Antenatal Diagnosis of Chromosomal Abnormalities

Antenatal diagnosis is an accepted component of prenatal care for women at increased risk for chromosomally abnormal offspring. The purpose of this chapter, which updates previous communications^{1, 2} is to review currently accepted indications and techniques for antenatal cytogenetic (chromosome) diagnosis, to consider additional indications that may prove valid, and to discuss the development of new techniques for detecting fetal chromosomal abnormalities.

Chromosome analysis can be performed on any of a variety of fetal tissues. Consideration of risks, technical expertise, and desire for rapid diagnosis figure in choice of method.

Amniotic fluid cells

Details of amniotic fluid cytology and cell culture methodology have been published elsewhere,^{3, 4} and the technique of amniocentesis is described in detail by Verp and Gerbie⁵ and elsewhere in this library. Therefore, features important to the clinician are emphasized in this chapter.

In early gestation, amniotic fluid resembles a dialysate of maternal serum; solutes are present in concentrations similar to those found in maternal serum. In addition, amniotic fluid contains fetal proteins (e.g., α-fetoprotein) and desquamated cells presumably derived from fetal skin, from gastrointestinal, genitourinary, and respiratory tracts, and from amnion. Cell concentration in amniotic fluid increases with gestation; however, not all of these cells are viable (35% at 15–17 weeks' gestation).^{4, 6} Cytogenetic analysis requires that cells be in mitosis; therefore, viable amniotic fluid cells must be cultivated in tissue culture before analysis is possible. With recent advances in culture media, laboratories routinely complete tissue culture and analysis of amniotic fluid cells in 2 weeks or less.

Because the interval from sampling to diagnosis is disconcerting to many patients, investigators have sought ways to shorten this period. An approach that uses interphase rather than mitotic cells offers significant advantages in this respect. Chromosome-specific DNA probes hybridize to chromosomes irrespective of cell cycle phase. Incubation of amniotic fluid cells with a labeled chromosome-specific DNA probe results in visual hybridization signals equal in number to the number of copies of that chromosome in the cell (e.g., fluorescence in situ hybridization [FISH]). Not only can the number of copies of one particular chromosome be detected but  cells can also be probed simultaneously for several chromosomes (e.g., chromosomes 21, 18, 13, X, and Y), thereby detecting all the common aneuploidies seen in newborns. Amniotic fluid cells, chorionic villus cells, fetal lymphocytes, or nucleated erythrocytes all are feasible targets for hybridization.⁷ This technique has proved useful for analysis of fetal cells detected in the maternal circulation. Many prospective studies of FISH analysis of amniotic fluid cells have been reported, with good predictive values and results available in 1 day.⁸ However, not all samples are informative, and not all chromosome abnormalities are detectable with this approach.⁹ Therefore, standard cytogenetic analysis should be performed in addition to FISH.

Chorionic villi

Because results from amniotic fluid cultures usually are not available until the middle of the second trimester, if an abnormality is diagnosed and pregnancy termination performed, maternal risk, psychological stress, and expense are considerably greater than they would have been for a first-trimester pregnancy termination. Such considerations led to the development of chorionic villus sampling (CVS), a technique in which a biopsy is performed on the placenta in the first trimester. Because early trophoblastic tissue contains many spontaneously dividing cells, results are usually available sooner than with cultured amniotic fluid cells. A full discussion of the methods and risks associated with CVS is given elsewhere in this library.

Fetal lymphocytes

It also is possible to perform chromosome analysis on fetal lymphocytes, historically obtained by fetoscopically directed aspiration or by placentesis. Results can be obtained from such cultures in less than 1 week. However, with fetoscopy and placentesis, there was a 5% risk of fetal loss; therefore, such procedures rarely were indicated for routine cytogenetic studies.

More recently, fetal lymphocytes have been aspirated in the second and third trimesters by ultrasound-directed umbilical cord blood sampling (PUBS).^{10, 11} This approach allows rapid diagnosis, particularly helpful in the case of late referral, with a complete cytogenetic analysis including structural chromosome rearrangements. Because only a narrow-gauge needle is inserted into the uterus, there is considerably less morbidity than that following fetoscopy.

Other tissues

Because cytogenetic analysis and FISH studies can be performed on most types of nucleated fetal cells, occasionally it is expedient to sample a different fetal tissue. For example, both cystic hygroma fluid and fetal urine (from a distended bladder) are amenable to cytogenetic evaluation.^{12, 13} Either of these fluids, if accessible, can be substituted for amniotic fluid.

Potential problems in interpretation

In most well-established laboratories, the success rate for amniotic fluid cultures is high, although variation naturally exists as a function of the experience and techniques of a given laboratory. Nonetheless, there are several potential sources of error or confusion. First, cells may not grow, or poor growth may provide insufficient cells for proper analysis. Some culture failures may be the result of insufficient numbers of viable cells in the original sample. Direct preparation of spontaneously dividing CVS cells usually yields some metaphases. These metaphases may, however, be of poorer quality than those obtained from cultures. A second source of error is that maternal rather than fetal cells may be cultured. Benn and Hsu¹⁴ have estimated that this phenomenon occurs in 0.3% of amniocenteses. Almost all cases of 46,XX/46,XY mosaicism in amniotic fluid cultures are caused by maternal cell contamination of a sample from a normal male fetus. The incidence of maternal cell contamination can be minimized by not using the first few drops of aspirated amniotic fluid for cell culture. Analysis of maternal rather than fetal cells is a particular concern in CVS because the sample obtained is usually a mixture of maternal decidua and chorionic villi. Careful attention must be given to separation of these tissues. Fetal rather than maternal origin of cultured cells can be verified by comparing DNA polymorphisms in maternal and paternal blood samples to those in the presumptive fetal specimen. However, because of the rarity of maternal cell contamination, a laboratory may elect not to perform such analyses in routine cases. Problems caused by cross-contamination of amniotic fluid in twin gestations are rare. My colleagues and I have cultured cells from both sacs of a large number of unlike sexed twins and have never observed cross-contamination. Cross-contamination is potentially a greater problem in CVS, unless completely separate placentas can be seen on ultrasound.

