Expanding multiple marker screening for Down's syndrome to include Edward'S syndrome

Information on maternal age and maternal serum alpha-fetoprotein, unconjugated oestriol (uE₃), and human chorionic gonadotrophin (hCG) levels was used to investigate retrospectively the effect of estimating Edward's syndrome risk in women having multi-marker screening for Down's syndrome. The screened population comprised 15 pregnancies affected by Edward's syndrome, 15 with Down's syndrome and 5472 unaffected pregnancies. The use of all three markers to estimate Edward's syndrome risk would have led to the detection of 10–12 (67–80 per cent) cases with a false-positive rate of 0.3–0.6 per cent depending on the risk cut-off. A further case would have been detected as a result of screening for Down's syndrome alone. Similar results were obtained when the Edward's syndrome risk was based on uE₃ and hCG only. These data suggest that extending Down's syndrome screening to include Edward's syndrome risk will yield a high detection rate with only a small increase in the false-positive rate.     

Repeat maternal serum testing in multiple marker down's syndrome screening programmes

The effect of repeat testing in maternal serum multiple marker screening for Down's syndrome was estimated using samples stored in an antenatal serum bank. Human chorionic gonadotropin (hCG) and unconjugated oestriol (uE₃) levels were determined in 142 pairs of routinely collected samples which had already been tested for alpha-fetoprotein (AFP). For each marker, about two-thirds of the pairs of values were within 20 per cent of each other and most were within 40 per cent. A multivariate Gaussian model was used to estimate the detection and false-positive rates for different repeat testing policies. A policy of repeat testing those with a high risk of a Down's syndrome term pregnancy given age and marker levels would reduce the false-positive rate but there would also be a reduction in the detection rate. For example, using all three markers and a 1 in 250 cut-off risk, the estimated false-positive rate would fall from 5·3 to 3·8 per cent but the detection rate would decrease from 58 to 55 per cent. A policy of repeating those with either high or borderline risks would produce a modest improvement in screening efficiency. Repeating the 11 per cent with a risk exceeding 1 in 500 yields an estimated false-positive rate of 5·0 per cent and a detection rate of 60 per cent. A policy of selective repeat testing is not recommended as it would not substantially improve screening efficiency. Nonetheless, if a repeat test has been performed, the parameters given in this paper will enable an unbiased estimate of the Down's syndrome risk to be calculated for individual women.     

Prospective maternal serum human chorionic gonadotropin screening for the risk of fetal chromosome anomalies and of subsequent fetal and neonatal deaths

A prospective study of maternal serum human chorionic gonadotrophin (hCG) measurement for the selection of pregnancies with an increased risk of fetal trisomy 21 was undertaken in 24 000 pregnancies from 1 January 1989 to 31 December 1990. Maternal serum was sampled at 15-18 weeks of gestation. hCG was measured in one laboratory, with one technique. This ‘hCG high level’ technique was developed for this screening. Amniocentesis was offered to each woman with a maternal serum hCG level above the cut-off. The follow-up of the pregnancies is known in 92 per cent of cases. The combination of hCG values and maternal age gave a detection efficiency of 63 per cent for trisomy 21 with rates of amniocentesis of 30 per cent for patients aged 37 years, 20 per cent for patients aged 35 or 36 years, and 5 per cent for patients under 35 years of age. Based on this prospective study, an individual risk was calculated combining the serum hCG value and maternal age. Seventy-four per cent of trisomy 13, trisomy 18, triploidy, and 5p- deletion were detected either in the same selected group of women or in combination with ultrasonography performed when hCG values were very low. The follow-up study showed that women who had high or low hCG values represented a group at high risk for fetal or perinatal death.     

Four-marker serum screening for Down's syndrome

The value of measuring the separate sub-units of human chorionic gonadotrophin (free α-hCG and free β-hCG) instead of total hCG together with alpha-fetoprotein (AFP) and unconjugated oestriol (uE₃) was examined to determine the effect on the performance of serum screening for Down's syndrome between 15 and 22 weeks of pregnancy. The study was based on stored serum samples relating to 75 singleton pregnancies with fetal Down's syndrome and 367 unaffected singleton pregnancies, matched for maternal age, gestational age, and duration of storage of the serum sample, supplemented by data from 970 white women with unaffected pregnancies. Using the four serum markers AFP, uE₃, free β-hCG, and free α-hCG, in addition to maternal age, 65 per cent of Down's syndrome pregnancies were detected for a 5 per cent false-positive rate compared with 59 per cent with the conventional triple test (AFP, uE₃, total hCG with maternal age). If gestation was based on an ultrasound scan examination, the detection rate was 72 per cent using the four serum markers compared with 67 per cent with the triple test. As an alternative illustration, if the detection rate was kept at 60 per cent and gestation was estimated by an ultrasound scan examination the four-marker test reduced the false-positive rate by one-third from 3 per cent using the triple test to 2 per cent with the four-marker test. Screening performance was hardly affected by adjusting marker levels for maternal weight. The four-marker test is, both from a medical and from a financial perspective, the most effective method of prenatal screening for Down's syndrome suitable for routine use.     

