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.     

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.     

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.     

Economic assessment of maternal serum screening for Down's syndrome using human chorionic gonadotropin

The effectiveness and costs of prenatal screening programmes for Down's syndrome using maternal serum markers will vary significantly depending on the biological cut-off values chosen in order to select women, at each maternal age, who will be sent for amniocentesis. On the basis of the first French prospective study of human chorionic gonadotropin (hCG) measurement in maternal serum, this paper shows that the screening protocol currently used in France, where hCG cut-off values are defined in order to offer amniocentesis to women of all ages with a 1 percent fetal risk of Down's syndrome, would detect 64·06 per cent of all cases of trisomy 21 at birth and would be highly profitable for the French social security system. On the basis of a representative sample of 100 000 pregnant women, the total costs of screening would reach $8 302 000 but would generate net potential savings of $32 186 000 in terms of life-long costs of care for trisomic 21 children which would be ‘avoided’ by termination of pregnancy following a positive diagnosis of Down's syndrome. Economic assessment shows that cost-benefit analysis would justify lower hCG cut-off values and a higher detection rate of fetal Down's syndrome (74·45 per cent) than the current French protocol. This paper concludes that it is ethical and value-laden issues, such as the consequences for women and couples of false positives and false negatives of screening, rather than economic and financial arguments that may set limits to the utilization of screening for Down's syndrome using maternal serum markers like hCG.     

Repeat maternal serum testing for down's syndrome screening using multiple markers with special reference to free α and free β-hCG

To determine the effect of routine repeat testing in serum screening for Down's syndrome, we compared estimates of the detection and false-positive rates. Five serum markers were measured-alpha-fetoprotein (AFP), unconjugated oestriol (uE₃), human chorionic gonadotrophin (hCG), and its two subunits, free α and free β-hCG. First and repeat test marker levels were available from 142 women whose samples had been routinely collected and stored in an antenatal serum bank. Different repeat testing policies were compared for various combinations of the markers. If all women had repeat tests using the four markers AFP, uE₃, and free α and free β-hCG, the detection rate for a 5 per cent false-positive rate was 69 per cent compared with 65 per cent if no women were repeated. Policies of repeating selected women gave similar results. The small gain in screening performance with repeat testing performed routinely is not worthwhile. If a woman does happen to have a repeat test, her risk estimate should, however, be based on both results, not just the second.     

Maternal serum free α-human chorionic gonadotrophin levels in twin pregnancies: Implications for screening for Down's syndrome

Maternal serum free α-human chorionic gonadotrophin (free α-hCG) levels were determined in twin and singleton pregnancies at 15–22 weeks of gestation using a set of stored serum samples relating to 200 twin pregnancies and 600 singleton control pregnancies matched for gestational age and duration of storage. Free α-hCG values are, on average, 1·66 times greater in twin pregnancies than in singleton pregnancies (95 per cent confidence interval 1·56–1·76). If maternal serum free α-hCG is used in screening for Down's syndrome, values in twin pregnancies can be adjusted using this result so that screening can be performed in twin pregnancies as well as in singleton pregnancies.     

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.     

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.     

Maternal serum free α- and free β-human chorionic gonadotrophin in pregnancies with insulin-dependent diabetes mellitus: Implications for screening for Down's syndrome

A study was performed to investigate the concentrations of the α and β free sub-units of human chorionic gonadotrophin (free α-hCG and free β-hCG) in maternal serum between 15 and 22 weeks of pregnancy in 126 pregnancies among 92 women with insulin-dependent diabetes mellitus (IDDM). Each IDDM pregnancy was matched with two control singleton pregnancies for gestational age (same completed week) and duration of sample storage (same calendar quarter). The median free α-hCG level in the IDDM pregnancies was 0·86 multiples of the median (MOM) for pregnancies without IDDM at the same gestational age (P<0·002) (95 per cent confidence interval 0·80–0·94). The corresponding free β-hCG level was 0·96 MOM (95 per cent confidence interval 0·85–1·09). These results enable free α-hCG values to be adjusted so that antenatal screening for Down's syndrome can be performed using this marker in IDDM pregnancies as well as in non-diabetic pregnancies.     