Table 1. Hypermodality in 1000 amniotic fluid specimens at Northwestern University

*A single chromosome broken into two to yield a spurious hypermodal count. (Simpson JL, Martin AO, Verp MS, et al: Hypermodal cells in amniotic fluid cultures: Frequency, interpretation, and clinical significance. Am J Obstet Gynecol 143: 250, 1982)

A third source of error is in vitro origin of aberrations. In vitro aberrations arise in all culture systems and should be suspected if many different aberrations are detected in the same specimen or if an abnormality is detected in only one of several cultures initiated from the same specimen. The problems of in vitro aberration are considered in more detail elsewhere,¹⁵ but briefly, cells containing an extra chromosome (N = 47) occur in approximately 5% of amniotic fluid specimens (Table 1). If the aberrant cells are confined to a single clone (in situ technique) or culture (flask technique), and multiple other clones or cultures do not contain cells with the identical aberration, the finding is termed pseudomosaicism and is without clinical significance in almost all cases. Detection of a single cell with a chromosomal trisomy associated with live birth (e.g., trisomy 21, polysomy X) should, however, be of greater concern than the finding of a cell with trisomy 2. Tetraploidy typically occurs in human amnion and should not cause concern.

In contrast to pseudomosaicism, true fetal mosaicism is likely to be present if cells with the same abnormal complement are detected in more than one flask or clone. True fetal mosaicism, defined by the presence of consistent abnormalities in multiple flasks, occurred in 0 of 1000 amniotic fluid specimens that our group analyzed¹⁵ and in only 0.25% of a large collaborative study.¹⁶ When consistent abnormalities are present, the neonate is subsequently confirmed to be a mosaic in 67% of cases. Autosomal mosaicism is much more frequently associated with phenotypic anomalies at birth or abortion (29%) than is sex chromosome mosaicism (11%).¹⁷ The finding of mosaic chromosomal abnormalities in chorionic villi is more common than in amniotic fluid cultures and occurs in approximately 1% of cases. Most of the time, the abnormality proves to be limited to villi and is not present in the fetus (confined placental mosaicism). Amniocentesis usually is recommended for clarification of fetal chromosome status in such cases, with uniparental disomy studies if the additional chromosome is one known to be clinically relevant.^{18, 19, 20} True mosaicism may not be detected prenatally if the minority cell line is limited to tissues not sampled or if the line is of low frequency. Detection of mosaicism always is a concern in cytogenetic analysis; however, the problem is of particular relevance to antenatal diagnosis because relatively few cells are of sufficient quality for reliable analysis. Despite potential for error, however, accuracy in cytogenetic diagnosis is 99% or greater.

In addition to errors, dilemmas in interpretation arise from time to time. For example, as mentioned previously, prediction of the phenotype is difficult when chromosomal mosaicism is diagnosed. The same is true when an apparently balanced translocation, an inversion, or a small supernumerary (marker) chromosome is detected. In such cases, parental chromosomes should be analyzed immediately. If a phenotypically normal parent has the identical translocation, inversion, or marker chromosome, the fetus also can be expected to be phenotypically normal, although in rare cases, inheritance of a parental translocation involving chromosomes 14 or 15 has resulted in a child with uniparental disomy.²¹ Conversely, if the translocation, inversion, or marker chromosome has arisen de novo in the fetus, pooled data indicate that 5–15% of such fetuses will be phenotypically abnormal.²² In the case of a marker chromosome, additional analysis with FISH and unique sequence chromosome-specific DNA probes, and special banding studies, usually allows the origin of the marker to be identified, aiding prognostication.²³ Comparative genomic hybridization (CGH) is a quantitative method of evaluating additional (or missing) chromosomal material. A digital image system analyses fluorescently labeled aberrant DNA, comparing it to the genomic DNA found in a normal cell. This approach can be used to further clarify the origin of supernumerary marker chromosomes.²⁴

The finding of a sex chromosome abnormality also creates a quandary. Although abnormal phenotype and slow development are associated with 45,X, 47,XXX, 47,XXY, and 47,XYY, most individuals with these complements are neither severely retarded nor grossly malformed.^{25, 26, 27, 28} Parents may have great difficulty in deciding whether to terminate such a pregnancy. At Northwestern University, we found that only 41% of pregnancies with sex chromosome abnormalities diagnosed at amniocentesis were terminated, in contrast to 88% of those with autosomal trisomies and none with de novo balanced structural abnormalities.²⁹

Cytogenetic studies can be performed readily from amniotic fluid cells, chorionic villus cells, or fetal lymphocytes. Thus, virtually all chromosomal disorders are potentially detectable in utero. Although technically feasible, however, it is not appropriate to determine the complement of every fetus because for many couples, the risks of prenatal diagnosis outweigh the potential benefits. Amniocentesis is considered to increase the risk of spontaneous abortion by approximately 0.25% over the background.^{30, 31} CVS increases the pregnancy loss rate by approximately 1%;³⁰ the risk associated with umbilical cord blood sampling probably is similar.^{10, 11} In this section, unequivocal indications for cytogenetic studies are considered.

