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We conducted a study to investigate ethnic group differences in levels of serum markers used in screening for Down's syndrome [serum alpha-fetoprotein (AFP), unconjugated oestriol (uE3), total human chorionic gonadotrophin (hCG), free alpha- and free beta-hCG, and dimeric inhibin-A], to estimate the extent to which maternal weight differences between ethnic groups explain these differences, and to estimate the effect of adjusting for ethnic group and maternal weight on screening performance. Serum measurements were taken from women who were screened prenatally for Down's syndrome. AFP, uE3, and hCG concentrations were available from 9462 white, 4215 black, and 4392 South Asian women with singleton pregnancies without Down's syndrome or neural tube defects between 15 and 22 weeks' gestational age. Frozen serum samples were available from a subset of 922 white, 449 black, and 135 South Asian women and were used for measurement of free alpha-hCG, free beta-hCG, and inhibin. Values were expressed as multiples of the median (MOM) for women of the same gestational age. There were statistically significant differences in the serum marker levels between ethnic groups that were not explained by differences in maternal weight. The main differences were found in black women compared with white women; black women had serum AFP levels 22 per cent higher (95 per cent confidence interval 20-24 per cent), total hCG levels 19 per cent higher (16-22 per cent), and free beta-hCG levels 12 per cent (3-21 per cent) higher. The other differences were less than 10 per cent. Adjusting for ethnic group only had a small estimated effect on screening performance: a maximum of about 0.5 per cent extra detection at a 5 per cent false-positive rate. At a fixed risk cut-off level, the false-positive rate will not be materially different between different ethnic groups. Adjusting serum markers for ethnic groups improves Down's syndrome screening performance to a very small extent. It is worthwhile because of its established value in AFP screening for open neural tube defects.

Objective To determine the value of serum screening for Down's syndrome at 8–14 weeks of pregnancy using seven potential serum markers (alpha-fetoprotein, unconjugated oestriol, total human chorionic gonadotrophin (hCG), free α-hCG, free P-hCG, pregnancy associated plasma protein A (PAPP-A), and dimeric inhibin A). Design Stored blood samples collected from women at about 10 weeks of pregnancy, prior to having a chorionic villus sampling procedure on account of advanced maternal age, were retrieved from pregnancies associated with Down's syndrome and from matched unaffected pregnancies. Setting Twenty-one obstetric centres in nine countries. Subjects Seventy-seven pregnancies associated with Down's syndrome each matched with five controls (except in two cases that were matched with four controls) for maternal age (same five year age groups), duration of storage of the serum sample (same calendar year), and gestational age (usually same week of pregnancy). Results The levels of two potential markers differed between affected and unaffected pregnancies sufficiently to be of value in screening—free β-hCG and PAPP-A. The median free P-hCG level in affected pregnancies was 1.79 times the median level for unaffected pregnancies, and the median PAPP-A level was 0.43 times the normal median. These two markers were combined with maternal age to estimate a woman's risk of having a fetus with Down's syndrome. A screening programme that used a risk cutoff level of 1:300 would detect 63 % of affected pregnancies and also classify 5.5% of unaffected pregnancies as screen positive. None of the other five markers added more than 2 % detection for the same false-positive rate. Conclusion The performance of screening using maternal age and serum-free β-hCG and PAPP-A at 10 weeks of pregnancy was better than the double test (alpha-fetoprotein and hCG with maternal age) and similar to the triple test (alpha-fetoprotein, unconjugated oestriol and hCG with maternal age) at 15–22 weeks.

The value of measuring inhibin-A (a beta A dimer) with human chorionic gonadotrophin (total or the sub-units free a-hCG and free beta-hCG separately), 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 from 77 Down's syndrome singleton pregnancies and 385 unaffected singleton pregnancies, matched for maternal age, gestational age, and duration of storage of the sample, supplemented by data from 970 white women with unaffected pregnancies. Inhibin-A was elevated in the serum of women with Down's syndrome pregnancies with a median of 1.79 multiples of the median (MOM). Using the four serum markers AFP, uE3, total hCG, and inhibin-A, in addition to maternal age, 70 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, and total hCG with maternal age). If the estimate of gestational age were based on an ultrasound scan examination, the detection rate would be 77 per cent [95 per cent confidence interval (CI) 69-85 per cent] using the four serum markers including inhibin-A, compared with 67 per cent with the triple test or 79 per cent (95 per cent CI 71-87 per cent) if marker values were adjusted for maternal weight. If the detection rate were kept at 70 per cent and the gestational age were estimated by an ultrasound scan examination, the four-marker test would reduce the false-positive rate from 6-1 per cent using the triple test to 2-9 per cent. The results were virtually the same if free beta-hCG was used instead of total hCG. The inhibin-A-based four-marker test is the most effective method of prenatal screening for Down's syndrome suitable for routine use. If the extra cost required to carry out the inhibin-A test were less than about [symbol: see text]3 per woman screened, the four-marker test including inhibin-A would be financially cost-effective.

