Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism

About 2 per cent of specimens from chorionic villus sampling (CVS) analysed either on direct preparation of cytotrophoblast cells or afterculture of mesenchymal stroma reveal confined placental mosaicism (CPM), most commonly involving chromosomal trisomy. A significantly higher rate of prenatal loss (22 per cent) as well as the presence of intrauterine growth retardation (IUGR) has been reported among pregnancies with CPM. To evaluate more precisely the effect of these aneuploid cell lines confined to the placenta on intrauterine fetal growth and fetal survival, we have studied 34 term placentae from pregnancies with CPM diagnosed on CVS and confirmed identical mosaicism in 17 of these placentae. There was a direct correlation between a high number of aneuploid cells present at CVS and a high likelihood of their detection in term placenta. Also, the proportion of aneuploid cells in the mosaic term placentae correlated with that observed in CVS specimens. Among 17 gestations with confirmed CPM at delivery, there were six cases of IUGR identified, five in liveborns and one associated with intrauterine death.     

The significance of trisomy 7 mosaicism in chorionic villus cultures

Two cases of mosaic trisomy 7 confined to the cultured cells and not found in direct preparation were detected from 200 consecutive first-trimester chorionic villus samples (CVS) analysed. The mosaicism was similar in the two cases, but the pregnancy outcome was different. In both cases, the direct metaphases from the CVS were 46, XY. Culture metaphases were mos46,XY/47,XY, + 7; the trisomy 7 was seen in 34 per cent of cells from case 1 and 53 per cent from case 2. A sonogram at 15¹/₂ weeks revealed fetal death in utero in case 1, and the patient declined amniocentesis. The fetal tissue failed to grow in culture, but the placental cultured cells were 47,XY, + 7 in 28 (100 per cent) cells analysed. In the second case, all the amniotic fluid cells were 46,XY and the pregnancy resulted in a normal male with a 46,XY karyotype in the cord blood and foreskin fibroblast cultures. The term placenta was mosaic with 13/163 (8 per cent) trisomy 7 cells. Extensive cytogenetic studies on the placenta for the first time confirmed trisomy 7 mosaicism confined to the villus cultures.     

Confined placental mosaicism in CVS and pregnancy outcome

The perinatal outcome of 26 patients with confined placental mosaicism (CPM) detected in chorionic villus sampling (CVS) who wished to continue their pregnancies was compared with that of two controls per patient matched for age and parity (n=52). There were no significant differences in birth weight or gestational age at delivery between patients with CPM and controls. There were no cases of intrauterine growth retardation (IUGR) in the CPM patients as compared with two (2/52, 3·8 per cent) in the control group (P>0·05). There was no significant increase in fetal loss between the study group (1/26, 3·6 per cent) and the controls (1/52, 1·9 per cent) (P>0·05).     

The European collaborative study on mosaicism in chorionic villus sampling: Data from 1986 to 1987

Data on a total of 11 855 diagnostic chorionic villus samples obtained in the years 1986 and 1987 were compiled from a questionnaire filled in by 36 European cytogenetic centres. Mosaicism was reported in 141 cases. The cytogenetic findings were followed by induced abortion in 24 cases. Spontaneous abortion was observed in nine mosaic pregnancies, a rate not significantly different from that observed for CVS in total. Mosaicism was found in 1.2 per cent of analyses by direct analysis/short-term culture, in contrast to the 0.6 per cent found after long-term culture. Evidence for fetal non-mosaicism was found in 99 of the 141 cases. The finding of mosaicism in first-trimester CVS should always elicit further analyses, preferably after amniocentesis, to substantiate the suspected fetal chromosome aberration.     

Is placental mosaicism associated with poor perinatal outcome?

In 37 pregnancies with placental mosaicism detected at first-trimester chorionic villus sampling in 2000 pregnancies, two pregnancies (5.4 per cent) ended in a spontaneous abortion at 15 weeks (46,XX/47,XX, + 16) and in a perinatal loss in a term pregnancy (46,XY/ 47,XY, +10), respectively. The outcome of the other 35 pregnancies was normal. The total pregnancy loss rate before 28 weeks in the first 1000 pregnancies was previously reported to be 3.6 per cent.     