Advanced maternal age

Traditionally, advanced maternal age has been an indication for antenatal cytogenetic studies.³² Although there still is no unequivocal explanation for the relationship between aneuploidy and advanced maternal age, one factor could be that with advancing maternal age, there is decreasing maternal selection against chromosomally abnormal conceptuses.³³ Given that approximately 7% of conceptuses but only 0.5% of liveborn infants are chromosomally abnormal, it is not unreasonable to suggest that maternal selection may be a factor in elimination of abnormal embryos. Another, more widely accepted, hypothesis is that chiasmata between homologous chromosomes decrease in aging oocytes, leading to nondisjunction and chromosomally abnormal ova. A more recent theory is that it is the decline in the oocyte pool, or in the number of maturing oocytes per cycle, that accounts for the increase in trisomies with advancing maternal age.^{34, 35} In any case, in contrast to the overall incidence of trisomy 21 (1:800 live births in the United States),³⁶ the likelihood of a 35-year-old mother having a child with trisomy 21 is 1:385; at 39 years of age, the risk is 1:137, and at 45 years of age, the risk is 1:30 (Table 2).³⁷

Table 2. Risk of having a liveborn child with Down syndrome or other chromosomal abnormality

Because sample size for some intervals is relatively small, 95% confidence limits are sometimes relatively large. Nonetheless, these figures are suitable for genetic counseling.
*47,XXX excluded for ages 20–32 (data not available). (Data from Hook EB: Rates of chromosome abnormalities at different maternal ages. Obstet Gynecol 58: 282, 1981; and Hook EB, Cross PK, Schreinemachers DM: Chromosomal abnormality rates at amniocentesis and in live-born infants. JAMA 249: 2034, 1983)

Trisomy 21 is not the only chromosomal abnormality that increases with maternal age. Trisomy 13, trisomy 18, 47,XXX, and 47,XXY also show an increased mean maternal age.³⁷ Based on these data, United States authorities believe that prenatal diagnosis should be offered, as an alternative to screening, to all women who will be 35 years of age or older when their infant is born. However, the choice of a particular age is largely arbitrary because the risk for a chromosomally abnormal child increases steadily year to year, even among younger women. Therefore, flexibility is desirable when confronted by an inquiry from a woman younger than 35 years of age. Some women younger that 35 years of age may be relatively less concerned about the risk of abortion than the risk of a chromosomally abnormal liveborn infant and may wish to have a diagnostic procedure, despite the ostensibly unfavorable risk-to-benefit ratio.

Finally, it is worth emphasizing that the aforementioned risk figures are based on detection in liveborn infants. In fact, the incidence of abnormalities in antenatal studies at 16–18 weeks' gestation is approximately 50% higher than that in liveborn infants,^{37, 38} and the incidence in first-trimester CVS studies is even greater.³⁹ The discrepancies between the frequencies in liveborn infants and in first-trimester and second-trimester fetuses are accounted for by the disproportionate number of chromosomally abnormal fetuses that abort spontaneously before live birth.^{37, 38, 39, 40}

Maternal serum screening

Maternal serum α-fetoprotein (MSAFP) screening initially was developed for detection of fetal neural tube defects, which are associated with elevated values of MSAFP. However, in 1984, Merkatz and associates observed that fetal autosomal trisomy was associated with low MSAFP values.⁴¹ Why MSAFP is decreased in such pregnancies still is uncertain but probably relates to decreased fetal production of α-fetoprotein. Complicating screening, however, is that the median MSAFP level is decreased only slightly in pregnant women carrying fetuses with Down syndrome. Fortunately, other fetal–placental products have also proved useful for prenatal screening for Down syndrome in both the first and second trimesters (see also Chapter 114). Initially studied in second trimester pregnancies, Bogart and colleagues showed that maternal serum human chorionic gonadotropin (hCG) levels were significantly higher in pregnancies complicated by chromosome abnormalities.⁴² In fact, hCG was superior to MSAFP as a screening tool for chromosomal abnormalities. Canick and colleagues reported decreased levels of maternal serum unconjugated estriol in the second trimester in women with Down syndrome pregnancies.⁴³ The authors suggested that decreased unconjugated estriol was related to immaturity of the fetal adrenal cortex, fetal liver, and placenta.

This suggested that screening would be most efficient if based on the aggregated values of maternal age, MSAFP, unconjugated estriol, and hCG. Using this combination of maternal serum markers and maternal age, Wald and colleagues retrospectively identified 60% of 77 Down syndrome pregnancies with a projected amniocentesis rate of 5%.⁴⁴ In the first prospective trial of this approach in the United States, Haddow and associates reported screening more than 25,000 women.⁴⁵ Patient-specific risk estimates were based on maternal age and serum screening, and patients were offered amniocentesis when the calculated risk for fetal Down syndrome was greater than or equal to 1 in 190 and gestational age was verified (3.8% of the population). Two thirds of known cases of Down syndrome were detected. Assuming that the prevalence of Down syndrome in the second trimester actually was higher but that some undetected abnormal fetuses were aborted spontaneously before live birth, the authors calculated a detection rate for Down syndrome of 58%. In a similar fashion, Phillips and colleagues have reported a prospective trial restricted to women younger than 35 years old.⁴⁶ In this study, the risk cut-off was 1 in 274, with identification of 57% (four of seven) of known cases of Down syndrome and an amniocentesis rate of 3.2%. A third prospective trial by Burton and colleagues used a cut-off of 1 in 270.⁴⁷ With a 10.4% initial positive rate and an offered amniocentesis rate of 5.9%, ten of 12 known cases of Down syndrome were identified. Increased risk for trisomy 18 also was identified with a different combination of cut-off values for the three serum markers (decreased MSAFP, unconjugated estriol, and hCG). One abnormality was detected for every 33 amniocenteses performed, including two cases of trisomy 18 and one case of triploidy. In addition, three of the five cases of 45,X in the screened population were detected.

More recently, addition of inhibin A to the Down syndrome serum screening panel has been shown to increase detection efficiency.⁴⁸ Further, urinary analytes, e.g., hyperglycosylated hCG, are being examined for usefulness in Down syndrome screening.⁴⁹

In the first trimester, median total β-hCG and free β-hCG are elevated and pregnancy-associated plasma protein A (PAPP-A) is decreased in women with Down syndrome fetuses and all three analytes are decreased with trisomy 18.⁵⁰ These markers, combined with measurement of fetal nuchal translucency, have been studied in Great Britain and the United States and the detection rates of first and second trimester screening have proved very similar.

