Clinical proceduress in prenatal diagnosis

Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 16, No. 5, pp. 611±627, 2002

doi:10.1053/beog.2002.0328, available online at http://www.idealibrary.com on

1 Clinical proceduress in prenatal diagnosis Barbara Eisenberg

BA

Medical Student

Ronald J. Wapner

MD

Professor, Obstetrics and Gynecology MCP Hahnemann University, Philadelphia, Pennsylvania, USA

The prenatal diagnosis of fetal genetic disease has become a routine part of obstetric care. Pregnancies at risk are identi®ed by a number of factors, including maternal age, positive serum screening, a history of a previous a€ected child, a parental chromosome rearrangement or an ultrasound-identi®ed anomaly. Invasive diagnostic testing can be performed in the ®rst trimester by chorionic villus sampling or in the second trimester by amniocentesis. Both procedures are safe, with an equivalent 0.5% risk of procedure-induced pregnancy loss. When performed prior to the routine sampling window of 15 weeks, amniocentesis may increase the risk of talipes equinovarus, the highest risk being encountered prior to 13 weeks' gestation. When chorionic villus sampling is performed prior to 9 weeks' gestation, there may be an increased risk of limb reduction defects. The laboratory analysis of both procedures is reliable. Chorionic villus sampling has a 1±2% incidence of con®ned placental mosaicism, requiring additional evaluation in some cases. Key words: prenatal diagnosis; chorionic villus sampling; amniocentesis; con®ned placental mosaicism; mosaicism.

Prenatal diagnostic procedures have become an indispensable tool-set for obtaining clinical information about a variety of fetal genetic, biochemical and physiological disorders. The most frequently used invasive diagnostic procedures are amniocentesis and chorionic villus sampling (CVS). Newly emerging procedures include cervical lavage and the isolation of fetal cells from the maternal blood, but these are not yet ready for clinical use. Individually or collectively, these tests are used for karyotyping as well as in an ever-expanding menu of hereditary, developmental, and infectious conditions. The results of these tests provide valuable information to the mother during genetic counselling. INDICATIONS FOR AMNIOCENTESIS AND CHORIONIC VILLUS SAMPLING In the USA, advanced maternal age is the primary indication for invasive prenatal testing.1 Amniocentesis is routinely o€ered as a standard practice to women who will be at least 35 years of age at the time of full-term delivery. This choice represents c 2002 Elsevier Science Ltd. All rights reserved. 1521±6934/02/$ - see front matter *

612 B. Eisenberg and R. J. Wapner

a reasonable balance between the hazards of an invasive diagnostic procedure and the probability of an undiagnosed fetal aneuploidy. This approach to aneuploidy screening is, however, relatively inecient since only 20±30% of Down syndrome births occur in women aged 35 years old or older. Since a women carrying a fetus with trisomy 21 has a unique pattern of serum biochemical analytes in the second trimester (decreased alpha-fetoprotein (AFP), increased human chorionic gonadotrophin [b-HCG]2 and decreased unconjugated serum oestriol) and ®rst trimester (increased free b-HCG and decreased pregnancy-associated plasma protein A [PAPP-A]), their values can be used to calculate a more accurate risk of Down syndrome than can be obtained from maternal age alone. In addition, a ®rsttrimester fetus with Down syndrome has increased ¯uid in the posterior cervical spine area (nuchal translucency). By combining maternal age with values of serum analytes in the second trimester, and with those of PAPP-A, free b-HCG and nuchal translucency in the ®rst trimester3, a more accurate risk assessment of a trisomy 21 fetus can be obtained. Using a screen-positive risk of 1:270, 60% of Down syndrome pregnancies can be identi®ed in the second trimester and 80±85% in the ®rst trimester compared with only 20±30% by limiting amniocentesis to women 35 years of age or older. Both approaches carry an approximately 5% amniocentesis rate. Other indications for invasive testing include a previous child with a chromosomal abnormality4, a parent carrying a balanced chromosomal rearrangement5, a fetus with a structural abnormality identi®ed on routine ultrasound6, a parental carrier of an autosomal recessive disorder and a mother carrying an X-linked disorder. Amniocentesis with measurement of the amniotic ¯uid AFP level was previously the de®nitive diagnostic tool for neural tube defects. More recently, ultrasound visualization of the fetal head and spine combined with maternal serum AFP evaluation has become a non-invasive alternative. Maternal serum AFP level can identify 95% of anencephalic fetuses and 85% of those with an open neural tube defect.7 By combining maternal screening with ultrasound, nearly 100% of anencephaly cases and nearly all cases of spina bi®da can be identi®ed in experienced centres using detailed ultrasound sectioning of the fetal spine and evaluation of the fetal cranium for signs of an Arnold± Chiari malformation.8 THE TECHNIQUE OF AMNIOCENTESIS Timing of the procedure Amniocentesis is usually performed after 15 weeks' gestation when the uterus is an abdominal organ that can be sampled with minimal risk of injury to the maternal bowel. Between 15 and 18 weeks' gestation, there are between 150 and 250 ml amniotic ¯uid in the uterus. Early in the second trimester, enough viable fetal cells are already available so that the required 20±30 cm3 can be removed without excessive risk in order to permit reliable analysis. It has recently been demonstrated with certainty that amniocentesis should not be performed until at least 14 weeks' gestation. Notwithstanding reports from the mid1970s suggesting that amniocentesis before 15 weeks is associated with an unacceptable procedural and cultural failure rate9,10, the advent of high-resolution ultrasound and direct needle guidance has made the amniotic sac accessible as early as 7 weeks. The development of improved tissue culture methods has simultaneously resulted in improved laboratory results requiring less ¯uid and fewer cells for cytogenetic analysis. These developments, when taken together, lead to a consideration of amniocentesis