Urinary multiple marker screening for down's syndrome

We have examined the possibility of using multiple markers in maternal urine rather than serum in order to screen for Down's syndrome. Urine samples were available from 36 cases (24 Down's syndrome, five Edwards' syndrome, three Turner's syndrome, one Klinefelter's syndrome, one triploidy, one triple-X, one twin discordant for Down's syndrome) and 294 controls, including three twins. Three markers were tested: the β-core fragment of human chorionic gonadotrophin (hCG), total oestrogen (tE) and the free a subunit of hCG. Levels were corrected for creatinine excretion and expressed as multiples of the gestation-specific median (MOM) level from the singleton controls. The median value for the singleton Down's syndrome cases was 6.02, 0.74, and 1.08 MOM for β-core-hCG, tE, and a-hCG, respectively. The increases in β-core-hCG and the reduction in tE levels were highly significant (P<0.0001 and 0.005, respectively; Wilcoxon rank sum test) but the increase in free a-hCG was not (P=0.40). On the basis of a mathematical model, the expected detection rate for a 5 per cent false-positive rate was 79.6 per cent for β-core-hCG alone, which increased to 82.3 per cent when combined with tE. Aneuploidies other than Down's syndrome were characterized by low levels of tE and either low or high β-core-hCG.     

The efficacy of maternal age screening for Down's syndrome in Wessex

The uptake of amniocentesis in Wessex for the period 1986–1988 was 36 per cent (2873 of 8004 births), a proportion that has not altered significantly since 1984. There is a large difference in uptake between women in the lower risk age group, 35–36 years, and those in the higher risk group, 37 + years, and very considerable differences in uptake among different districts. The prenatal detection over the 3-year study period for women aged 35 or more, after correction for spontaneous loss of Down's syndrome fetuses between prenatal detection and birth, is 53 per cent, a figure that must be inflated due to our failure to ascertain all liveborn Down's syndrome patients.     

Down's syndrome screening in multiple pregnancies using alpha-fetoprotein and free beta hCG

Second-trimester distributions of the free beta human chorionic gonadotrophin (hCG) and alpha-fetoprotein (AFP) levels in 420 twin and 19 triplet pregnancies were measured and compared with the distributions in 6661 singleton pregnancies. On average, the levels of both analytes were twice as high in twins and over three times as high in triplets. Eight sets of twins discordant for Down's syndrome showed elevated levels of free beta hCG and reduced levels of AFP after correction of the multiple of the median for the presence of a twin pregnancy. Screening for Down's syndrome using the twin correction of the multiple of the median is expected to achieve a 51 per cent detection rate at a 5 per cent false-positive rate using these two markers.     

Maternal serum inhibin levels in second-trimester down's syndrome pregnancies

Maternal serum inhibin levels were measured in 19 second-trimester pregnancies affected by fetal Down's syndrome and 95 unaffected control pregnancies matched for gestational age. A statistically significant elevation was found in the affected pregnancies compared with the controls (Wilcoxon rank sum test: one-tail P=0·02). The median level in the cases was 1·3 times that in the controls, with 95 per cent confidence limits of 0·9–1·9. Although the inhibin levels were unrelated to those of alpha-fetoprotein and unconjugated oestriol in the same samples, there was a statistically significant correlation with human chorionic gonadotropin. This together with the relatively small elevation in cases suggests that inhibin would be of limited value in maternal serum screening for Down's syndrome.     

Flow cytometric method to measure urea-resistant alkaline phosphatase from blood neutrophils for use in the prenatal screening of Down's syndrome

The histochemical measurement of urea-resistant alkaline phosphatase from maternal blood neutrophils is known to have a high detection rate for the prenatal detection of Down's syndrome pregnancies. However, because the histochemical method is laborious and subjective to use, it has not gained widespread acceptance in prenatal screening programmes. We present a simple and objective method for the measurement of urea-resistant alkaline phosphatase by flow cytometry. The method should allow the design of larger studies aimed at evaluating the role of neutrophil urea-resistant alkaline phosphatase in the prenatal screening for Down's syndrome.     

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 unconjugated oestriol and alpha-fetoprotein in fetal Down's syndrome

Maternal sera (MS) taken from 1396 women prior to chorionic villus sampling at 9–12 menstrual weeks were assayed for unconjugated oestriol (uE3) and alpha-fetoprotein (AFP). Median levels increased by 41 and 26 per cent per week respectively in normal pregnancies. There were 32 pregnancies with a chromosome abnormality. The median MS uE3 and AFP were 0.73 and 0.75 multiples of the median (MoM) respectively in the ten cases of Down's syndrome (DS) but not decreased in the other abnormalities. These results suggest that both uE3 and AFP may be useful in identifying DS in the first trimester. Additional prospective studies are needed to confirm these findings.     

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 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.     

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.     

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.