Both first and second trimester serum screening have proved very useful for providing an individual, patient-specific risk for women younger than 35 years of age who typically would not be offered prenatal diagnosis. Whether maternal serum screening should be substituted for amniocentesis or CVS for women 35 years of age and older has been a controversial issue.^{51, 52, 53} For example, if invasive testing is offered only to women with abnormal second trimester serum screens rather than to all women older than 34 years of age, the detection rate of fetal Down syndrome is lower (89%), but only 25% of this population requires amniocentesis. Conversely, if all older women are offered and accept amniocentesis or CVS, the detection rate is 100%. Additionally, multiple marker screening is not highly sensitive in detecting chromosome abnormalities other than trisomy 21 and trisomy 18, which also increase with advancing maternal age. Thus, a significant number of aneuploidies are missed when using serum screening alone rather than invasive testing. Awaiting the results of second trimester multiple marker screening before offering invasive testing also frequently results in delay in the diagnosis of chromosome abnormalities until approximately 20 weeks' gestation, rather than detection in the late first or early second trimester after CVS or amniocentesis. Moreover, screening is less sensitive in multiple gestations compared to singleton gestations. Finally, justly or not, obstetricians are concerned about their legal liability should an older woman have a child with Down syndrome after a normal serum screen. For these reasons, the advantages and limitations of using serum screening as an alternative to invasive testing for cytogenetic abnormalities should be discussed carefully with patients before a choice is made.⁵⁴

Previous child With chromosomal abnormality

After the birth of one child with either an autosomal trisomy or a sex chromosome abnormality, the likelihood that subsequent progeny will have a chromosomal abnormality traditionally has been considered increased, even if parental chromosome complements are normal. However, the risk for a second offspring with Down syndrome or another chromosomal abnormality appears to be substantially increased primarily for mothers 29 years of age or younger at the time of the birth of the proband with Down syndrome (Table 3).⁵⁵⁵⁶ Nonetheless, parental anxiety dictates that antenatal chromosomal studies at least be offered to all couples who have previously had a child with Down syndrome.

Table 3. Relationship of recurrence risk to maternal age at birth of proband with Down syndrome

*Not significant. (Data from Mikkelsen M: Down syndrome: Current stage of cytogenic epidemiology. In Bonne-Tamir B, Cohen T, Goodman RM, (eds): Human Genetics, Part B: Medical Aspects, pp 297–309. New York, Alan R. Liss, 1982)

Information concerning recurrence risk after the birth of a child with a chromosomal abnormality other than trisomy 21 is very limited, but data from five collaborative studies indicate that the risk is 1–2% for either the same or a different chromosomal abnormality (Table 4).^{57, 58, 59, 60, 61} Thus, antenatal studies also should be offered to such couples.

Table 4. Recurrence risk for a chromosomal abnormality after birth of a child with a chromosomal abnormality other than trisomy 21

CVS, chorionic villus sampling
(Data from Mikkelsen M, Stene J: Previous child with Down syndrome and other chromosome aberrations. In Murken JD, Stengel-Rutdowski S, Schwinger E, [eds]: Prenatal Diagnosis: Proceedings of the Third European Conference on Prenatal Diagnosis of Genetic Disorders, pp 22–23. Stuttgart, F Enke, 1979; Simoni G, Fraccaro M, Arslanian A, et al: Cytogenetic findings in 4952 prenatal diagnoses: An Italian collaborative study. Hum Genet 60: 63, 1982; Stene J, Stene E, Mikkelsen M: Risk for chromosome abnormality at amniocentesis following a child with a noninherited chromosome aberration. Prenat Diagn 4 [special issue]: 81, 1984; Mikkelsen M: In Jackson L [ed]: CVS Newsletter, No. 19, pp 7–10. December 1, 1986; Jewell AF, Keene WE, Ferre MM, et al: Analysis of the recurrence risks for trisomy 13 and 18. Am J Hum Genet 59: A121, 1996

Parental translocation, inversion, or aneuploidy

TRANSLOCATION

A third, less common, cytogenetic indication for antenatal diagnosis is the presence of a balanced translocation in a parent. The rare detection of an inversion or a numerical chromosomal abnormality (aneuploidy) warrants similar attention. The significance of a translocation can be illustrated by considering the most common type of translocation Down syndrome, a Robertsonian translocation between chromosomes 14 and 21 (Robertsonian translocations involve the acrocentric chromosomes: 13, 14, 15, 21, 22). If a child has Down syndrome resulting from such a translocation (e.g., 46,XX, −14, +t[14q;21q]), the rearrangement originates de novo in 50–75% of cases (e.g., it is present in neither parent). The likelihood of Down syndrome recurring in the progeny of parents whose previous offspring had a de novo translocation probably is minimal, although recurrence of apparently de novo translocations (21q;21q) has been reported.⁶² Conversely, in 25–50% of subjects who have Down syndrome as a result of a translocation, one parent has the same translocation chromosome in a balanced state (e.g., 45,XX, −14,−21, +t[14q;21q]). The theoretical risk that a parent carrying a t(14q;21q) chromosome will have a child with Down syndrome is 33%. However, empirical risks are considerably less. If the father carries the translocation, the risk is approximately 3%, whereas if the mother carries the translocation, the risk is approximately 10–15%. This sex-specific difference has been found in cases ascertained through chromosomally abnormal liveborn infants,⁶³ as well as in collaborative reports of amniotic fluid studies⁶⁴ and CVS⁶⁰ (Table 5). Risks are considered similar for other Robertsonian translocations involving chromosome 21 (e.g., t[13q;21q], t[15q;21q], t[21q;22q]), but Robertsonian translocations that do not include chromosome 21 apparently carry much lower risks for unbalanced offspring. In fact, t(13q;14q), the most common Robertsonian translocation found in normal persons, apparently confers 1–2% risk (see Table 5).⁶⁴ Liveborn offspring of individuals with balanced homologous translocations (e.g., 21q;21q or 13q;13q) will virtually all be trisomic for the involved chromosome.