Clinical procedures in prenatal diagnosis 613

earlier in gestation.11±17 Unfortunately, recent randomized trials have demonstrated a number of concerns related to amniocentesis performed prior to 14 weeks, the most important being a 10-fold increase in the risk of severe talipes equinovarus.18,19 Club feet occur at a rate of approximately 1±3 per 1000 births in the general population, which rises to approximately 1.5% following early amniocentesis. This dramatic increase appears to be closely related to a greater propensity for amniotic ¯uid leakage with earlier sampling. The Canadian randomized comparison of early amniocentesis and mid-trimester sampling demonstrated a twofold increased risk of ¯uid leakage with the earlier procedure. When this occurred, there was a 15% incidence of talipes equinovarus. Without ¯uid leakage, the incidence of talipes was still approximately 10-fold higher than had been expected. Although the exact mechanism by which early amniocentesis contributes to the formation of club feet remains uncertain, fetal compression or an alteration of the normal fetal leg relationship because of a loss of ¯uid appears to be a signi®cant factor. From an anatomical perspective, this increased risk of leakage may occur because the amnion and chorion have not yet fused. When this does occur, at around 14 weeks' gestation, the risk of leakage and subsequent complication appears to be minimized. In addition to the higher incidence of fetal deformities, early amniocentesis demonstrates a greater risk of failed procedures, failed tissue culture and pregnancy loss than is seen with standard amniocentesis.18 These observations have been consistent in all trials, and because of this the procedure should only be performed under extenuating circumstances after complete risk counselling. The procedure The actual needle insertion should be performed under continuous ultrasound monitoring, which lowers the incidence of bloody and dry taps, and decreases the need for multiple insertions.20 Continuous monitoring will also reduce the incidence of inadvertent fetal injury since acute changes in fetal position occur, on average, 200 hundred times every 30 minutes. Over the years, there has been a trend toward the use of smaller needles for aspiration. The use of 20±22 gauge needles is not only less painful, but also carries fewer complications than does the use of the previously used 19 and higher gauged needles.21,22 It has also been postulated that even smaller needles (i.e. 23±25 gauge) might be safer, especially when transversing an anterior placenta. The longer sampling times required to collect the required sample size may, however, o€set any bene®ts these o€er. The use of 20±22 gauge spinal needles is therefore currently recommended. Complications and risks The most common complications of amniocentesis ± vaginal bleeding, rupture of the membranes, chorio-amnionitis and pregnancy loss ± also occur in unsampled pregnancies. Thus, to understand the additional risk imposed by the procedure, it is necessary to compare sampled and unsampled populations. Pregnancy loss The primary concern for patients undergoing prenatal testing is the chance that the procedure will lead to the loss of the desired pregnancy.

614 B. Eisenberg and R. J. Wapner

Only the study by Tabor et al is based on a prospective, randomized, controlled evaluation.23 All study participants were between the ages of 25 and 34 years. None had a genetic indication for testing, and all were willing to be randomized into groups undergoing amniocentesis or receiving no procedure. The procedure itself was performed by a small number of experienced operators using standardized, accepted techniques, including ultrasound guidance. The sampled group experienced a fetal loss rate of 1.7% compared with 0.7% for the control (unsampled) group (P 5 0.01) (95% con®dence intervals 0.3±1.5, relative risk 2.2). The authors commented that the 1% di€erence in fetal loss might represent an underestimate of the true di€erence since the identi®cation and elective termination of karyotypically abnormal fetuses from the amniocentesis group might have reduced the spontaneous loss rate. Estimates of pregnancy loss given to patients during pre-testing counselling have traditionally been somewhat lower than those reported by Tabor et al. Unless additional studies of comparable size, quality and design are performed, patients should be informed that the risk of pregnancy loss from an amniocentesis performed at 15±20 weeks' gestation may be as high as 1%. Most centres continue to counsel a somewhat lower loss rate, typically 0.5%, which lies within the 95% con®dence interval identi®ed by Tabor, and some recent studies indeed support this lower risk.24 Amniotic ¯uid leakage Leakage of amniotic ¯uid following a second-trimester amniocentesis occurs in 0.8±2% of sampled pregnancies and may be 1% higher than that observed in unsampled pregnancies.23,25 Unlike spontaneous rupture, the ¯uid leakage is in most cases selflimiting and is associated with excellent pregnancy outcomes.26,27 Infection Post-amniocentesis chorio-amnionitis is quite rare, occurring in 0.5±1.5 cases per 1000 procedures performed, and is most often attributed to the accidental introduction of either skin or bowel ¯ora into the amniotic sac.21,28 Ascending infection is possible, especially if chronic amniotic ¯uid leakage occurs. The initial signs of a second trimester intra-uterine infection can be quite understated, the patient presenting with only a low-grade fever and in¯uenza-like symptoms. If ignored, these symptoms can progress rapidly to severe chorio-amnionitis and maternal sepsis. Although uterine tenderness may not initially be present, a high index of suspicion is required to make an early diagnosis. In a patient who has recently undergone an amniocentesis and subsequently presents with a fever of no other obvious origin, a repeat amniocentesis for Gram staining and culture is indicated. If organisms are present, the prognosis is uniformly bleak and there is no justi®cation for a delay in emptying the uterus. Rhesus isoimmunization In 2±3% of second trimester amniocenteses, a fetal-to-maternal haemorrhage of at least 0.1 ml will occur, and this haemorrhage will lead to rhesus isoimmunization in 2.1±5.4% of the at-risk pregnancies.29 Sensitization can be prevented by the administration of rhesus (D) immunoglobulin, and we believe that all rhesus (D)-negative, non-sensitized women undergoing amniocentesis should receive rhesus (D) immunoglobulin. A dose of 100 mg is sucient and less expensive than the standard 300 mg dosage.30

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Congenital anomalies The possibility that the incidence of congenital anomalies may be increased continues to be studied. Several reports, including animal studies, suggest a higher incidence of neonatal respiratory diculties in sampled pregnancies. Tabor et al demonstrated a signi®cant increase in the incidence of both neonatal respiratory distress syndrome and congenital pneumonia following amniocentesis23, the relative risks, compared with the control group, being 2.1 and 2.5 respectively. In a primate study in which amniocentesis was performed at a comparable gestational age, Hislop et al demonstrated an altered alveolar number and size and a reduction in the number of respiratory airways.31 Other studies, both controlled25 and uncontrolled32±35, have also demonstrated pulmonary alterations in sampled pregnancies, although similar investigations have failed to con®rm this.21,26,36 There has also been a recurrent but less well-substantiated observation that amniocentesis is associated with neonatal orthopaedic deformities. In the UK Medical Research Council study higher incidences of severe talipes equinovarus and congenital hip dislocation were seen in the sample group than in the control group25, although other studies have not con®rmed these observations. Despite the ongoing debate concerning neonatal e€ects, a 1990 longitudinal investigation on the long-term pathology of genetic amniocentesis that compared the development, behaviour and physical status of the 4-year-old children of mothers who had either chosen or declined amniocentesis may allay at least some fears.37 The only observed di€erence between the two groups was a somewhat higher incidence of bilateral middle ear impedance abnormalities and recurrent ear infections in children exposed to amniocentesis. The authors hypothesized that the reduction in intraamniotic ¯uid volume had led to pressure changes within the ear that disturbed normal development. Perinatal complications No pattern of increased perinatal complications, including in the incidence of preeclampsia, abruption, placenta praevia and dysfunctional labour, has been observed in patients undergoing amniocentesis.26 Success and accuracy of laboratory analysis Following successful amniotic ¯uid aspiration, the retrieved cells are concentrated by centrifugation and cultured for a minimum of 5±7 days, the ®nal laboratory results usually being available within 10±14 weeks. Although the details of the laboratory procedures are beyond the scope of this chapter and are available elsewhere, it is important to understand that, even under ideal laboratory conditions, amniocentesis can occasionally fail to yield results or, worse still, may yield a diagnostic error or falsepositive result. Tissue culture failure occurs infrequently (0.2±0.6%)9,23, usually resulting from a de®cit of viable cells, a failure of the cells to grow in culture or contamination of the culture with infectious organisms. These failures can be minimized by aspirating sucient ¯uid (20±30 ml) and by mid-second trimester sampling.9,23 Signi®cant diagnostic and clinical errors following amniocentesis are exceedingly rare but, in addition to clerical, sampling, handling and labelling mistakes, may occur from maternal cell contamination or the misinterpretation of mosaic results. Pooled data from US and European centres show that although maternal cells may be present