Second-trimester maternal serum screening using alpha-fetoprotein,human chorionic gonadotrophin,and unconjugated oestriol: Experience of a regional programme

Over a 2-year period from January 1991 to December 1992, second-trimester maternal serum screening for Down's syndrome using alpha-fetoprotein (aFP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE₃) was made available to five health districts in East Anglia, with a total population of 1·2 million. Amniocentesis was offered when the risk of Down's syndrome at term was 1:200 or greater. 25359 singleton pregnancies were screened, representing an uptake of 77 per cent. The recall rate for the 24 per cent of women who had not had a dating scan prior to the test was 9·4 per cent compared with 3·9 per cent for those who had been scanned (P<0·0005). Seventy-five per cent (36/48) of Down's syndrome pregnancies were detected for a false-positive rate of 4·0 per cent. Twenty-five out of 36 of detected Down's syndrome pregnancies were dated by scan prior to sampling, and in the 11 remaining cases, the dates were confirmed by scan after a high-risk result was obtained. The exclusion of uE₃ from the screening protocol would have reduced the detection rate to 52 per cent (25/48) for the same false-positive rate. Eighty-five per cent of women identified at high risk accepted the offer of an amniocentesis. Other fetal abnormalities detected were trisomy 18 (3), trisomy 13 (2), 45,X (6), 69,XXX (5), other chromosome abnormalities (9), open neural tube defects (26), hydrocephalus (7), abdominal wall defects (4), and steroid sulphatase deficiency (6).     

Prenatal screening for chromosome abnormalities using maternal serum chorionic gonadotrophin,alpha-fetoprotein,and age

Human chorionic gonadotrophin (hCG) levels were assayed retrospectively in stored maternal serum samples from 78 chromosomally abnormal pregnancies and 410 controls matched for gestation and maternal age. The median serum hCG concentration in 49 pregnancies with Down's syndrome was significantly elevated, at 2.18 multiples of the normal median. Significantly reduced hCG concentrations were found in a group of four trisomy 18 pregnancies (all less than 0.4 multiples of the median). Eight cases of unbalanced chromosome rearrangements appeared to show some lowering of hCG levels, while there was no significant difference in the levels in the cases of trisomy 13, balanced translocations, and sex chromosome abnormalities. Maternal serum hCG alone is a better indicator of Down's syndrome pregnancies than maternal age or maternal serum alpha-fetoprotein (AFP), either individually or in combination, and provides a further virtually independent measure of risk. On the basis of our findings, screening for Down's syndrome using hCG and AFP results combined with maternal age risks is predicted to result in a higher detection rate (57 per cent) for a lower false-positive rate (5.0 per cent) than would be attainable by combined AFP and age screening (37 per cent detection at a 6.6 per cent false-positive rate).     

Screening for Down's syndrome in older women based on maternal serum alpha-fetoprotein levels and age: Preliminary results

We report the results of screening for Down's syndrome (DS) in older women using published rate schedules based on maternal serum alpha-fetoprotein (MSAFP) and age. Five hundred and seventeen patients aged 35 years and older, who were referred for a mid-trimester genetic amniocentesis, were first tested for MSAFP and then underwent an amniocentesis. Individual risks for DS, combining MSAFP and age, were derived using three different published rate schedules. Theoretical selection for amniocentesis was made using the cut-off level of the average collective risk for a 35-year-old woman (1:380 at live birth or 1:270 at amniocentesis). Six affected pregnancies (five with DS and one with trisomy 18), which were diagnosed prenatally, were all found to be at a higher risk than the specified cut-off. These cases would have been diagnosed in any event, using any of the published rate schedules. According to these rate schedules, between 39 and 45 per cent of the patients would be in the lower risk group and therefore would have been counselled not to undergo amniocentesis. Further studies should be conducted in order to reach conclusive screening policies for DS in older women.     

