in the fetus or child and to delineate the mechanism(s) that gave rise to the trisomy. In two cases (cases A and C), the mosaicism was confined to the placenta, resulting in normal liveborns. Although, in one case, the molecular results suggested an apparent duplication of one paternal chromosome 12 in the placenta, normal biparental inheritance was found in the diploid fetal cell line in both cases. In two other cases (cases B and D), trisomy 12 was observed in both extraembryonic and fetal tissues. In one of these pregnancies, a child was born by Caesarean section at 37 weeks because of intrauterine growth retardation and oligohydramnios, and resulted in neonatal death. Molecular markers and fluorescence in situ hybridization (FISH) revealed low-level trisomy 12 mosaicism in the spleen. In the fourth case, fetal abnormalities were detected on ultrasound and low-level trisomy 12 mosaicism was observed in amniotic fluid cells using conventional cytogenetics and FISH. Molecular markers revealed a maternal meiosis I non-disjunction of chromosome 12 in DNA from a cultured placental biopsy. Although predicting the outcomes of pregnancies involving confined placental mosaicism remains difficult, molecular techniques are valuable tools for distinguishing uniparental from biparental disomy and mechanisms of mosaicism.     

Fetoplacental discrepancy involving structural abnormalities of chromosome 8 detected by prenatal diagnosis

We describe the finding of three cell lines involving different structural abnormalities of chromosome 8 detected in a prenatal diagnosis. Chorionic villi sampling (CVS) was performed on a pregnant woman because of advanced maternal age. Semidirect cytogenetic analysis showed a mos46,XX,i(8q)/46,XX,del(8)(p11.2) karyotype, confirmed by fluorescence in situ hybridization (FISH). Amniocentesis was subsequently performed, and the karyotype obtained was 46,XX,dup(8)(p23p11.2). The pregnancy was terminated; pathologic findings included clubfeet, clenched left hand, subcutaneous edema and bilateral hydrocephalus. Molecular studies using chromosome 8 microsatellites performed on parents' blood and fetal tissues revealed a maternal meiotic origin of the inv dup(8p) with deletion of the distal p23 region and duplication of the remaining 8p. We propose a model to explain the cytogenetic findings, which includes a first maternal meiotic error giving rise to a large dicentric isochromosome 8 present in the ovum, a second error in one of the first zygote divisions with misdivision of the dicentric 8 giving rise to a cell line with del(8p) confined to the trophoblast and another cell line with inv dup(8p) confined to the fetal tissue and a third error in the trophoblast giving rise to a further cell line with isochromosome 8q. Copyright © 2003 John Wiley & Sons, Ltd.     

The fetal intrahepatic umbilical vein as an alternative to cord needling for prenatal diagnosis and therapy

Seventy-one fetal blood samplings (FBS) were attempted from the intrahepatic portion of the umbilical vein (IHV) at 18–34 weeks; 54 were attempted primarily and 17 secondarily after a failed attempt at the placental cord insertion. Fetal blood was obtained in 89 per cent of the cases. Intravascular transfusion (IVT) was attempted on 31 occasions and successful in 24 (77 per cent). In all cases of failed sampling or transfusion via the IHV, prenatal diagnosis and/or therapy was accomplished using alternative procedures. On only one occasion was the procedure postponed. There were no losses or neonatal morbidity attributable to the procedure. FBS from the IHV may be considered as an alternative approach to sampling the placental cord insertion. It is recommended in cases where the approach to the placental cord insertion is difficult or hazardous.     

‘False-negative’ and ‘false-positive’ prenatal cytogenetic results due to ‘true’ mosaicism

A 37-year-old gravida was referred for CVS because of advanced maternal age. A trisomy 21 was present in all cells after short-term incubation (direct processing (DP)) and long-term culture. According to our policy, a retap was offered for confirmation of the result during the legally required 3-day waiting period between communication of the result and termination of pregnancy. Unexpectedly all cells after DP showed a normal male chromosome complement. Further investigations revealed mosaicism in trophoblast tissue and a normal karyotype in amniotic fluid cells and fetal blood (50 mitoses each). The parents elected to continue the pregnancy after extensive ultrasound examinations did not show suspicious findings. After the birth of a healthy child, cell cultures from ten different placental sites confirmed mosaicism. Four out of 100 mitoses from a lymphocyte culture showed an additional chromosome 21. The child had no dysmorphic features and the development was normal at the age of 10 weeks. This case demonstrates the restricted validity of prenatal cytogenetic analysis in the presence of true fetal mosaicism. It also stresses the benefit of our policy to offer a retap in cases with abnormal cytogenetic results prior to termination of pregnancy which is considered unnecessary by many cytogeneticists.     

Complete karyotype discrepancy between placental and fetal cells in a case of ring chromosome 18

A case of complete karyotype discrepancy between cultured chorionic villi and amniotic in addition to fetal cells is reported. Ring chromosome 18 and monosomy 18 mosaicism was detected after amniocentesis. The pregnancy was terminated in the 23rd gestational week. Cytogenetic analysis of cultured umbilical cord tissue after termination confirmed the finding of ring chromosome 18/monosomy 18 mosaicism. In cultured umbilical blood lymphocytes monosomic cells 45,-18 were not detected and the karyotype was 46,XY,r(18). In contrast, short-term and long-term cultured chorionic villi showed a normal male karyotype of 46,XY. Ultrasonographic examination revealed amniotic band syndrome and scoliosis in the caudal region of the spine. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis of thalassemia major by fetal blood analysis: Experience with 1000 cases

In this report we have summarized our experience with the prenatal diagnosis of β-thalassemia in 1000 pregnancies followed at least until 12 months after birth. In the majority of these cases, the thalassemia lesion was the nonsense mutation at the codon corresponding to amino acid 39, which produces the hematological phenotype of β-thalassemia. Fetal blood sampling was carried out by placental aspiration, by which a sufficient amount of fetal blood for analysis was obtained in the majority of cases (99 per cent). The fetal mortality associated with fetal blood sampling was 6·3 per cent. Those placental samples contaminated by maternal cells were successfully purified by Ørskov lysis. Fetal blood was analysed by globin chain synthesis on CM–52 columns, which gave reliable results. Two misdiagnoses (0·2 per cent) have been made of which one was due to a non-globin protein co-migrating with the β-chains while the other resulted from a misclassification of the type of thalassemia segregating in the family.     

The vanishing twin: An explanation for discordance between chorionic villus karyotype and fetal phenotype

One ‘erroneous’ diagnosis occurred in 200 first-trimester chorionic villus samples (CVS) analysed. In direct preparations following 24 h incubation as well as in long-term cultures, a 46.XX karyotype was observed in the villi (28 and 25 cells, respectively). At 20 weeks of gestation, labour was induced because of fetal death in utero. An autopsy performed on the fetus revealed a male phenotype. Placenta and fetal tissues were not submitted for cytogenetic studies. The discordant CVS karyotype (46,XX), in view of the male fetal phenotype, prompted further cytogenetic and molecular studies. Chromosome marker studies on the parents' blood and chorionic villi confirmed both maternal and paternal inheritance of chromosomes in the CVS. DNA studies on formalin-fixed skin using a Y-specific probe, DYZ1, confirmed the presence of a Y chromosome in the fetus. The most likely cause of the discrepant CVS karyotype is the presence of an undetected degenerating dizygotic twin.