616 B. Eisenberg and R. J. Wapner

in 0.1±0.2% of cases, the risk of major diagnostic error is extremely small and can be further reduced by discarding the ®rst few millilitres of amniotic ¯uid during sampling. The ®nding of two or more cell lines with di€erent chromosomal constitutions in an amniotic ¯uid is called mosaicism and requires careful clinical interpretation. Although the abnormal cell line may represent a signi®cant fetal chromosomal abnormality, it occurs most often secondary to in vitro tissue culture artefacts or a chromosomal error existing in the extra-embryonic membrane but not in the fetus. The clinical signi®cance is in most cases determined by the speci®c chromosome abnormality or the distribution of the cells in the culture. To facilitate the interpretation of mosaicism, multiple cultures of the cells are prepared. If the aberrant cell line is found in only a single culture, this is considered to be an in vitro clonal event of no clinical signi®cance to the fetus. If, however, the aberrant cell line is found in multiple cultures, the possibility that the fetus may carry the abnormal cell line is appreciably increased. Such a ®nding is present in 0.1±0.2% of amniotic ¯uid samples analysed38,39 and requires further analysis for the de®nitive determination of its clinical signi®cance. Finding mosaicism in which the abnormal karyotype is reported in dysmorphic liveborns requires the sampling of additional fetal cell lines, such as blood or skin, to evaluate the implications. The abnormal cell line will, however, be con®rmed in the fetus on only 60±70% of occasions.39,40 Chromosome-speci®c probes and ¯uorescence in situ hybridization (FISH) have recently been used to detect numerical aberrations in interphase, non-dividing cells.41 Since the viable cells in amniotic ¯uid are nucleated, this process eliminates the need for cell cultivation and makes more cells available for screening than for cytogenetic analysis. Most commonly, chromosome-speci®c probes for the diagnosis of the common autosomal trisomies (13, 18 and 21) and sex chromosome aneuploidy are used. When necessary and clinically appropriate, probes for speci®c chromosome regions are helpful. Examples include probes for chromosome 22q deletions with heart defects, evaluation of translocations and the improved identi®cation of chromosome markers. FISH is most useful when a rapid diagnosis is required and there is strong clinical suspicion of a common autosomal trisomy. CHORIONIC VILLUS SAMPLING First-trimester prenatal diagnosis can be safely performed by CVS, which is performed between 70 and 97 days after the last menstrual period. This window is chosen to minimize the high background spontaneous miscarriage rate of early pregnancy, yet it still allows time to obtain laboratory results within the ®rst trimester. Sampling earlier in gestation may be associated with an increased risk of fetal abnormalities and should not be routinely carried out. There are two routes used for CVS: transcervical and transabdominal (Figure 1). Both are safe and equally ecacious, and the majority of patients can be sampled using either.42 In most cases, physician and patient preference will dictate which technique is used, but in 3±5% of patients, clinical circumstances will support one approach over the other, requiring operators to be pro®cient in both.42,43 Transcervical CVS is preferred when the placenta is located on the posterior uterine wall, whereas transabdominal sampling is particularly useful when the placenta is implanted in a fundal or high anterior location. Transcervical sampling has the advantage of minimal patient discomfort but is somewhat more dicult to learn.44 Both approaches are usually performed by using a two-person technique,

Clinical procedures in prenatal diagnosis 617

Figure 1. Diagrammatic representation of transabdominal and transcervical chorionic villus sampling. RPMI, Roswell Park collection medium.

one person performing the sampling and one guiding with ultrasound. Communication between the sonographer and the sampler is crucial, and the best results have come from centres in which a limited number of samplers and sonographers

618 B. Eisenberg and R. J. Wapner

perform CVS. By having fewer people performing more procedures, both the operator and the sampler become more experienced, which improves success and safety rates.45 Complications and risks of chorionic villus sampling The common post-CVS complications are summarized to Table 1. These occur infrequently. Pregnancy loss Multiple reports from individual centres have repeatedly demonstrated the high degree of safety and low rate of pregnancy loss post-CVS.47±55 In experienced centres, the rate of miscarriage from the time of CVS through to 28 weeks' gestation is approximately 2±3%.42 To determine the incidence of procedure-induced pregnancy loss, however, adjustments for the relatively high background loss rate of 2±3% at this gestational age must be made.56 Since it would be inappropriate to expose women with no indication for a ®rst-trimester invasive procedure to CVS, no randomized comparison of sampled with unsampled patients is likely, but comparisons with amniocentesis have been done. Because the background loss rate is higher in the ®rst trimester than in the second, all study patients must be enrolled before the gestational age at which CVS is performed. If all losses, whether from spontaneous miscarriage or induced terminations for abnormal results, are included, the total loss rates can be compared. This eliminates any bias that might occur when comparing procedures performed at signi®cantly di€erent gestational ages and includes cytogenetically abnormal embryos that miscarry before amniocentesis, which would be electively terminated following CVS. The largest collection of data evaluating the relative safety of CVS and amniocentesis comes from three collaborative reports. A 1989 Canadian study showed 7.6% fetal losses (spontaneous abortion, induced abortions and late losses) in the CVS group and 7.0% in the amniocentesis group, i.e. an excess loss rate for desired pregnancies of 0.6%.57 This di€erence was considered statistically insigni®cant. An American report published shortly thereafter showed a 0.8% di€erence, which was also considered to be statistically insigni®cant.58 A third study, carried out in Europe, showed a 4.6% greater pregnancy loss rate following CVS (95% con®dence interval 1.6±7.5%).59 This di€erence re¯ected more spontaneous deaths before 28 weeks of gestation (2.9%), more terminations of pregnancy for chromosomal abnormalities (1.0%) and more neonatal deaths (0.3%) in the CVS group. The factors responsible for the discrepancy in results between the North American and European studies remain uncertain, but it is probable that inadequate operator experience with CVS might account for a large part of the di€erence. Whereas the Canadian trial consisted of 11 centres and the US trial of 7, the European trial included 31. There were, on average, 325 cases per centre in the US study and 106 in the Canadian study, but only 52 cases per centre in the European study. Although no signi®cant change in the pregnancy loss rate was seen during the course of the European trial, it appears that the learning curve for both transcervical and transabdominal CVS may exceed 400 or more cases.45,60 Operators who have performed fewer than 100 cases may have 2±3 times the post-procedural loss rate of operators who have performed more than 1000 procedures.