Improved performance in a prenatal screening programme for Down's syndrome incorporating serum-free hCG subunit analyses

A prenatal screening programme for Down's syndrome potentially detecting 76 per cent of affected pregnancies in the South Australian general population at an amniocentesis rate of 3.9 per cent was designed following analysis of mid-trimester serum samples from 57 women who carried an affected fetus. This equates to one affected pregnancy being detected for 41 chromosomal analyses performed. For the experimental series, 75.4 per cent of affected pregnancies were detected, while 4.1 per cent of control specimens produced estimated risk odds consistent with further action. A maternal risk odds of birth of a Down's syndrome fetus of 1:420 was taken as the decision value, which is the prevalence of Down's syndrome births to 35-year-old mothers in South Australia. This screening performance was achieved by investigating combinations of serum analytes not previously reported and by refining the calculation of maternal risk odds to include selective weighting of indicator analytes. Combination of the measurements of free α-subunits and β-subunits of chorionic gonadotrophin, α-fetoprotein, unconjugated oestriol, and placental lactogen was found to be most effective in indicating Down's syndrome fetuses. In all combinations of analytes tested, replacing the measurements of free α-subunits and free β-subunits of chorionic gonadotrophin with the measurement of intact chorionic gonadotropin produced a less effective screen.     

Second-trimester maternal serum immunoreactive inhibin as a marker for fetal Down's syndrome

We measured immunoreactive inhibin in the maternal serum of 80 pregnancies with a chromosomally normal fetus and ten Down's syndrome pregnancies in the second trimester. The inhibin level in all Down's syndrome pregnancies was above the normal median; the multiple of the normal median (MoM) was 1.9. We found a statistically significant difference between the levels of inhibin in unaffected and affected pregnancies (Kolmogorov–Smirnov test: p <0.002). Using an arbitrarily chosen cut-off of 2.4 MoM, 40 per cent of Down's syndrome and 5 per cent of the normal pregnancies were found. We conclude that immunoreactive inhibin may be useful as a marker for fetal Down's syndrome.     

The influence of gravidity on Down's syndrome screening with free beta hCG

Total human chorionic gonadotropin (hCG) levels have been shown in one previous small study to vary with gravidity. In order to establish if maternal serum free beta hCG was similarly affected, approximately 4500 pregnancies of varying gravidity were analysed. The results indicated that there was a 6 per cent higher median MOM in primigravid compared with multigravid women. The impact of such elevation in Down's syndrome screening programmes will be minimal.     

Maternal smoking habits and Down's syndrome

Two series of pregnancies were studied to investigate the relationship between maternal smoking and the risk of fetal Down' s syndrome. In the first series, ascertained in the 1960s, in which smoking habits were determined after the outcome of pregnancy was known, the proportion of smokers (47 per cent) among the 461 women whose pregnancies ended in the birth of an infant with Down' s syndrome was similar to that in the 461 controls (46 per cent) who had pregnancies affected by other congenital disorders. In the second series, ascertained between 1973 and 1984, smoking habits were determined by measurement of cotinine in antenatal serum samples that were routinely collected and stored or, if a serum sample was not available, from information in the antenatal notes. In this series, the proportion of smokers (14 per cent) among the 91 women who had pregnancies associated with Down' s syndrome was lower than that among 413 controls (19 per cent), though this was not statistically significant. Collectively, our results provide no evidence for an association between fetal Down' s syndrome and smoking. Other published studies found a deficit of smokers among women who had pregnancies associated with Down' s syndrome. This may be partly due to some studies not taking adequate account of maternal age (older women are more likely to have had a Down' s syndrome pregnancy but are less likely to be smokers) and partly due to the greater tendency for positive findings to be published than negative ones.     

Analysis of maternal serum alpha-fetoprotein and free beta human chorionic gonadotrophin in the first trimester: Implications for down's syndrome screening

