Maternal cell contamination in cultured chorionic villi: Comparison of chromosome Q-polymorphisms derived from villi,fetal skin,and maternal lymphocytes

Maternal cell contamination of chorionic villi (CV) samples used for first trimester prenatal diagnosis can cause obvious and/or unrecognized diagnostic dilemmas. The purpose of this investigation is to assess the frequency of maternal cell contamination (MCC) in chorionic villus samples and to evaluate selected parameters which might predict where contamination is more likely to have occurred. Maternal lymphocytes, chorionic villi from ultrasonically directed transcervical catheter aspiration, and fetal tissue were obtained at 8–11 weeks gestation from 45 patients undergoing elective termination. Quinacrine (Q) banded metaphases were compared from duplicate direct preparations of chorionic villi; cultured chorionic villi, fetal fibroblast tissue cultures, and maternal lymphocyte cultures. Q-polymorphisms in metaphase chromosomes were 100 per cent concordant between fetal tissue and direct CV preparation. However, evidence for maternal cell contamination occurred in 13.1 per cent of cultured chorionic villi preparations where polymorphisms were found to be identical between maternal and cultured CV and both distinct from fetal tissue preparations. Where MCC was identified, it was noted that CV cell cultivation interval was prolonged (24.2±6.8 days) compared with non-contaminated cultures (14.1±4.4 days) (p <0.05). We conclude that maternal cell contamination is a significant problem with chorionic villus sampling. Where direct preparations are not employed or when cultures are ‘slow growing’, MCC may be a significant and unrecognized complication re: fetal diagnosis. Direct preparations, multiple cultures, quinacrine banding, and maternal Q-polymorphism comparisons can minimize diagnostic dilemmas secondary to maternal cell contamination. Q-polymorphism comparisons between maternal and fetal chromosomes should be included in all instances where cultured chorionic villi are utilized for fetal diagnosis and where direct preparations are not available.     

A case of paternal uniparental disomy for chromosome 11

We report a case of paternal uniparental disomy for chromosome 11 that presented as severe intrauterine growth retardation. Autopsy following intrauterine death also revealed aberrant intestinal rotation and hypospadias. Chromosome analysis of direct preparations from placental biopsy showed an abnormal 47,XY,+11 karyotype. Analysis of long-term cultures from the placenta revealed 46,XY/47,XY,+11 mosaicism. Fluorescence in situ hybridization (FISH) studies on interphase nuclei confirmed trisomy 11 in multiple placental sites but detected only disomic cells in fetal skin. Investigation using microsatellite polymorphisms demonstrated paternal isodisomy at loci D11S909, D11S956, and D11S488, and paternal heterodisomy at locus D11S928.     

An embryogenic model to explain cytogenetic inconsistencies observed in chorionic villus versus fetal tissue

While the fetus and placenta have a common ancestry, chorionic villus tissue does not always reflect fetal genotype. Data are presented from 15 CVS subjects in whom cytogenetic inconsistencies were observed when comparing (1) cultured chorionic villi, (2) direct chromosome preparations of intact villi, and (3) cultured fetal tissue. Embryogenic models are presented to explain these discrepancies. Mosaicism confined to direct chromosome preparations was the most commonly observed inconsistency. This can be explained by postzygotic non-disjunction limited to cytotrophoblast. In all but one instance, the abnormal cell line was limited to the placenta, with the normal cell line reflecting fetal genotype. Analysis of direct chromosome preparations from multiple individually processed villus fragments may be helpful in recognizing mosaicism confined to the placenta. While both direct chromosome preparations and villus cultures can be misleading, the latter are more likely to reflect fetal genetic status since they are derived from the extraembryonic mesoderm.     

Chromosome mosaicism and maternal cell contamination in chorionic villi

While chorionic villus sampling allows both early and rapid prenatal diagnosis of chromosome disorders, the accuracy of this technique has not been fully established. Maternal cell contamination and pseudomosaicism represent two major sources of diagnostic error. Combined use of both direct chromosome preparations and villus cultures is important in overcoming these problems. Direct preparations of villus tissue allow recognition of maternal cell contamination of villus cultures. Conversely, villus cultures yield higher resolution chromosomes and may be helpful in differentiating between true versus pseudomosaicism when two or more cell lines are identified in direct chromosome preparations. Preliminary data suggest that analysis of direct preparations from multiple individually processed villus fragments may also be of value in this regard. Until more experience is gained, mid-trimester amniocentesis should be offered to CVS patients when mosaicism is encountered.     