Perinatal complications Preterm labour Premature rupture of membranes Small for gestational age Obstetric complications

Rhesus sensitization

Acute rupture of membranes Delayed ¯uid leakage ± oligohydramnios

Chorio-amnionitis

Vaginal bleeding

Fetal±maternal haemorrhage possible Rhogam suggested post-procedure; may exacerbate pre-existing sensitization No increase post-CVS Not evaluated to date

No increase post-CVS Normal

Rare 51:1000 cases Usually bowel ¯ora Very rare Very rare

Rare

Transabdominal

7±10% of cases Almost always self-limiting Rare 51:1000 cases Usually vaginal ¯ora Very rare Oligohydramnios ± 0.3%, rare Frequently associated with post-procedural bleeding and raised alpha-fetoprotein level Fetal±maternal haemorrhage possible Rhogam suggested post-procedure; may exacerbate pre-existing sensitization

Transcervical

Table 1. Complications associated with transabdominal and transcervical chorionic villus sampling (CVS)46.

Clinical procedures in prenatal diagnosis 619

620 B. Eisenberg and R. J. Wapner

Risk of fetal abnormalities after chorionic villus sampling Firth et al suggested that CVS might be associated with the occurrence of speci®c fetal malformations.61 In a series of 539 CVS-exposed pregnancies, they identi®ed ®ve infants with severe limb abnormalities, all of whom came from a cohort of 289 pregnancies sampled at 66 days of gestation or less. Four of these infants had the unusual and rare oromandibular±limb hypogenesis syndrome and the ®fth a terminal transverse limb reduction defect. Oromandibular±limb hypogenesis syndrome occurs with a birth prevalence of 1 per 175 000 live births62 and limb reduction defects occur in 1 per 1690 births.63 Therefore, the occurrence of these abnormalities in more than 1% of CVS-sampled cases raised the strong suspicion of an association. In this initial report, all of the limb abnormalities followed transabdominal sampling performed between 55 and 66 days' gestation. Subsequent to this initial report, others added supporting cases to the list. Using the Italian multicentre birth defects registry, Mastroiacovo et al reported, in a casecontrol study, an odds ratio of 11.3 for transverse limb abnormalities after ®rsttrimester CVS.64 When strati®ed by gestational age at sampling, pregnancies sampled before 70 days had a 19.7% increased risk of transverse limb reduction defects, whereas patients sampled later did not demonstrate a signi®cantly increased risk. Other single-centre and case-control studies have, however, been inconclusive about an association of CVS with limb reduction defects, the majority demonstrating no increased risk. There is support for the notion that CVS may increase the risk of limb defects when sampling is performed before 63 days' gestation. Most notably, Brambati et al, an extremely experienced group who have reported no increased risk of limb defects in patients sampled after 9 weeks, have recorded a 1.6% incidence of severe limb reduction defects in patients sampled at 6 and 7 weeks.65 This rate decreased to 0.1% for sampling at 8±9 weeks. The question remains of whether CVS sampling after 70 days has the potential to cause more subtle defects, such as shortening of the distal phalanx or nail hypoplasia.66 Few data substantiate this concern. Most experienced centres performing CVS after 10 weeks have not seen an increase in limb defects of any type. A review of more than 200 000 CVS procedures reported to the World Health Organization registry reported and demonstrated no increase in the overall incidence of limb reduction defects after CVS or in any speci®c type or pattern of defect.67 In a similar review of more than 65 000 procedures performed in 10 of the most experienced centres in the world, no increase in limb reduction defects was identi®ed.68 Patients planning to undergo CVS can be counselled that there is no increased risk of severe limb defects if CVS is performed after 70 days' gestation. They should be made aware of the present controversy concerning more subtle defects and reassured that this has not been seen in most experienced centres. If such a risk does exist, the magnitude, based on case-control studies, can be estimated to be no higher than 1 in 3000.69 Centres performing CVS should ideally have aggressive follow-up systems in place and be capable of giving patients information about the rate of congenital abnormalities in their centre. Sampling before 10 weeks of gestation should be limited to very exceptional cases, and these patients must be informed of a 1% or higher risk of limb reduction defects.