Chorionic mosaicism: Association with fetal loss but not with adverse perinatal outcome

Cytogenetic data from the United States NICHD collaborative study of chorionic villus sampling (CVS) were used to evaluate the clinical significance of chorionic mosaicism. The 10 754 patients with normal cytogenetic results were compared wtih 108 patients (1.0 per cent) with placental mosaicism and 181 patients (1.6 per cent) with pseudomosaicism. Of the pregnancies intended to continue, the pregnancy loss rate was significantly greater in patients with placental mosaicism than in the cytogenetically normal cohort (8.6 vs. 3.4 per cent, p <0.05). However, there was no difference in the frequencies of abruptio placenta, preterm labour or delivery, small-for-gestational-age newborns, pregnancy-induced hypertension, or neonates with Apgar scores less than 7.     

Prenatal diagnosis of chromosome mosaicism

The frequency of mosaicism and pseudomosaicism in the prenatal diagnosis of cytogenetic disorders is reported, based on 3000 pregnancies studied in our laboratory. Diagnosis of true mosaicism was only made when an abnomality was detected in two or more independent cultures established from an amniotic fluid sample. On this basis, 0.37 per cent of all cases were diagnosed as true mosaics. 1.07 per cent of all cases had pseudomosaicism involving more than one cell from the same culture with an identical abnormality. 4.13 per cent of cases had a single abnormal cell with an extra chromosome, loss of a sex chromosome (or part of a sex chromosome), or translocation. Details of the outcome and follow-up of cases is given. Particularly problematical were cases where multiple cells from one culture contained an abnormality which could have been clinically significant. A crude estimate of the extent to which true mosaicism might currently be misinterpreted as pseudomosaicism or entirely missed has been made, based on data from the U.S. survey (Hsu and Perlis, in press). It was concluded that even when two, and if necessary a third culture is extensively analysed with an average of 24 cells per culture counted, at least 4.5 per cent of cases of true mosaicism may be completely missed and at least 7 per cent could be misdiagnosed as pseudomosaicism. There is an urgent need for improved laboratory techniques which allow growth of a greater number of cell colonies and therefore a more broadly based analysis. Detailed long term follow-up of prenatally diagnosed mosaics is also essential for assessing the clinical significance of the laboratory findings.     

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.     

Fetal blood sampling and cytogenetic abnormalities

From September 1984 to April 1991, we performed cytogenetic analysis on fetal blood samples from 214 second-and third-trimester pregnancies. One hundred and thirty-four cases were referred to consider the possibility of chromosomal mosaicism following amniocyte studies. The confirmation rate of mosaicism is at 0 per cent (0/9), 1·4 per cent (1/70), and 40 per cent (22/55) for cases of level I, level II, and level III mosaicism, respectively. Four out of 17 cases were positive for the diagnosis of fragile X syndrome. Of 63 cases with abnormal ultrasound findings, blood disorders, or other genetically related clinical conditions, 11 were found to have a chromosome abnormality. Fetal blood sampling is a valuable adjunct to other methods in the prenatal diagnosis of chromosomal mosaicism or pseudomosaicism. It is also useful when rapid cytogenetic diagnosis is desired because of malformations detected in pregnancies at a late gestational age.     

Follow-up and pregnancy outcome after a diagnosis of mosaicism in CVS

In 2103 consecutive diagnostic chorionic villus samples, examined in a 4-year period in our clinical genetics unit, 26 samples (1.2 per cent) presented chromosomal mosaicism in the direct and/or long-term culture preparations. Only once (46,XX/47,XX,+9) was the mosaicism confirmed in the fetus. In the cytogenetic follow-up studies of the remaining 25 pregnancies, in no cases could the aberration be confirmed in amniotic fluid or fetal tissue. One patient requested a termination after the CVS result. Of the remaining 24 pregnancies, four (16.7 per cent) ended in a spontaneous abortion. These findings suggest an association between placental mosaicism and fetal loss.     