As part of the Medical Research Council randomized trial of vitamin supplementation in the prevention of neural tube defects (NTDs), maternal serum alpha-fetoprotein (AFP) was available for 19 NTD pregnancies. Each of these was matched with four unaffected controls, by maternal age, participating centre, and duration of sample storage. The samples came from women whose gestational age ranged from 6 to 14 completed weeks. The median AFP level in the affected pregnancies was 1.2 multiples of the median value in unaffected pregnancies of the same gestational age (95 per cent confidence interval (CI) 0.83-1.59). This confirmed the view that serum AFP measurement is of no practical value in the detection of NTDs in the first trimester of pregnancy. The study also showed that folic acid supplementation, used as a method of preventing NTDs, had no effect on the concentrations of maternal serum AFP up to 14 weeks of pregnancy.

Objective: To investigate the value of the measurement of free beta human chorionic gonadotrophin (hCG) as a serum marker of Down's syndrome in the second trimester of pregnancy. Design: A prospective observational study using stored antenatal serum samples. Setting: Serum samples collected from women receiving routine antenatal care in Oxford. Subjects: Seventy-five singleton pregnancies with fetal Down's syndrome and 367 unaffected singleton pregnancies. Each affected pregnancy was matched with five control pregnancies for maternal age, gestational age, and duration of storage of the serum sample. None of the pregnancies were associated with neural tube defects. Main study measures: Maternal serum free beta-hCG levels. These were compared with total hCG levels in the same pregnancies. The performance of screening using free beta-hCG was compared with that using the principal markers, namely alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and total hCG together with maternal age. Results: The median free beta-hCG level in the affected pregnancies was 2.22 multiples of the normal median (MoM), significantly higher than in the unaffected pregnancies (95% confidence interval, 1.84-2.68 MoM). The discriminatory performances of free beta-hCG and total hCG, each considered separately, were similar; with a cut-off level of 2.5 MoM the detection rate was 43% and 5.7 of unaffected pregnancies had raised free beta-hCG levels (likelihood ratio of 7.5 (43/5.7)), somewhat better discrimination than the 32% and 4.6% respectively using total hCG (likelihood ratio of 7.0 (32/4.6)). With a higher cut-off level of 3.5 MoM, the rates were 19% and 2.7% respectively (likelihood ratio of 7.0), using free beta-hCG, worse than the 19% and 1.4% using total hCG (likelihood ratio of 13.6). Screening using maternal age, AFP, uE3 and free beta-hCG (instead of total hCG) yielded a detection rate of 62% (instead of 58%) at a screening risk cut-off level corresponding to a 5% false-positive rated). Conclusion: The main advantage in using free beta-hCG instead of total hCG is that there is a small increase in the detection rate (4%) for a given false-positive rate when used with maternal age, AFP and uE3. The main disadvantage is that there is less practical experience with free beta-hCG measurement and insufficient data to screen in certain categories of pregnancy (e.g. twins). The best practical advice is to use total hCG for the present but consider changing to free beta-hCG either (i) after further data are available that will permit the interpretation of screening results in the same way as is currently available with total hCG, or (ii) if its use with another marker confers a worthwhile increase in the detection rate for a given false-positive rate.

A study was performed to investigate the concentrations of the alpha and beta free sub-units of human chorionic gonadotrophin (free alpha-hCG and free beta-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 alpha-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 beta-hCG level was 0.96 MOM (95 per cent confidence interval 0.85-1.09). These results enable free alpha-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.