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Laboratory aspects of chorionic villus sampling CVS is now considered to be a reliable method of prenatal diagnosis, but incorrect results were reported early in its development.70±72 The major sources of these errors included maternal cell contamination and the misinterpretation of mosaicism con®ned to the placenta. Today, the genetic evaluation of chorionic villi provides a high degree of success and accuracy, particularly related to the diagnosis of common trisomies.73,74 The US collaborative study reported a 99.7% rate of successful cytogenetic diagnosis, with 1.1% of the patients requiring a second diagnostic test, such as amniocentesis or fetal blood analysis, to interpret the results further.75 In most cases, the additional testing was required to delineate the clinical signi®cance of mosaic or other ambiguous results (76%); laboratory failure (21%) and maternal cell contamination (3%) also required follow-up testing. Continued experience has almost eliminated maternal cell contamination as a source of clinical error. In addition, we now have a better understanding of the biology of the placenta so that con®ned placental mosaicism no longer leads to incorrect diagnosis but instead provides us with information predictive of pregnancy outcome and can serve as a clue to the presence of uniparental disomy. Con®ned placental mosaicism One of the most interesting and biologically illustrative observations seen with CVS is mosaicism con®ned to the placenta.76 Although there was initially concern that this might invalidate CVS as a prenatal diagnostic tool, subsequent investigations have led to a clearer understanding of villus biology, revealed new information about the aetiology of pregnancy loss, discovered a new cause of intra-uterine growth retardation and clari®ed the basic mechanism of uniparental disomy. Con®ned placental mosaicism secondary to trisomic rescue can lead to uniparental disomy (UPD), in which both members of a chromosome pair come from the same parent. This occurs when the trisomic cell, which undergoes anaphase lag, loses the one chromosome contained by the parent that is not involved in the initial nondysjunction. After rescue, there is a theoretical 1 in 3 chance that the resulting pair of chromosomes will come from the same parent. UPD has clinical consequences if the chromosomes involved carry imprinted genes in which expression is based on the parent of origin. Prader±Willi syndrome, for example, may result from uniparental maternal disomy for chromosome 15. Therefore, a CVS diagnosis of con®ned placental mosaicism for trisomy 15 may be the initial clue that UPD could be present and lead to an a€ected child.77,78 Consequently, all cases in which CVS reveals trisomy 15 (either complete or mosaic) should be evaluated for UPD by subsequent amniotic ¯uid analysis. In addition to chromosome 15, chromosomes 7, 11, 14 and 22 are felt to be imprinted and require follow-up if implicated.79 There is also evidence that con®ned placental mosaicism (unassociated with UPD) can alter placental function and lead to fetal growth failure or perinatal death.40,80±85 The exact mechanism underlying this is unknown, but the e€ect may be limited to speci®c chromosomes. Con®ned placental mosaicism for chromosome 16 leads, for example, to severe intra-uterine growth restriction, prematurity or perinatal death, fewer than 30% of pregnancies resulting in normal full-term infants appropriate for their gestational age.86±91 Whereas CVS mosaic results may be con®ned to the placenta, diligent follow-up by amniocentesis or fetal sampling is frequently required to eliminate a true fetal

622 B. Eisenberg and R. J. Wapner

mosaicism that may have signi®cant phenotypic consequences. Mosaicism occurs in about 1% of all CVS samples73,76,88,89 but is con®rmed in the fetus in only 10±40% of these cases. The probability of fetal involvement is related to the tissue source in which the aneuploid cells were detected and the speci®c chromosome involved.79 For reasons previously discussed, mesenchymal core culture results are more likely than direct preparations to re¯ect a true fetal mosaicism. Phillips et al demonstrated that autosomal mosaicism involving common trisomies (i.e. 21, 18 and 13) was con®rmed in the fetus in 19% of cases, whereas uncommon trisomies involved the fetus in only 3% of instances.89 When sex chromosome mosaicism was found in the placenta, the abnormal cell line was con®rmed in the fetus in 16% of cases. When a non-familial marker chromosome was involved, it was con®rmed in the fetus in more than one- quarter of cases, whereas mosaic polyploidy was con®rmed in only 1 out of 28 cases. Chromosomal structural abnormalities were con®rmed in 8.6% of cases. When placental mosaicism is discovered, amniocentesis is frequently performed to elucidate the extent of fetal involvement. When mosaicism is limited to the direct preparation, amniocentesis appears to correlate perfectly with fetal genotype.89 When, however, a mosaicism is observed in tissue culture, both false-positive and falsenegative amniocentesis results occur. In these cases, amniocentesis will predict the true fetal karyotype in approximately 94% of cases.89 These discrepancies may involve the common autosomal trisomies. There have been three reported cases of mosaic trisomy 21 on villus culture with a normal amniotic ¯uid analysis that have been followed by a fetus or newborn with mosaic aneuploidy.75 The following clinical recommendations may be used to assist in the evaluation of CVS mosaicism. The analysis of CVS samples should, if possible, include both direct preparation and tissue culture. Although the direct preparation is less likely to be representative of the fetus, its use will minimize the likelihood of maternal cell contamination. In addition, if culture fails, a non-mosaic, normal direct preparation result can be considered to be conclusive, although rare cases of false-negative results for trisomies 21 and 18 have been reported.90±94 If mosaicism is found on either culture or direct preparation, follow-up amniocentesis should be o€ered. In no circumstances should a decision to terminate a pregnancy be based entirely on a CVS mosaic result. For CVS mosaicism involving sex chromosome abnormalities, polyploidy, marker chromosomes, structural rearrangements and uncommon trisomies, the patient can be reassured if the amniocentesis results are euploid and detailed ultrasonographic examination is normal. No guarantees should, however, be made, and, as described above, testing for UPD will be indicated in certain cases. If the common trisomies 21, 18 and 13 are involved, amniocentesis should be o€ered, but the patient must be advised of the possibility of a false-negative result. Follow-up may include detailed ultrasonography, fetal blood sampling and fetal skin biopsy. The predictive accuracy of these additional tests is currently uncertain. Biochemical and DNA procedures Most biochemical and molecular diagnoses that can be made from amnionic ¯uid or cultured amniocytes can also be made from chorionic villi. In many cases, the results will be available more rapidly and more eciently by using villi because sucient enzyme or DNA is present to allow direct analysis rather than waiting for tissue culture. The analysis of Tay-Sachs disease can, for example, be performed in less than 30 minutes using fresh villi.95