Table 5. Risk of an unbalanced rearrangement in a second trimester fetus if a parent has a balanced rearrangement (carrier)

(Data from Mikkelsen M: In Jackson L (ed): CVS Newsletter, No. 19, pp 7–10. December 1, 1986 and Daniel A, Hook EB, Wulf G: Risks of unbalanced pregnancy at amniocentesis to carriers of chromosomal rearrangements: Data from United States and Canadian laboratories. Am J Med Genet 31: 14, 1989)

Reciprocal translocations do not involve centromeric fusion and, hence, usually do not involve acrocentric chromosomes. Unfortunately, because of their individual rarity, specific empirical data for most translocations are not available, and generalizations must be made on the basis of pooled data derived from many different translocations. Knowledge of the length of the translocated segment provides some additional guidance in predicting risk of a fetus with an unbalanced translocation, in other words, a longer translocation segment is associated with a lower risk.⁶⁴ However, overall theoretical risks for abnormal (unbalanced) offspring are greater than empirical risks, which are approximately 10% for either maternal or paternal carriers (see Table 5).⁶⁴

INVERSIONS

In a chromosomal inversion, the normal sequence of genes on the chromosome is altered. Subjects with such inversions are phenotypically normal; however, they may produce unbalanced gametes if, during meiosis I, crossing over (recombination) occurs within the inverted sequence. Thus, certain genes would be duplicated and others would be deficient in the unbalanced gamete (see Fig. 1). Pericentric inversions and inversions involving long segments are more likely to be associated with anomalous offspring than are paracentric or short inversion segments.⁶⁵ Empirical data are not available for specific inversions, but pooled data for all inversions indicate approximately a 3% risk for abnormal progeny, with maternal carriers again at greater risk than paternal carriers⁶⁴ (see Table 5). An exception is inv(9), which is a common variant and is thought to be without clinical significance.

ANEUPLOIDY

If a parent has a numerical chromosomal abnormality (aneuploidy), the risk to offspring is increased. For example, approximately 35% (but not 50%) of offspring of females with 47,XX, + 21 (Down syndrome) are aneuploid;⁶⁶ therefore, antenatal chromosomal studies are indicated in a pregnant female with Down syndrome. Males with Down syndrome are sterile. If a parent is mosaic for trisomy 21, antenatal diagnosis is again in order.^{67, 68} Although risk figures are plainly biased by the method of ascertainment, approximately 20% of offspring of fertile 45,X; 45,X/46,XX; and 45,X/46,XX/47,XXX subjects are said to show abnormalities.⁶⁶ Women with 47,XXX or 46,XX/47,XXX also have produced children with chromosomal abnormalities, although most of the offspring are normal. Theoretically, 47,XYY men are also at increased risk for chromosomally abnormal offspring, and several abnormal offspring have been reported. Men with 47,XXY (Klinefelter syndrome) are sterile, but those with mosaicism (46,XY/47,XXY) may be fertile. Antenatal diagnosis should be offered to all aneuploid parents.

RELATIONSHIP OF ASCERTAINMENT TO EMPIRICAL RISK

Mode of ascertainment is a significant determinant of empirical risk for an unbalanced liveborn infant. Thus, when a family with a translocation is ascertained through a balanced proband, the risk for an unbalanced liveborn infant is very low. In contrast, if ascertainment is through an unbalanced individual, the risk for unbalanced offspring is significantly greater.^{69, 70} Presumably, then, with some translocations, unbalanced gametes do not arise during meiosis, or, alternatively, unbalanced products are selected against at the gametic or embryonic level. If a rearrangement has been ascertained during an evaluation for repetitive abortions, the risk for an unbalanced liveborn infant is lower than the risk expected after ascertainment through an anomalous liveborn infant but still is substantial.⁷⁰

Fetuses manifesting intrauterine growth retardation or anomalies on ultrasound examination

The potential indications considered previously are based on the premise that abnormal fetal outcome can be predicted on the basis of certain parental characteristics. However, trisomic fetuses, especially fetuses with trisomy 13 or 18, often show intrauterine growth retardation, which may be clinically evident during the second trimester. Clinical suspicion of intrauterine growth retardation can be followed with ultrasound monitoring to confirm intrauterine growth retardation. Gross anomalies also frequently can be visualized in fetuses with chromosomal abnormalities (Table 6).^{71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9192, 93, 94} Antenatal chromosomal studies are appropriate if an abnormal fetus is detected on ultrasound examination. In addition to routine cytogenetic studies, particular defects suggest the need for more specific studies. For example, conotruncal heart defects are frequently associated with deletion of a small portion of chromosome 22 (del22q.11.2). FISH with specific probes will be diagnostic of this microdeletion that also implies the presence of other defects, for example, absent thymus and parathyroids (DiGeorge/velocardiofacial syndrome).^{95, 96} Even if chromosomal studies cannot be obtained sufficiently early to permit pregnancy termination or termination is not desired, cesarean section for fetal distress in a fetus with a lethal abnormality might be avoided.

Table 6. Chromosomal abnormalities in pregnancies with anomalies detected by ultrasonography

*Listed under primary defect indicated by the investigator. In some cases, multiple abnormalities were present.

Several authors have reported that certain biometric findings (e.g., short femur, short humerus, pyelectasis, thickened nuchal fold, nuchal translucency, echogenic bowel, absent nasal bone) are indicative of an increased risk of fetal Down syndrome.^{97, 98, 99, 100} The positive predictive value of the ultrasound findings depends on the patient's a priori risk based on maternal age or biochemical serum screening. Different recommendations have been made as to how best to estimate the absolute risk of Down syndrome.¹⁰¹ Antenatal diagnosis should be offered when the risk estimate is greater than the procedure-associated risk of pregnancy loss.