An audit of trisomy 16 in man

Sufficient information is now available from the literature to produce an audit of trisomy 16, in a theoretical cohort of 100 000 recognized pregnancies, from gametogenesis to term and onwards. Recent reports of premature separation of chromosome 16 bivalents during maternal meiosis I provide a novel mechanism for generation of this aneuploidy. Most, if not all, errors resulting in recognized mosaic and non-mosaic trisomy 16 pregnancies investigated using polymorphic DNA markers appear to originate at that stage. The incidence of this maternally derived trisomy 16 in the late first trimester is equivalent to 1500 cases in 100 000 recognized pregnancies, a figure which now corresponds very closely to the reinterpreted oogenesis data. Most trisomy 16 pregnancies are lost around 12 weeks' gestation, but of the order of 10 per cent (120–150 in this audit) undergo reduction to disomy, with 30 of these excluding aneuploidy from the fetal cell lineage (trisomic zygote rescue) and continuing into the second trimester. Maternal uniparental disomy (UPD) in one-third of this latter group is associated with loss later in pregnancy or severe intrauterine growth retardation, but can be compatible with a viable pregnancy. Adverse pregnancy outcomes are not restricted to those with UPD. Analysis of reports of confined placental mosaicism for chromosome 16 without associated UPD indicates that the presence of high levels of trisomic cells in the placenta alone consistently produces a more variable inhibition of fetal growth, which may also, in cases, be associated with late pregnancy loss.     

A canadian collaborative study of mosaicism in amniotic fluid cell cultures

Data on chromosomal mosaicism was collected retrospectively on 12 386 amniotic fluid samples cultured over a 10 year period in 14 Canadian centres. Level I mosaicism (a single abnormal cell—excluding single cell monosomy) was encountered in 863 cases (7.1 per cent). Level II mosaicism (multiple cells with the same abnormality in a single flask or colony) was encountered in 138 cases (1.1 per cent). Level III mosaicism (multiple cells distributed over multiple flasks or colonies) was encountered in 34 cases (0.3 per cent). Analysis of the details of these cases allowed five major conclusions to be drawn: (1) Single cell abnormalities should not be taken as an indication of true fetal mosaicism. Only rarely will this interpretation prove to be incorrect. (2) Mosaicism involving multiple cells confined to a single flask should not be regarded as an indication of true fetal mosaicism. Only occasionally will this interpretation prove to be incorrect. (3) Mosaicism involving multiple cells distributed over more than one flask should be regarded as a strong indication of true fetal mosaicism. Sixty per cent will be confirmed by karyotype analysis of the fetus or infant. (4) Mosaicism of the XX/XY type is usually due to maternal cell contamination. Occasionally it can be a female fetus with XY cells from an unknown source. (5) The in situ or colony method of chromosome analysis has no clear advantage over the flask method for either the detection of true fetal mosaicism or for the ability to distinguish true mosaics from false positives.     

Postnatal placental confirmation of trisomy 2 and trisomy 16 detected at chorionic villus sampling: A possible association with intrauterine growth retardation and elevated maternal serum alpha-fetoprotein

Detection of trisomy 2 and trisomy 16 mosaicism through chorionic villus sampling (CVS) is not an infrequent finding. We describe here two cases, one of non-mosaic trisomy 2 and the other of high level mosaicism for trisorny 16. Amniocentesis in both cases demonstrated non-mosaic 46,XY karyotypes. Each pregnancy continued to delivery of liveborn, normal-appearing boys; both pregnancies were complicated by severe intrauterine growth retardation (IUGR). Postnatal studies of placental biopsies in both cases confirmed the original CVS findings, whereas cord blood karyotypes were normal in both boys. Both children have demonstrated adequate catch-up growth.     