Clinical procedures in prenatal diagnosis 623

It cannot always be assumed that biochemical or molecular results from villus tissue will be a true re¯ection of the fetal state. A misdiagnosis of the peroxisomal disorder X-linked adrenoleukodystrophy from cultured villus cells has recently been reported.96 In addition, tests requiring the determination of DNA methylation status, such as that for fragile X,97 are also not always reliable in villus tissue. This does not preclude CVS when making these prenatal diagnoses because other molecular approaches can be used, but it does emphasize that all tests on villus tissue must be validated by testing sucient numbers of a€ected and una€ected pregnancies before being used clinically. Because of the rarity and unique aspects of most biochemical and molecular disorders, only a few laboratories usually perform speci®c diagnoses. Before performing a CVS, the clinician should contact the centre analysing the tissue so that the details of testing can be discussed. SUMMARY CVS sampling is a safe technique for the ®rst-trimester prenatal diagnosis of genetic disorders and is clearly preferred to early amniocentesis. Real-time sonography and technological advances in the sampling instruments have resulted in a safe and reliable technique for retrieving villus tissue for genetic analysis. Clinical trials suggest that CVS carries a very low risk of pregnancy loss, a risk that is comparable to that of second-trimester amniocentesis. An understanding of laboratory techniques and human embryology is essential in avoiding diagnostic errors related to con®ned placental mosaicism. To maximize outcome, an experienced team of physicians, ultrasonographers and genetic laboratory technicians should perform CVS. Studies and our experience show that the higher the number of procedures performed, the better the success rate and the fewer the number of complications. Before initiating a CVS programme, operators should have considerable experience in the placement of the catheter, which can be achieved either in a formal training programme with the observation of 50 procedures followed by the close hands-on supervision of another 100 cases47 or by supervised practice on pregnancies undergoing subsequent abortion. In addition, it is advisable to limit the number of ultrasonographers assigned to assist in this procedure because sampling success is equally dependent on skilful ultrasound guidance. As with the physician retrieving the villi, the guiding sonographer should be knowledgeable in the didactics of CVS sampling and should obtain adequate hands-on training before beginning work in this area. This can be achieved in a formal training programme or by visiting centres performing this procedure.47 In this setting, CVS will continue to be an important, reliable and safe contributor to prenatal genetic diagnosis. REFERENCES 1. D'Alton ME. Operative Obstetrics. East Norwalk, CT: Appleton & Lange, 1995. 2. Wald N, Cuckle H & Densem J. Maternal serum screening for Down syndrome in early pregnancy. British Medical Journal 1988; 297: 883. 3. Ong C, Liao A, Spencer K et al. First trimester maternal serum free beta human chorionic gonadotrophin and pregnancy associated plasma protein A as predictors of pregnancy complications. British Journal of Obstetrics and Gynaecology 2000; 107: 1265±1270. 4. Stene J, Stene E & Mikkelsen M. Risk for chromosome abnormality at amniocentesis following a child with a non-inherited chromosome aberration. Prenatal Diagnosis 1984 (special issue); 4: 81.

624 B. Eisenberg and R. J. Wapner 5. Daniel A, Hook E & Wulf G. Risks of unbalanced progeny at amniocentesis to carrier of chromosome rearrangements: data from United States and Canadian laboratories. American Journal of Medical Genetics 1989; 33: 14. 6. Nicolaides K, Snijders R & Gorden C. Ultrasonographically detectable markers of fetal chromosomal abnormalities. Lancet 1992; 340: 704±707. 7. Genetics Disease Branch, Department of Health Services, State of California. AFP screening. California Department of Health Services, 1991. 8. Nadel A, Green J & Holmes L. Absence of need for amniocentesis in patients with elevated levels of maternal serum alpha-fetoprotein and normal ultrasonographic examinations. New England Journal of Medicine 1991; 232: 557±561. 9. Golbus M, Loughman W & Epstein C. Prenatal genetic diagnosis in 3000 amnincenteses. New England Journal of Medicine 1979; 300: 157. 10. Carlan S, Papenhausen P & O'Brien W. E€ect of maternal±fetal movement on concentration of cells obtained at genetic amniocentesis. American Journal of Obstetrics and Gynecology 1990; 163: 490. 11. Hanson F & Tennant F. Ultrasonography-guided early amniocentesis in singleton pregnancies. American Journal of Obstetrics and Gynecology 1990; 162: 1376. 12. Hanson F, Tennant F & Hune S. Early amniocentesis: outcome, risks, and technical problems at 412.8 weeks. American Journal of Obstetrics and Gynecology 1992; 166: 1707. 13. Jorgensen F, Bang J & Lind A. Genetic amniocentesis at 7±14 weeks of gestation. Prenatal Diagnosis 1992; 12: 227±283. 14. Nevin J, Nevin N & Dornan J. Early amniocentesis: experience of 222 consecutive patients, 1987±1988. Prenatal Diagnosis 1990; 10: 79. 15. Penso C, Sanstrom M & Garber M. Early amniocentesis: report of 407 cases with neonatal follow-up. Obstetrics and Gynecology 1990; 76: 1032. 16. Stripparo L, Buscaglia M & Longatti L. Genetic amniocentesis: 505 cases performed before the sixteenth week of gestation. Prenatal Diagnosis 1990; 10: 359. 17. Thayer B, Braddock B & Spitzer K. Clinical and laboratory experience with early amniocentesis. Birth Defects 1990; 26: 58. *18. Cemat G. Randomized trial to assess safety and fetal outcomes of early and midtrimester amniocentesis. Lancet 1998; 351: 1435. 19. Nicolaides K, Brizot MdL, Patel F & Snijders R. Comparison of chorionic villus sampling and amniocentesis for fetal karyotyping at 10±13 weeks' gestation. Lancet 1994; 344: 435. 20. Romero R, Jeanty P & Reece E.. Sonographically monitored amniocentesis to decrease intraoperative complications. Obstetrics and Gynecology 1985; 65: 426. 21. Lowe C, Alexander D & Bryla D. WICHD Amniocentesis Registry. The Safety and Accuracy of Midtrimester Amniocentesis. Washington, DC: US Department of Health, Education, and Welfare, 1978. 22. Simpson N, Dallaise L & Millen J. Prenatal diagnosis of genetic disease in Canada: report of a collaborative study. Canadian Medical Association Journal 1976; 115: 739. *23. Tabor A, Philip J & Madsen U. Randomized controlled trial of genetic amniocentesis in 4606 low-risk women. Lancet 1986; i: 1287. 24. Scott F, Peters H, Boogert T et al. The loss rates for invasive prenatal testing in a specialised obstetric ultrasound practice. Australian and New Zealand Journal of Obstetrics and Gynaecology 2002; 42: 55±58. 25. Medical Research Council Working Party of Amniocentesis. An assessment of the hazards of amniocentesis. British Journal of Obstetrics and Gynaecology 1978; 85: 1. 26. Crandall B, Howard J & Lebherz T. Follow-up of 2,000 second-trimester amniocenteses. Obstetrics and Gynecology 1980; 56: 625. 27. Gold R, Goyert G & Schwartz D. Conservative management of second-trimester post-amniocentesis ¯uid leakage. Obstetrics and Gynecology 1989; 74: 745. 28. Porreco R, Young P & Resnik R. Reproductive outcome following amniocentesis for genetic indications. American Journal of Obstetrics and Gynecology 1982; 43: 653. 29. Bowman J & Pollock J. Transplacental fetal hemorrhage after amniocentesis. Obstetrics and Gynecology 1985; 66: 749±754. 30. Brandenberg H, Jahoda MG & Pijpers L. Rhesus sensitization after mid-trimester genetic amniocentesis. American Journal of Medicine and Genetics 1989; 32: 225. 31. Hislop A, Fairweather D & Blackwell R. The e€ect of amniocentesis and drainage of amniotic ¯uid on lung development in Macaca fascicularis. British Journal of Obstetrics and Gynaecology 1984; 91: 835. 32. Milner A, Hoskyns E & Hopkin I. The e€ects of midtrimester amniocentesis on lung function in the neonatal period. European Journal of Pediatrics 1992; 151: 458±460. 33. Sant-Cassia L, MacPherson M & Tyack A. Mid-trimester amniocentesis: is it safe? A single centre controlled prospective study of 517 consecutive amniocentesis. British Journal of Obstetrics and Gynaecology 1984; 91: 736.