Mendelian disorders associated with chromosome breakage

Several inherited disorders are characterized by chromosome breakage in vivo and in vitro. Persons with these disorders often show increased propensity for neoplasia, growth retardation, and various somatic anomalies. Bloom's syndrome, ataxia-telangiectasia, and Fanconi's anemia are examples of such disorders. In some of these disorders, when the precise molecular defect is not known in individual families, distinctive cytogenetic features may permit antenatal diagnosis. For example, Voss and colleagues diagnosed Fanconi's anemia in a second-trimester fetus on the basis of high frequencies of spontaneous and clastogen-induced chromosome breakage in amniotic fluid cells.¹⁰² Similar studies have been performed with chorionic villus tissue¹⁰³ and fetal blood.¹⁰⁴ Parallel cultures of cells from other family members are required to distinguish affected fetuses from heterozygotes.¹⁰⁵

Knowledge that patients with ataxia-telangiectasia (A-T) have an increased rate of spontaneous chromosome breakage has historically facilitated the diagnosis in the second trimester.^{106, 107} However, localization of the A-T gene now allows more reliable molecular genetic testing.¹⁰⁸ In Bloom's syndrome, the rate of sister chromatid exchanges is increased in peripheral lymphocytes, fibroblasts, and bone marrow cells, making prenatal diagnosis feasible even in families in which the gene mutation is unidentified. All these disorders are inherited in autosomal recessive fashion; therefore, couples who have had an affected child have a 25% recurrence risk in each pregnancy. Antenatal diagnosis should be offered to such families.

Fragile X syndrome and other X-linked recessive disorders

The fragile X syndrome is an X-linked disorder characterized in males by moderate mental retardation, macroorchidism, and a long face with a prominent jaw. Approximately one third of female carriers (heterozygotes) are mildly retarded, and the others have a normal phenotype. This syndrome accounts for a significant proportion of cases of familial X-linked mental retardation. The gene responsible for the condition is linked to a fragile site on the long arm of the X chromosome, visible as a break, or gap, in the chromosome structure. The fragile site is seen only when cells are grown in special medium deficient in folic acid and thymidine or when an antimetabolite such as 5-fluorodeoxyuridine or methotrexate is added to the culture medium.

Prenatal diagnosis has been accomplished by visualization of the fragile site in amniotic fluid cells, chorionic villus tissue, and fetal blood.^{109, 110, 111, 112, 113} Unfortunately, both false-negative and false-positive results have occurred in amniotic fluid and CVS samples.^{111, 112, 113} Molecular (DNA) methods also have been used to diagnose fragile X syndrome and have proven the more reliable approach.^{114, 115}

In other X-linked recessive, or male-limited autosomal dominant traits, only males are affected. It is possible to distinguish affected from unaffected male fetuses in some but not all of the sex-limited disorders. In others, affected infants can be avoided consistently only by terminating all pregnancies in which the fetus is male. In these cases, antenatal chromosomal studies to determine fetal sex may be indicated.

Even if the use of antenatal cytogenetic studies increased greatly, the incidence of liveborn infants with chromosomal abnormalities would not be decreased greatly as long as these studies are performed only for the aforementioned indications. Offering antenatal diagnosis only to women 35 years of age and older decreases the frequency of trisomy 21 by less than 20%.¹¹⁶ Even maternal serum screening does not detect the majority of chromosome abnormalities. Monitoring on the basis of the other cytogenetic indications also results in detection of only a minority of fetuses with chromosomal abnormalities. Conversely, offering antenatal diagnosis to all women does not seem justified because of the small, yet finite, risk of invasive procedures. Therefore, one would hope to identify categories of younger women whose risk of having a chromosomally abnormal fetus justifies the risk of prenatal diagnosis. That is, such women would constitute a special high-risk group based on factors such as previous reproductive or medical history or fetal characteristics. Following are potential indications for antenatal studies.

Advanced paternal age

Although the relationship between aneuploidy and increased maternal age is better recognized and more established, Down syndrome also has been associated in some studies with advanced paternal age.¹¹⁷ Stene and colleagues¹¹⁸ and Matsunaga and associates¹¹⁹ found that the risk of siring offspring with trisomy 21 increased by paternal age 55 years and perhaps as early as 41 years of age.¹²⁰ Other investigators, however, have found a much smaller or no effect.^{121, 122, 123, 124} Therefore, advanced paternal age alone is not a sufficient indication for prenatal cytogenetic studies.

Previous stillborn or spontaneous abortions

Couples experiencing repetitive abortions should undergo cytogenetic studies to exclude the presence of a parental translocation or inversion, either of which clearly justifies antenatal chromosomal studies. By virtue of the following argument, antenatal diagnosis also might be considered for couples who have had one or more spontaneous abortions or stillborn infants but who have not been found to have a parental chromosomal rearrangement.

Approximately 50–60% of all first-trimester abortuses show chromosomal abnormalities, as do 5% of stillborn infants. Of abortuses with chromosomal abnormalities, 50% are trisomic; thus, 25% of all abortuses are trisomic.

If women who have chromosomally abnormal abortuses have an increased risk for a subsequent trisomic conceptus, it would be reasonable to offer antenatal cytogenetic studies on the presumption that the couple's next trisomic conceptus might not abort, but rather continue to the liveborn stage. Indeed, pooled results of several small studies, albeit not corrected for maternal age, suggest that couples with a trisomic abortus have approximately a 1% risk for an aneuploid live birth (Table 7).^{125, 126, 127, 128, 129} Other investigators disagree, however, on the validity of such studies and on whether aneuploidy recurs more often than would be expected based on maternal age alone.^{130, 131, 132, 133} In practice, chromosomal studies rarely are performed on abortuses or stillborn infants. Therefore, one usually cannot say in a particular case whether a couple with several abortuses or stillborn fetuses experienced recurrent aneuploid conceptions and, hence, might benefit from antenatal diagnosis. To be considered is whether the theoretical risk for an aneuploid livebirth in such circumstances outweighs the empirical risk of an invasive diagnostic procedure.