Randomized trial comparing first-trimester transcervical chorionic villus sampling and second-trimester amniocentesis

A total of 800 patients were randomized at the 9th to 11th week of pregnancy either for transcervical chorionic villus sampling (CVS) on the day of trial entry or for amniocentesis (AC) at the 16th week. The indication for fetal karyotyping was maternal age in 94 per cent of the cases; the mean maternal age was 39.2 years. An adequate sample was obtained in 98.3 per cent of the cases in the CVS group and in all cases in the AC group. Retesting was indicated in 3.3 per cent of the CVS cases. An abnormal karyotype was found in 6.1 per cent of the CV samples and in 4.5 per cent of the amniotic fluid samples. There was one false-positive chromosome result in both groups. Twelve (3.1 per cent) miscarriages occurred by the 22nd week of pregnancy in the CVS group in pregnancies intended to continue. No difference was seen between the groups for total fetal loss rates. The number of surviving infants in the CVS group was 92.2 per cent and in the AC group 91.7 per cent (rate difference 0.5 per cent (95 per cent confidence interval − 3.3 to 4.3)). In our study, both the diagnostic accuracy and the risk of fetal loss were equal in the CVS and AC groups.     

Confined placental mosaicism in term placentae: Analysis of 125 cases

In order to determine the incidence of confined placental mosaicism (CPM) in term placentae and to show the presence of specific sites and the effect on fetal development, 125 placentae from uneventful pregnancies were analysed by cytogenetic methods. The incidence was at least 4.8 per cent and there were no specific sites on the placenta. Although the number of cases is still too small, we found CPM to be associated with intrauterine growth retardation in six cases.     

Evaluation of transcervical chorionic villus sampling with a completed follow-up of 1550 consecutive pregnancies

Data from 1550 consecutive pregnancies after first-trimester prenatal diagnosis by transcervical chorionic villus sampling (TC-CVS) are presented. The sampling efficacy was 97.8 per cent; the mean amount of collected villus tissue was 23 mg (range 5–100 mg). There were 97 affected fetuses, mainly (73.2 per cent) with a chromosomal abnormality or a male karyotype in carriers of X-linked disease. Pregnancy termination in these and four other women for social reasons resulted in 1449 continuing pregnancies. In these pregnancies, the fetal loss rate up to 28 weeks of gestation was 5.1 per cent with the highest loss rate (3.9 per cent) before 16 weeks. When relating this fetal loss rate to maternal age, this was 6.1 per cent in the advanced maternal age group (⩾36 years) against 3.1 per cent in the younger age group. In 1376 pregnancies continuing beyond 28 weeks, the perinatal mortality rate was 1.1 per cent; the percentage of non-genetic congenital anomalies was 0.9 per cent. The reproductive pattern of women at high genetic risk after CVS followed by pregnancy termination was evaluated. Within 12 months after the first CVS followed by pregnancy termination, 70 percent of women again requested CVS in a subsequent pregnancy.     

Prenatal detection of chromosomal mosaicism

A significant problem associated with cytogenetic prenatal diagnosis is distinguishing between true and pseudomosaicism. This becomes a diagnostic dilemma when fetal mosaicism corresponds with a known clinical entity. True mosaicism reportedly occurs with a frequency of 0·2 per cent and pseudomosaicism in 0·7 per cent to 2·7 per cent of cases. In the past 12 months, our laboratory has completed 522 fetal karyotypes. Nine cases were found to demonstrate mosaicism, 4 true mosaics (0·8 per cent) and 6 pseudomosaics (1·1 per cent). One case demonstrated both true and pseudomosaicism. In all cases of true mosaicism, the pregnancy was continued and karyotypes completed at birth. Our results demonstrate a danger of rigid adherence to the criteria for true and pseudomosaicism in the examination of amniotic fluids. It is suggested that the criteria established for true and pseudomosaicism may not be valid when an aberrant cell line is found in a single flask and when that aberrant cell line is compatible with a known clinical entity due to a chromosome anomaly.     