Clinical procedures in prenatal diagnosis 625 34. Thompson P & Greenough A. Lung volume measured by functional residual capacity in infants following ®rst trimester amniocentesis or chorion villus sampling. British Journal of Obstetrics and Gynaecology 1992; 99: 479±482. 35. Vyas H, Milner A & Hopkin I. Amniocentesis and fetal lung development. Archives of Disease in Childhood 1982; 57: 627. 36. Hunter A. Neonatal lung function following mid-trimester amniocentesis. Prenatal Diagnosis 1987; 7: 433. 37. Finegan J. Child outcome following mid-trimester amniocentesis: development, behavior, and physical status at age 4 years. British Journal of Obstetrics and Gynaecology 1990; 97: 32. 38. Bui T, Iselius L & Linsten J. European collaborative study on prenatal diagnosis: mosaicism, pseudomosaicism and single abnormal cells in amniotic ¯uid cell cultures. Prenatal Diagnosis 1984; 4: 145. 39. Hsu L & Perlis T. United States survey on chromosome mosaicism and pseudomosaicism in prenatal diagnosis. Prenatal Diagnosis 1984; 4: 97. 40. Worton R & Stern R. A Canadian collaborative study of mosaicism in amniotic ¯uid cell cultures. Prenatal Diagnosis 1984; 4: 131. 41. Klinger K, Landes G & Shook D. Rapid detection of chromosome aneuploidies in uncultured amniocytes by using ¯uorescence in situ hybridization (FISH). American Journal of Human Genetics 1992; 51: 55. *42. Jackson L, Zachary J & Fowler S. Randomized comparison of transcervical and transabdominal chorionic villus sampling. New England Journal of Medicine 1992; 327: 594±598. 43. Silver R, MacGregor S & Sholl J. Initiating a chorionic villus sampling program. Relying on placental location as the primary determinant of the sampling route. Journal of Reproductive Medicine 1990; 35: 964±968. 44. Brambati B, Oldrini A & Lanzani A. Transabdominal and trans-cervical chorionic villus sampling: Eciency and risk evaluation of 2,411 cases. American Journal of Medicine and Genetics 1990; 35: 160±164. 45. Saura R, Gauthier B & Taine L. Operator experiences and fetal loss rate in transabdominal CVS. Prenatal Diagnosis 1994; 14: 70. 46. Weiner S & Kurjak A. Interventional Ultrasound. New York: Parthenon, 1999. 47. Boehm F, Salyer S & Dev V. Chorionic villus sampling: quality control ± a continuous improvement model. American Journal of Obstetrics and Gynecology 1993; 168: 1766±1777. 48. Clark B, Bissonnette J & Olson S. Pregnency loss in a small chorionic villus sampling series. American Journal of Obstetrics and Gynecology 1989; 161: 301. 49. Green J, Dorfman A & Jones S. Chorionic villus sampling: Experience with an initial 940 cases. Obstetrics and Gynecology 1988; 71: 208. 50. Gustavii B, Claesson V & Kristo€ersson U. Risk of miscarriage after chorionic biopsy is probably not higher than after amniocentesis. Lakartidningen 1989; 86: 4221. 51. Jahoda M, Pijpers L & Reuss A. Evaluation of transcervical chorionic villus sampling with a completed follow-up of 1550 consecutive pregnancies. Prenatal Diagnosis 1989; 9: 621. 52. Young S, Shipley C & Wade R. Single-center comparison of results of 1000 prenatal diagnoses with chorionic villus sampling and 1000 diagnoses with amniocentesis. American Journal of Obstetrics and Gynecology 1991; 165: 255. 53. Hogge W, Schonberg S & Golbus M. Chorionic villus sampling: experience of the ®rst 1000 cases. American Journal of Obstetrics and Gynecology 1986; 154: 1249. 54. Jackson LCVS Newsletter 1988; 25±27. 55. Wade R & Young S. Analysis of fetal loss after transcervical chorionic villus sampling: a review of 719 patients. American Journal of Obstetrics and Gynecology 1989; 161: 513±519. 56. Wilson R, Kendrick V & Wirtman B. Spontaneous abortion and pregnancy outcome after normal ®rsttrimester ultrasound examination. Obstetrics and Gynecology 1986; 67: 352±355. 57. Group CCC-ACT. Multicentre randomized clinical trial of chorion villus sampling and amniocentesis. Lancet 1989; i: 1. *58. Rhoads G, Jackson I & Schlesselman S. The safety and ecacy of chorionic villus sampling for early prenatal diagnosis of cytogenetic abnormalities. New England Journal of Medicine 1989; 320: 609±619. 59. Blakemore K, Mahoney M & Hobbins J. Infection and chorionic villus sampling. Lancet 1985; 2: 339. 60. Wapner R, Barr M & Heeger S. Chorionic villus sampling: a 10-year, over 13,000 consecutive case experience. Presented at the First Annual Meeting of the American College of Medical Genetics, Orlando, Florida, March 1994. 61. Firth H, Boyd P & Chamberlain P. Severe limb abnormalities after chorion villus sampling at 56±66 days gestation. Lancet 1991; 337: 726. 62. Hoyme F, Jones K & Van Allen M. Vascular pathogenesis of transverse limb reduction defects. Journal of Pediatrics 1982; 101: 839. 63. Foster-Iskenius U & Baird P. Limb reduction defects in over 1,000,000 consecutive live births. Teratology 1989; 39: 127.