Table 7. Risk of trisomic live-born fetus preceding or following a trisomic abortus

Exposure to irradiation or chemotherapeutic agents

Retrospective case–control studies by several independent groups have found that women whose pregnancies terminated in liveborn offspring with Down syndrome received significantly more X-irradiation before conception than had controls. The irradiation occurred 2–10 years before conception, and doses as small as 2 rads appeared to predispose to aneuploidy.^{134, 135} These studies are highly suggestive; however, other studies revealed no such correlation.^{136, 137, 138} Although additional data clearly are necessary, it seems inappropriate to recommend antenatal diagnosis solely on the basis of diagnostic X-irradiation before or especially during gestation. Therapeutic radiation, however, is a more complex issue. Theoretical risk for gametic chromosomal and genetic damage is a serious consideration, and men treated with testicular radiation doses of 0.4–5 grays (40–500 rads) have shown increased chromosomal abnormalities in sperm.¹³⁹ However, empirically, no increase in congenital anomalies has been found in the offspring of persons treated with X-irradiation for Hodgkin's disease or other cancers.^{140, 141, 142, 143, 144, 145} Japanese women exposed to X-irradiation through proximity to the atomic bomb explosions also did not show an increased prevalence of Down syndrome offspring.¹⁴⁶ Therefore, antenatal cytogenetic studies might be discussed but not encouraged for women (or men) who have undergone radiation therapy. Couples electing to undergo antenatal diagnostic studies must realize that only numerical and structural chromosomal abnormalities can be assessed. There is no possibility of monitoring for gene mutations, which also would be predicted to increase after irradiation.

Similar reasoning also might apply to men and women who have received chemotherapeutic agents because many agents used to treat neoplasia produce in vitro chromosomal damage and induce mutations. A person who previously has received such agents should thus theoretically be at increased risk for chromosomally abnormal progeny. Again, and analogous to irradiation data, no increase in the actual rate of anomalies has been observed in liveborn infants of such couples.^{141, 142, 143, 144, 145, 146, 147, 148} Thus, antenatal cytogenetic studies are not necessarily indicated in this situation, although the issues may be worthy of discussion.

Parental metabolic Derangements

Parental, especially maternal, metabolic derangements could predispose to aneuploidy. Although once considered suspect, diabetes mellitus,¹⁴⁹ infectious hepatitis, and other infectious diseases¹⁵⁰ do not seem to have this effect. Whether parental α₁-antitrypsin (α₁-protease inhibitor) phenotype is a significant risk factor is still controversial.^{151, 152, 153} Conversely, data showing a correlation between the presence of maternal antithyroid antibodies or hyperthyroidism and offspring with Down syndrome are more convincing.^{154, 155, 156} No conclusive studies, however, have been published; therefore, the presence of antithyroid antibodies or hyperthyroidism alone should not be considered an obligatory indication for antenatal cytogenetic studies. More recently, James has reviewed the evidence for an association between a polymorphism in the maternal methyltetrahydrofolate reductase (MTHFR) gene and the likelihood of a child with Down syndrome. She concluded that it may be the maternal metabolic phenotype, rather than the maternal genotype, which more closely predicts risk.¹⁵⁷

In a similar vein, based on the hypothesis that both Alzheimer's disease and chromosomal nondisjunction may be caused by failure of microtubular organization, Heston purported to demonstrate an excessive incidence of trisomy 21 in relatives of probands with Alzheimer's disease.¹⁵⁸ However, the expected incidence was not adjusted for maternal age, nor were karyotypes performed on all subjects with “trisomy 21” to exclude the possibility of a familial translocation. Therefore, further verification is required before these findings are accepted.

Parental chromosomal variants

Chromosomal variants are structural polymorphisms believed to be without phenotypic effect. However, it has been proposed that such variants or other in vitro findings (e.g., increased satellite association) may predispose to chromosomal nondisjunction and thereby aneuploid gametes. There is evidence both for and against this position;^{159, 160, 161, 162} biases of ascertainment and publication make evaluation of the available data difficult. An association between the presence of a particular variant, a double nucleolar organizing region on an acrocentric chromosome, and predisposition to aneuploid gametes has been claimed¹⁶³ but not uniformly corroborated.^{164, 165, 166} Therefore, routine studies on all couples to detect such variants or in vitro aberrations are not indicated.

Periconceptive events and assisted reproduction

Certain periconceptive events might predispose a couple to liveborn infants with chromosomal abnormalities. For example, in a questionnaire survey of pregnancy outcome after artificial insemination by donor, Forse and colleagues found three aneuploid liveborn infants (two with trisomy 21 and one with trisomy 13) in a population of 400 term offspring (p <0.05).¹⁶⁷ In contrast, neither the experience of my colleagues and I nor that of other investigators reveals an increase in autosomal trisomy in artificial insemination by donor pregnancies.^{168, 169} However, some men with severe oligospermia and nonobstructive azoospermia have microdeletions of the Y chromosome. This will be transmitted to their male offspring if conception occurs, either naturally or with the assistance of in vitro fertilization/intracytoplasmic sperm insemination (IVF/ICSI). Presumably couples undergoing IVF/ICSI are willing to accept potential infertility in their sons, but if not, prenatal chromosome analysis is possible. A more serious concern is the finding that children born after IVF or ICSI have an increased rate of autosomal and sex chromosome anomalies (1–3%).^{170, 171} Couples should be counseled accordingly and prenatal diagnosis considered.

Aging gametes and asynchrony between oocyte and sperm have also been associated with chromosome abnormalities. An example is that in animals fertilization of an aged oocyte results in polyploidy and sometimes aneuploidy.¹⁷² In humans, intrafollicular (preovulatory) delays may contribute to polyploidy but not to aneuploidy.¹⁷³ Asynchrony between an oocyte and its fertilizing sperm also might occur in pregnancies associated with ovulation induction (clomiphene or human menopausal gonadotropins), intercourse after a period of abstinence, or intercourse occurring more than 1 day before or after ovulation.¹⁷⁴ Boué and Boué found a higher frequency of chromosomal abnormalities in abortuses recovered from pregnancies associated with ovulation induction than in abortuses recovered from pregnancies in which ovulation was not induced.¹⁷⁵ Other investigators have reported an increased incidence of liveborn infants with Down syndrome after pregnancies associated with ovulation induction;¹⁷⁶ however, these findings have not been verified.