The aim of this study was to determine the maternal population, pregnancy, serum alpha-fetoprotein (AFP) and free β subunit of human chorionic gonadotrophin (FβhCG) parameters in a large series of women attending prenatal clinics before 15 weeks' gestation and to assess the practical problems of population screening for Down's syndrome in the first trimester using these markers. Serum samples were collected from 8600 women attending prenatal clinic booking visits. Maternal serum AFP and FβhCG medians were calculated for each day of gestation (49–104 days), using both dates and ultrasound estimates of gestation. The effects of maternal weight, twin pregnancies, and threatened abortion on AFP and FβhCG levels were analysed. The median age of the population was 27.1 years and the median weight 62.1 kg. Twenty-six per cent of samples were collected before 70 days and 50 per cent before 78 days' gestation. Eighty-nine per cent of all samples had gestational estimates by dates, 60 per cent by ultrasound and 52 per cent by both dates and ultrasound. The AFP median was 5 kU/1 at 49 days, 5.9 kU/1 at 70 days, and 17.9 kU/1 at 100 days. The peak median FβhCG level was 66.4 ng/ml at 64 days, falling to 20.6 ng/ml at 100 days' gestation. Both AFP and FβhCG levels showed log Gaussian distributions but the standard deviation for AFP was 20 per cent greater than that found in the second trimester. AFP and FβhCG levels showed an inverse relationship with maternal weight and were increased in twin pregnancies (1.68 and 1.97 multiples of the median, respectively). AFP and FβhCG can be readily measured in a large screening population in the first trimester. Down's syndrome screening protocols based on these markers could be refined by the use of gestations in individual days but AFP is likely to be a less effective marker and detection rates are likely to be lower than in the second trimester. To realize the potential of first-trimester screening, more women should be encouraged to attend the prenatal clinic in early pregnancy and ultrasound dating should be carried out for all pregnancies at this stage.     

A single chorionic gonadotropin assay for maternal serum screening for Down's syndrome

A simple enzyme immunoassay measuring human chorionic gonadotropin in undiluted maternal serum has been developed in order to be used as a prenatal screening test for Down' s syndrome. A retrospective study of maternal serum sampled during pregnancies associated with trisomy 21 shows that with a 5% amniocentesis rate determined on a single test, the detection rate of trisomy 21 would be around two-thirds of the affected pregnancies. A prospective study of 9040 pregnant women under 38 years has confirmed the usefulness of the assay.     

Prospective intervention trial of a screening protocol to identify fetal trisomy 18 using maternal serum alpha-fetoprotein,unconjugated oestriol,and human chorionic gonadotropin

Two prenatal centres in New England, routinely using a screening protocol for fetal Down syndrome that included maternal serum alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and human chorionic gonadotropin (hCG) measurements in combination with maternal age, adopted a separate screening protocol for trisomy 18. That protocol identified a pregnancy as being at high risk when AFP, uE3, and hCG measurements all fell at or below specified cut-offs (0.75, 0.60, and 0.55 multiples of the median, respectively), regardless of maternal age. Among the first 19 491 women screened, 98 (0.5 per cent) were found to have values which placed them in the high-risk category. Four of these women were subsequently found not to be pregnant. In two others, samples from non-pregnant individuals were found to have been incorrectly submitted for analysis in place of the samples from the pregnant women. All of the remaining 92 women were counselled and offered amniocentesis and fetal karyotyping. Eighty-eight (96 per cent) accepted. Karyotypes or birth outcomes were available on all 92 pregnancies. Six cases of trisomy 18 and one case of Turner syndrome were identified by karyotype. One case of trisomy 18 was identified for every 14 unaffected pregnancies offered amniocentesis. In the present prospective study, an estimated 85 per cent of the cases of trisomy 18 were identified. However, given the small number ofcases (six), the 95 per cent confidence interval for the detection rate is broad (40–95 per cent).     

Evaluation of maternal serum dimeric inhibin a as a first-trimester marker of down's syndrome

While second-trimester prenatal screening programmes for Down's syndrome have become established in prenatal care, it would be advantageous to be able to offer screening in earlier preganancy. To this end, we have evaluated a new potential maternal serum marker, dimeric inhibin A, as a possible first-trimester marker. Dimeric inhibin A was measured in propsectively collected maternal serum from 23 cases of Down's syndrome and matched chromosomally normal controls, at 11–13 weeks' gestation. Levels of this protein were significantly elevated in the Down's pregnancies compared with the control pregnancies. The median multiple of the normal median (MOM) for the Down's samples was 2.46 (95 per cent confidence interval: 2.11–3.26, P<0.0001 vs. controls). These results suggest that dimeric inhibin A is a useful discriminator of Down's-affected pregnancies from normal pregnancies in the first trimester and that sensitive screening in combination with maternal age and other possible markers may be practicable in the first trimester.     