626 B. Eisenberg and R. J. Wapner 64. Mastroiacovo P, Botto L & Cavalcanti D. Limb anomalies following chorionic villus sampling: a registry based case control study. American Journal of Medicine and Genetics 1992; 44: 856±863. 65. Brambati B, Simoni G & Traui M. Genetic diagnosis by chorionic villus sampling before 8 gestational weeks: eciency, reliability, and risks on 317 completed pregnancies. Prenatal Diagnosis 1992; 12: 784±799. 66. Burton B, Schultz C & Burd L. Spectrum of limb disruption defects associated with chorionic villus sampling. Pediatrics 1993; 91: 989±993. *67. Froster U & Jackson L. Limb defects and chorionic villus sampling: results from an international registry, 1992±1994. Lancet 1996; 347: 489. 68. Brent R. Relationship between uterine vascular clamping, vascular disruption syndrome and cocaine teratology. Teratology 1990; 41: 757. 69. Olney R, Khoury M & Alo C. Increased risk for transverse digital de®ciency after chorionic villus sampling: results of the United States Multistate Case-Control Study, 1988±1992. Teratology 1995; 1: 20±29. 70. Martin A, Simpson J & Rosinksy B. Chorionic villus sampling in continuing pregnancies. II. Cytogenetic reliability. American Journal of Obstetrics and Gynecology 1986; 154: 1653±1662. 71. Simoni G, Girnelli G & Cuoco C. Discordance between prenatal cytogenetic diagnosis after chorionic villi sampling and chromosomal constitution of the fetus. In Brambati B (ed.) First Trimester Fetal Diagnosis, pp 137±143. Heidelberg: Springer-Verlag, 1985. 72. Cheung S, Crane J & Beaver H. Chromosome mosaicism and maternal cell contamination in chorionic villi. Prenatal Diagnosis 1987; 7: 535±542. 73. Mikkelsen M & Ayme S. Chromosomal ®ndings in chorionic villi. In Sperling K (ed.) Human Genetics, p 597. Heidelberg: Springer-Verlag, 1987. 74. Simoni G, Brambati B & Danesino C. Ecient direct chromosome analysis and enzyme determinations from chorionic villi samples in the ®rst trimester of pregnancy. Human Genetics 1983; 63: 349. *75. Ledbetter D, Martin A & Verlinsky Y. Cytogenetic results of chorionic villus sampling: high success rate and diagnostic accuracy in the United States collaborative study. American Journal of Obstetrics and Gynecology 1990; 162: 495. 76. Karkut I, Zakrzewski S & Sperling K. Mixed karyotypes obtained by chorionic villi analysis: mosaicism and maternal contamination. In Brambati B (ed.) First Trimester Fetal Diagnosis, pp 144±146. Heidelberg: Springer-Verlag, 1985. *77. Cassidy S, Lai L & Erickson R. Trisomy 15 with loss of the paternal 15 as a cause of Prader±Willi syndrome due to maternal disomy. American Journal of Human Genetics 1992; 51: 701. 78. Purvis-Smith S, Sayille T & Manass S. Uniparental disomy 15 resulting from ``correction'' of an initial trisomy 15. American Journal of Human Genetics 1992; 50: 1348. 79. Ledbetter D & Engel E. Uniparental disomy in humans: development of an imprinting map and its implications for prenatal diagnosis. Human Molecular Genetics 1995; 4: 1757±1764. *80. Wolstenholine J. Con®ned placental mosaicism for trisomies 2, 3, 7, 8, 9, 16, and 22: their incidence, most likely origins, and mechanisms for cell lineage compartmentalization. Prenatal Diagnosis 1996; 16: 511. 81. Kalousek D, Dill F & Pantzat T. Con®ned chorionic mosaicism in prenatal diagnosis. Human Genetics 1987; 77: 163. 82. Johnson A, Wapner R & Davis G. Mosaicism in chorionic villus sampling: an association with poor prenatal outcome. Obstetrics and Gynecology 1990; 75: 573. 83. Wapner R, Simpsom M & Golbus M. Chorionic mosaicism: association with fetal loss but not with adverse perinatal outcome. Prenatal Diagnosis 1992; 12: 347. 84. Goldberg J, Proter A & Golbus M. Current assessment of fetal loss as a direct consequence of chorionic villus sampling. American Journal of Medicine and Genetics 1990; 35: 174. *85. Kalousek D, Howard-Pebbles P & Olson S. Con®rmation of CVS mosaicism in term placentae and high frequency of interuterine growth retardation association with con®ned placental mosaicism. Prenatal Diagnosis 1991; 11: 743. 86. Benn P. Trisomy 16 and trisomy 16 mosaicism: a review. American Journal of Human Genetics 1998; 79: 121±133. 87. Vejerslev L & Mikkelsen M. The European collaborative study on mosaicism in chorionic villus sampling: data from 1986±1987. Prenatal Diagnosis 1989; 9. 88. Breed A, Mantingh A & Vosters R. Follow-up and pregnancy outcome after a diagnosis of mosaicism in CVS. Prenatal Diagnosis 1991; 11: 577. 89. Phillips O, Tharapel A & Lerner J. Risk of fetal mosaicism when placental mosaicism is disgnosed by chorionic villus sampling. American Journal of Obstetrics and Gynecology 1996; 174: 850. 90. Bartels I, Hansmann I & UH. Down syndrome at birth not detected by ®rst trimester chorionic villus sampling. American Journal of Medicine and Genetics 1989; 34: 606.

Clinical procedures in prenatal diagnosis 627 91. Lilford R, Caine A & Linton G. Short-term culture and false-negative results for Down's syndrome on chorionic villus sampling. Lancet 1991; 337: 861. 92. Miny P, Basaran S & Holzgreve W. False-negative cytogenetic result in direct preparations after CVS. Prenatal Diagnosis 1988; 8: 663. 93. Simoni G, Fraccaro M & Girnelli G. False-positive and false-negative ®ndings on chorionic villus sampling. Prenatal Diagnosis 1987; 7: 671. 94. Martin A, Elias S & Rosinksy B. False-negative ®ndings on chorion villus sampling. Lancet 1986; 2: 391. 95. Grebner E & Jackson L. Prenatal diagnosis for Tay±Sachs disease using chorionic villus sampling. Prenatal Diagnosis 1985; 5: 313. 96. Gray R, Green A & T C. A misdiagnosis of X-linked adrenoleukodystrophy in cultured villus cells by the measurement of very long fatty acids. Prenatal Diagnosis 1995; 15: 486. 97. Castellvi-Bel S, Mila M & Solar A. Prenatal diagnosis of fragile X syndrome: expansion and methylation of chorionic villus samples. Prenatal Diagnosis 1995; 15: 801.