Prenatal detection of multiple copies of a familial supernumerary marker chromosome

Direct preparations and long-term cultures from a chorion villus biopsy, taken because of a known maternal additional marker chromosome, showed a 48,XX,+mar,+mar karyotype in all cells examined. The same karyotype was revealed in a subsequent amniotic fluid sample and in cord blood at delivery. Detailed examination of the child at delivery and at 1 year of age showed no evidence of phenotypic abnormality or developmental problems.     

Proliferation kinetics in native chorionic villus cells

In accordance with observations by other groups direct CVS preparations reveal only a few mitoses, whereas semi-direct cultures (overnight incubation) give good results. By monitoring the BrdU uptake with an immunological method, a delay in the proliferation activity in native villi could be observed up to 7 h after biopsy.     

Maternal cell contamination in chorionic villus samples assessed by direct preparations and three different culture methods

Presumptive maternal cell contamination (MCC) was monitored in identified male cases during cytogenetic comparison of direct techniques and three different culture regimes from 140 thoroughly dissected chorionic villus samples. Of the 66 identified male cases, 11(16·7 per cent) showed MCC, the mean number of cells examined per case being 8·2 (direct) and 14·5 (cultures); in the direct preparations only one of a total of 457 cells examined was female, while preparations from cultures revealed MCC in 11 cases. Four of these had MCC in more than one culture regime and four had only a single female cell. The results showed that (1) dependence on the culture system alone would have given a diagnosis based on maternal cells in one (1·5 per cent) male case, thus underlining the danger of this approach (a similar undetected rate of misdiagnosis being expected in the female cases) and (2) MCC was significantly lower in cultures grown in Chang medium as compared with the other two regimes, McCoy's 5A + 15 per cent fetal calf serum and 1 per cent Ultroser G, and McCoy's 5A + 25 per cent fetal calf serum, the latter expressing the highest level of MCC.     

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.     

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.     

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.     

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.     

Discordant findings in chorionic villus direct preparation and long term culture—mosaicism in the fetus

Prenatal cytogenetic study of chorionic villi showed a discrepancy between a normal female karyotype 46,XX in the direct preparation after short-term incubation, and a 45,X karyotype in the long-term culture. The subsequent amniocentesis revealed a normal karyotype in three cultures and a 45,X/46,XX mosaicism in one culture. Cytogenetic analysis of chorionic villi after termination of the pregnancy showed a normal karyotype in the direct preparation and a 45,X/46,XX mosaicism in the long-term culture. Fetal lymphocytes showed normal karyotypes, whereas fibroblast cultures revealed a 45,X/46,XX mosaicism.     

Comparison of maternal and fetal chromosome heteromorphisms to monitor maternal cell contamination in chorionic villus samples

Maternal cell contamination (MCC) presents a potential problem in the analysis of chorionic villus sampling (CVS) preparations for early prenatal diagnosis by chromosomal, biochemical and molecular methods. Through the comparison of fluorescent chromosome variants from CVS and maternal cells, we found three out of 50 samples to have MCC. One of these was observed on a direct preparation. Routine chromosome heteromorphism analysis suggested as a reliable method for monitoring MCC in CVS specimens.     

Chromosome banding in direct preparations of chorionic villi

Chorionic villus sampling (CVS) is now currently offered for first trimester prenatal diagnosis of genetic disorders. Chromosome analysis of CVS in direct and culture preparations is possible using modifications of standard banding techniques. We summarize our experience in applying QFQ, GTG, RBG, CBG, DA/DAPI, NOR, and SC differentiation protocols to direct preparations. Characteristic chromosome regions are properly labelled by these techniques, and analysis of 300 band stage karyotypes is consistently achievable on GTG banded direct preparations. However, banding of CVS direct chromosomes has proved to be difficult, and the analysis needs to be backed up by culture preparations.     

Cytogenetic analysis of chorionic villi for prenatal diagnosis: An ACC collaborative study of U.K. data