Urinary β-core human chorionic gonadotrophin: A new approach to Down's syndrome screening

Human chorionic gonadotrophin (hCG) is the most discriminatory maternal serum marker of Down's syndrome. We have carried out a study to establish whether urinary β-core-hCG, a major metabolic product of hCG, might be an even better marker. Urine samples were available from seven singleton pregnancies with Down's syndrome, and one each of Edwards' syndrome, triploidy, and twins discordant for Down's syndrome. β-Core-hCG levels were corrected for creatinine and expressed as multiples of the normal gestation-specific median (MOM) level derived from 67 singleton controls. There was a highly statistically significant elevation in level among the singleton Down's syndrome cases (P<0·0005; Wilcoxon rank sum test). All had levels exceeding 2 MOM with a median of 6·11 MOM (95 per cent confidence interval 3·7–10·0). The levels were extremely low in Edwards' syndrome (0·08 MOM) and triploidy (0·02 MOM), but the twin pregnancy discordant for Down's syndrome did not have a raised β-core-hCG level (0·64 MOM). The findings are sufficiently encouraging to investigate the possibility of urinalysis as a routine modality in the prenatal screening for Down's syndrome and other common serious aneuploidies.     

Maternal serum markers in second-trimester oligohydramnios

The levels of the maternal serum markers alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE₃) in 35 pregnant women with early second-trimester oligohydramnios differed from those in a reference population of 1699 singleton pregnancies. Maternal serum AFP levels above the 95th centile of the population distribution were observed in 80 per cent (16/20) of oligohydramnios cases with a normal fetus and in only 20 per cent (3/15) of the cases with a fetus displaying urogenital tract malformations. Elevated levels of hCG (above the 95th centile) and decreased levels of uE₃ (below the fifth centile) were encountered in 26 per cent (9/35) and 17 per cent (6/35) of the women, irrespective of the fetal condition. The abnormal profile of the serum markers in early second-trimester oligohydramnios resulted in 57 per cent (20 out of 35) of screen-positive cases for either fetal Down's syndrome or neural tube defects, compared with 8·4 per cent (143 out of 1699) in the reference population.     

Value of maternal serum unconjugated oestriol measurement in prenatal screening for Down's syndrome

We compared the medical and financial cost-effectiveness of prenatal serum screening for Down's syndrome using maternal age, serum alpha-fetoprotein and human chorionic gonadotrophin with and without the use of unconjugated oestriol. The use of unconjugated oestriol is medically more cost-effective than screening without it at all levels of detection. The actual performance depends on whether gestational age is estimated using ‘dates’ or an ultrasound scan. At a detection rate of 60 per cent, the proportion of unaffected fetal losses per case diagnosed at amniocentesis is about 22 per cent less if gestational age is estimated using dates (time since the first day of the last menstrual period) and about 47 per cent less if it is based on an ultrasound scan examination. At this detection rate, the inclusion of unconjugated oestriol increases costs by about £2k per case diagnosed (£36k instead of £34k) if gestational age is estimated using dates, but it is no more expensive if gestational age is measured from an ultrasound scan examination (indeed, it is more cost-effective at detection rates above 60 per cent). Since there is little change in the financial cost with the inclusion of unconjugated oestriol, for the improved medical performance of screening, it is worthwhile including it in the screening test.     

Maternal serum thyroid antibodies in early pregnancy and fetal Down's syndrome

Thyroid antibodies were measured in mid-trimester antenatal serum samples from 77 pregnancies affected by fetal Down's syndrome and 385 unaffected control pregnancies. Using a haemagglutination technique, thyroglobulin antibodies were detected in 5·2 per cent of cases (4) and 2·9 per cent of controls (11), and thyroid microsomal antibodies were detected in 22 per cent (17) and 15 per cent (59), respectively. Using an enzyme-linked immunosorbent assay (ELISA) for thyroglobulin antibodies and a cut-off level of 50 KIU/1, positive results were found in 25 per cent of cases (19) and 22 per cent of controls (84). Using an ELISA for thyroid microsomal antibodies and the same cut-off level, the proportions were 52 per cent (40) and 39 per cent (149), respectively. While not statistically significant, the differences were consistent with the previously reported increased levels of thyroid antibody found in nonpregnant women who had had pregnancies associated with Down's syndrome.     

First-trimester maternal serum alpha-fetoprotein and human chorionic gonadotropin screening for chromosome defects

The aim of this study was to determine the efficacy of combined maternal serum alpha-fetoprotein (MSAFP) and maternal serum human chorionic gonadotropin (MShCG) screening in detecting chromosome defects in the first trimester of pregnancy. Sera of 492 women (previously assayed for MSAFP) were analysed for MShCG under code without knowledge of cytogenetic results. Overall, 48 of 492 patients (9·8 per cent) had either an MSAFP multiple of the median ⩽0·5 or an MShCG β/a z ratio multiple of the median ⩽ 0·25, eight of whom had a fetus with a serious chromosome defect. A third of fetuses with Down' s syndrome and 83 per cent with trisomy 18 were detected at a potential‘cost’ of providing chorionic villus sampling or amniocentesis in 8·6 percent of women screened.