Simpson and associates have examined data from women who conceived while using natural family planning methods and recording coital events.¹⁷⁷ No association was found between conception outside the optimal period immediately surrounding ovulation and aneuploidy (Down syndrome) in the resultant offspring.

Conversely, in one study, women with a reduced ovarian complement based on previous unilateral oophorectomy or congenital absence of one ovary, were significantly more likely to have a child with Down syndrome than were the controls who did not have absence of an ovary.³⁵ This finding is consistent with Warburton's hypothesis that a suboptimally developed oocyte may become the dominant follicle in situations in which only a small number of oocytes are available (e.g., in older women).¹⁷⁸ This hypothesis is further supported by the finding that age of menopause is 1 year earlier in women with trisomic losses compared to women with euploid losses or euploid livebirths.³⁴ This implies that women with trisomic conceptions have accelerated oocyte atresia or a smaller number of oocytes. If the findings of Freeman and associates³⁵ are confirmed, discussion of prenatal diagnosis may be indicated in women with unilateral ovaries.

Preimplantation diagnosis

Diagnosis of a chromosome disorder before implantation of the zygote in the uterus offers the ability to select and transfer only normal conceptuses. For some families who are at very high risk (e.g., a parental chromosome translocation), avoiding the need for repeated pregnancy terminations of affected fetuses is a significant advantage. Details of preimplantation diagnosis are found elsewhere in this library.

Fetal cells in the maternal circulation

For many years, obstetricians have wished for a method of prenatal diagnosis that is risk-free, in other words, noninvasive. In 1975, Schroder and associates showed that fetal lymphocytes are present in the maternal circulation during pregnancy.¹⁷⁹ However, practical exploitation of this knowledge required the ability to separate fetal from maternal lymphocytes, thereby enriching the sample for fetal cells. A number of enrichment schemes were tried; however, poor specificity and the difficulty in inducing mitosis in fetal lymphocytes isolated in this fashion made this approach impractical.

More recently, however, with new developments in cell sorting techniques, in situ hybridization with chromosome-specific probes, and polymerase chain reaction for amplification of small amounts of DNA, several groups have been successful in isolating and analyzing fetal cells and fetal DNA from maternal blood samples. Fetal trophoblasts, lymphocytes, granulocytes, and erythroblasts (nucleated erythrocytes) all have been potential target cells. In one example, Lo and colleagues obtained blood samples from 27 women at 6–41 weeks' gestation and, without sorting fetal from maternal cells, performed polymerase chain reaction with primers for a single-copy sequence on the Y chromosome.¹⁸⁰ Of 17 women carrying a male fetus, 13 showed a signal for Y DNA. Of ten pregnancies with female fetuses, eight were negative for male (Y) DNA. Other investigators have used monoclonal antibodies to separate fetal from maternal cells, followed by polymerase chain reaction to detect Y sequences. In one study, fetal sex prediction was correct in 11 of 12 women studied, with one female fetus misdiagnosed as a male.¹⁸¹

Using techniques such as these, several groups have reported diagnosis of fetal aneuploidy and Mendelian traits from maternal blood samples obtained before or after amniocentesis or CVS.^{182, 183} Chromosome diagnosis takes advantage of the presumption that virtually all pregnant women have a 46,XX karyotype. Any other chromosomal complement in a maternal blood sample can be ascribed to a conceptus, either from the current or from a previous pregnancy.

In a demonstration of the possibilities of this approach, Price and associates described the detection of fetal chromosome abnormalities in maternal blood samples.¹⁸² The authors first flow-sorted nucleated erythrocytes from maternal samples on the basis of cell size, granularity, and presence of transferrin receptors and glycophorin A cell surface antigens. In a series of samples from first- and second-trimester pregnancies, approximately 10% of the final sorted samples were fetal cells (2–20 x 10³ nuclei). The authors used both polymerase chain reaction with single copy Y-specific sequences and in situ hybridization with a Y-specific probe to confirm that they were enriching for fetal cells.

In one of two cases with fetal aneuploidy, blood was drawn from the mother 1 week after CVS was performed. After enrichment, the sample was analyzed by in situ hybridization with a chromosome 21-specific probe. Thirty-nine per cent of the cells showed three signals, indicative of trisomy 21, and consistent with the findings in villi and in abortus tissue.

In the second case, maternal blood was drawn before CVS, enriched, and analyzed with probes for chromosomes X, Y, 18 and 21. Nine percent of cells showed hybridization with the Y probe, indicating that male fetal cells constituted approximately 9% of the sample. The same percentage of cells in the sample showed three hybridization signals with the probe for chromosome 18, thereby diagnosing a male fetus with trisomy 18. This diagnosis was confirmed with abortus tissue.

Subsequently, a collaborative study of 69 maternal blood samples, obtained before or after an invasive procedure and analyzed with chromosome-specific probes, showed that it was possible to detect both sex chromosome polysomy, as well as autosomal trisomy.¹⁸⁴ Other investigators have shown that the number of fetal cells and fetal DNA in the maternal circulation is increased in aneuploid pregnancies.^{185, 186} This finding may prove useful as an initial screen.

Thus, noninvasive prenatal diagnosis with maternal blood samples appears feasible; however, the sensitivity and specificity still are not adequate for clinical application. The National Institute of Child Health and Human Development (NICHD) funded a multicenter clinical trial of this technology (NIFTY). An analysis of 5 years of data showed correct diagnosis of fetal sex in 41% of women carrying male fetuses and 89% of women carrying female fetuses. One or more aneuploid cells were detected in 74% of cases in which aneuploidy was truly present, with a false-positive rate of 0.6–4%.¹⁸⁷

If difficulties can be worked out, screening for chromosome disorders early in gestation with maternal blood samples might be offered to all pregnant women, similar to the screening now performed with biochemical analytes. Initially, the test might be viewed as a screening test only, with follow up by CVS or amniocentesis for definitive diagnosis if abnormal cells are detected. Eventually, however, if maternal blood screening proves sensitive and specific enough, it could be the hoped for noninvasive diagnostic test.