Increased incidence of cytogenetic abnormalities in chorionic villus samples from pregnancies established by in vitro fertilization and embryo transfer (IVF–ET)

We studied 201 pregnancies that were established by in vitro fertilization and embryo transfer (IVF–ET) and compared the frequency of cytogenetic abnormalities with that found in a large control population matched for indication group (advanced maternal age) and time of sampling. A total of 252 IVF–ET fetuses were cytogenetically analysed by either chorionic villus sampling (CVS; n = 80) or amniocentesis (n = 172). Eleven chromosome abnormalities were found in the CVS group (13·8 per cent); among them, a 45, X/46, X, dic(q11)/46, X, del(Y)(q11) mosaic that was found in an IVF pregnancy established by intracytoplasmic sperm injection (ICSI), four cases of trisomy 21, and three cases of trisomy 7 confined to the placenta. The results indicate a statistically significant three-to five-fold increase in both confined placental abnormalities (P<0·008) and true fetal chromosome anomalies (P<0·04). In the amniocentesis group, identical rates (1·7 per cent) of chromosome abnormalities were found in the IVF–ET and control groups. It is concluded that late first trimester, but not early second trimester, IVF–ET pregnancies are characterized by an increased frequency of cytogenetic abnormalities found at prenatal diagnosis.     

Cytogenetic results from the U.S. collaborative study on CVS

Cytogenetic data are presented for 11 473 chorionic villus sampling (CVS) procedures from nine centres in the U.S. NICHD collaborative study. A successful cytogenetic diagnosis was obtained in 99.7 per cent of cases, with data obtained from the direct method only (26 per cent), culture method only (42 per cent), or a combination of both (32 per cent). A total of 1.1 per cent of patients had a second CVS or amniocentesis procedure for reasons related to the cytogenetic diagnostic procedure, including laboratory failures (27 cases), maternal cell contamination (4 cases), or mosaic or ambiguous cytogenetic results (98 cases). There were no diagnostic errors involving trisomies for chromosomes 21, 18, and 13. For sex chromosome aneuploidies, one patient terminated her pregnancy on the basis of non-mosaic 47,XXX in the direct method prior to the availability of results from cultured cells. Subsequent analysis of the CVS cultures and fetal tissues showed only normal female cells. Other false-positive predictions involving non-mosaic aneuploidies (n = 13) were observed in the direct or culture method, but these cases involved rare aneuploidies: four cases of tetraploidy, two cases of trisomy 7, and one case each of trisomies 3, 8, 11, 15, 16,20, and 22. This indicates that rare aneuploidies observed in the direct or culture method should be subjected to follow-up by amniocentesis. Two cases of unbalanced structural abnormalities detected in the direct method were not confirmed in cultured CVS or amniotic fluid. In addition, one structural rearrangement was misinterpreted as unbalanced from the direct method, leading to pregnancy termination prior to results from cultured cells showing a balanced, inherited translocation. False-negative results (n = 8) were observed only in the direct method, including one non-mosaic fetal abnormality (trisomy 18) detected by the culture method and seven cases of fetal mosaicism (all detected by the culture method). Mosaicism was observed in 0.8 per cent of all cases, while pseudomosaicism (including single trisomic cells) was observed in 1.6 per cent of cases. Mosaicism was observed with equal frequency in the direct and culture methods, but was confirmed as fetal mosaicism more often in cases from the culture method (24 per cent) than in cases from the direct method (10 per cent). The overall rate of maternal cell contamination was 1.8 per cent for the culture method, but there was only one case of incorrect sex prediction due to complete maternal cell contamination which resulted in the birth of a normal male. The rate of maternal cell contamination was significantly higher in samples obtained by the transcervical sampling method (2. 16 per cent) than in samples obtained by the transabdominal method (0.79 per cent). From these data, it is clear that the culture method has a higher degree of diagnostic accuracy than the direct method, which should not be used as the sole diagnostic technique. The direct method can be a useful adjunct to the culture method, in which maternal cell contamination can lead to incorrect sex prediction and potentially to false-negative diagnostic results.     

The dilemma of chromosomal mosaicism in chorionic villus sampling—‘direct’ versus long-term cultures

Chromosomal mosaicism is one of several unanswered dilemmas in first-trimester prenatal diagnosis. We report the course of a pregnancy in which a normal karyotype was detected on direct CVS preparation and fetal blood, 100 per cent trisomy 21 in one long-term CVS culture, and low-rate trisomy 21 mosaicism in a second long-term CVS culture and amniocentesis. The phenotypically normal infant had a 6 per cent mosaicism of trisomy 21. It appears that a persistent low-rate mosaicism in different tissues may be indicative of the true status of the fetus.