A prospective 3-year collaborative study was undertaken in 1987 to collect cytogenetic data from diagnostic chorionic villus samples (CVS) in the U.K. in order to determine the predictive value of the chromosome abnormalities encountered. Twenty-seven laboratories contributed a total number of 7595 cases, of which 97·6 per cent were successful. Excluding single cell anomalies, a total of 480 cytogenetic abnormalities were reported, of which 137 were familial structural rearrangements and 343 were de novo problems. Non-mosaic trisomies of chromosomes 13, 18, and 21 (n=157), non-mosaic sex chromosome abnormalities (n=33), and triploidy (n=6) were all confirmed in cells of fetal origin where follow-up information was available. Of the nine remaining non-mosaics including tetraploidy, trisomies of other autosomes, and extra markers, only a trisomy 16 and a case of a supernumerary marker proved genuine. Eighty-eight cases of mosaicism were reported to the study, of which only nine were confirmed as genuine: two cases involving chromosome 13, one trisomy 18, two examples of extra marker chromosomes, three 45,X, and one 47,XXX. There were no reports of false-negative findings. Presumptive maternal cell contamination was encountered in 39 cases, a detected incidence of 0·5 per cent. Four cases of presumptive ‘vanishing twin’ were recorded: in three of these, direct preparations showed a female karyotype, whereas cultures indicated a male (with male fetuses in two cases). The fourth case was of a female fetus with male and female cells in the CVS cultures. Subtle structural chromosome abnormalities were missed in three instances. Accurate prediction of the fetal karyotype was shown to require detailed knowledge of both the nature and the distribution of abnormal cells in the extra-embryonic tissues. In many cases, this could only be made where results from direct preparations and cultured cells were available. A number of conclusions were reached from these and similar data in the literature regarding the reliability of chromosome findings in CVS.     

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.     

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.     

退役工业固废填埋场地再利用的全寿命环境风险特征

通过现场采样和过程模型模拟等方法研究了典型退役工业固废填埋场地（DISWL）原位开发条件下的健康风险及长期演化规律.结果表明,经过近20a的浸出和降解,86%的废物浸出浓度依然处于有害水平,70%的废物不宜直接作为建设用地土壤.直接作为建筑用地开发利用条件下,由于DISWL的性能退化会导致有害组分的浸出和渗漏增加,由此导致地下水水质超标概率经历从无（短期）到有（中期,个别物质如总氰化物T-CN和易释放氰化物F-CN）,再到后期的较大概率超标（T-CN和F-CN）的渐变过程;同时,场地利用过程的健康风险也逐步增加,来自于As的致癌风险和自于T-CN的非致癌风险,分别超过风险可接受水平的81~179倍和55.32~224.3倍.上述结果提示DISWL场地开发再利用的风险评估和管控策略应重点考虑长期风险.对于长期风险不可接受的场地,通过降低废物中毒性物质的浸出浓度可实现长期风险可接受,并提出了相应浸出浓度限值的计算框架和方法.     

退役工业固废填埋场地再利用的全寿命环境风险特征

通过现场采样和过程模型模拟等方法研究了典型退役工业固废填埋场地（DISWL）原位开发条件下的健康风险及长期演化规律.结果表明,经过近20a的浸出和降解,86%的废物浸出浓度依然处于有害水平,70%的废物不宜直接作为建设用地土壤.直接作为建筑用地开发利用条件下,由于DISWL的性能退化会导致有害组分的浸出和渗漏增加,由此导致地下水水质超标概率经历从无（短期）到有（中期,个别物质如总氰化物T-CN和易释放氰化物F-CN）,再到后期的较大概率超标（T-CN和F-CN）的渐变过程;同时,场地利用过程的健康风险也逐步增加,来自于As的致癌风险和自于T-CN的非致癌风险,分别超过风险可接受水平的81~179倍和55.32~224.3倍.上述结果提示DISWL场地开发再利用的风险评估和管控策略应重点考虑长期风险.对于长期风险不可接受的场地,通过降低废物中毒性物质的浸出浓度可实现长期风险可接受,并提出了相应浸出浓度限值的计算框架和方法.     

46,XY,dup(10q) in direct CVS preparation and mosaic 48,XXXY,dup(10q) in CVS long-term culture and fetal tissue

Chorionic villus sampling (CVS) was performed on a 40-year-old woman at 9 1/2 menstrual weeks because of advanced maternal age. The direct preparation showed 46,XY,dup(10)(q11.2q23.2). CVS long-term culture and fetal tissue revealed a rare additional abnormality: 48,XXXY,dup(10)(q11.2q23.2). This abnormality represented the major cell line (>85 per cent in 691 cells) in an (XY)/XXY/XXXY/(XXXXY) mosaic (all cell lines presumably bearing the dup(10q); the presence of XY and XXXXY cell lines is uncertain). To our knowledge, this is the first report of trisomy 10q11-q23 and of prenatally detected 48,XXXY in chorionic villi. The mosaic could have resulted from early post-zygotic non-disjunctions in a 46,XY,dup(10q) or 47,XXY,dup(10q) zygote. The results from DNA studies of four polymorphisms, mapped to Xp and Xq, support this theory. The literature on prenatally detected cases with sex chromosome tetrasomy and pentasomy and those with additional autosomal abnormalities is reviewed. The reported case underlines the problem of false-negative findings when only direct CVS preparations are karyotyped.