Objective To determine how frequently hydatidiform mole will be detected in a maternal serum Down's syndrome screening programme. Design Affected pregnancies were identified using a national register. Unconjugated oestriol (μE3) and human chorionic gonadotrophin (hCG) were measured in stored serum samples and alphafetoprotein (AFP) levels were available from previous neural tube defect screening at 15–20 weeks gestation. Subjects Ten pregnancies with a complete mole (i.e., hydropic placenta without a fetus), nine with stored serum samples and one with an AFP level only. Results The median values were 0.08, 0.13 and 1–83 multiples of the normal median for AFP, uE3 and hCG respectively. Six out of nine (67%) tested for all three markers had a high risk of Down's syndrome given maternal age and the marker levels. Conclusion Many molar pregnancies that have not presented clinically before 15 weeks will be detected through Down's syndrome screening.

Objective To investigate maternal serum unconjugated oestriol (uE3) and human chorionic gonadotrophin (hCG) levels in pregnant women with insulin-dependent diabetes mellitus and to consider the implications of the results for antenatal screening for Down's syndrome. Design Descriptive study using stored antenatal serum samples. Setting Stored serum samples collected from women receiving routine antenatal care in Oxford. Subjects 126 singleton pregnancies in 92 women with insulin-dependent diabetes mellitus and for each pregnancy, two pregnancies without diabetes matched for gestational age and duration of storage of the serum sample. None of the pregnancies was associated with fetal neural tube defect or Down's syndrome. Main study measures Maternal serum uE3 and hCG levels at 15–22 weeks gestation. Alpha-fetoprotein (AFP) levels were also measured for comparison. Results The median uE3 level in the diabetic pregnancies was 0.92 multiples of the median (MoM) for pregnancies without diabetes at the same gestational age (P

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 alpha and free beta-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 alpha and free beta-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 alpha-human chorionic gonadotrophin (free alpha-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 alpha-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 alpha-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.

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.

Objective To investigate the effect of using a routine ultrasound estimate of gestational age and maternal weight adjustment on maternal serum alpha-fetoprotein (AFP), unconjugated oestriol (uE3) and human chorionic gonadotrophin (hCG) levels in antenatal screening for Down's syndrome. Design Women with a singleton pregnancy without Down's syndrome were screened using the three serum markers and an estimate of gestational age based on ‘dates’ (time since first day of the last menstrual period) and one based on an ultrasound scan examination was recorded together with maternal weight. Setting Women attending the Homerton Hospital, Hackney, for their antenatal care between February 1989 and January 1990. Subjects 2113 women with a singleton pregnancy without Down's syndrome. Results The use of ultrasound to estimate gestational age (usually based on the biparietal diameter of the fetal skull) led to a significant reduction in the variance of each marker at a given week of pregnancy. The level of each marker was negatively associated with maternal weight, so that adjustment for weight also led to a reduction in variance. These data on gestational age and maternal weight, taken together with published data on pregnancies associated with Down's syndrome, indicate that the routine use of ultrasound to estimate gestational age will increase the detection rate from 58% to 67% while maintaining the false-positive rate at 5%, or reduce the false-positive rate from 5.7% to 3.1% while maintaining the detection rate at 60%. Routine maternal weight adjustment for the serum marker levels was much less useful, increasing the detection rate by about 0.5% for a given false-positive rate, or reducing the false-positive rate about 0.1% for a given detection rate. Conclusion An ultrasound gestational age estimate available at the time of Down's syndrome screening confers a substantial advantage to screening performance with a further small benefit resulting from maternal weight adjustment, which is worth adopting if it can be done without difficulty or extra cost.

Objective— To investigatc the rcason for low matcrnal scrum unconjugated oestriol (uE3) and raised human chorionic gonadotrophin (hCG) levels in Down's syndrome pregnancies. Design— Mcasurement of uE3, total oestriol (tE3), dehydroepiandrosterone sulphate (DHEAS), a precursor of oestriol, and hCG in 15–20 week amniotic fluid samples from pregnancies with and without Down's syndrome. Setting— The retrieval and use of stored amniotic fluid samples collected from women who had had an amniocentesis for antenatal diagnosis. Subjects— 45 women with a Down's syndrome pregnancy and 224 unaffected controls of the same gestational age. Results— The median level of amniotic fluid in affected pregnancies was low for uE3, tE3 and DHEAS but high for hCG: 0.50,0.46,0.35 and 1–58 multiples of the normal median, respectively. Conclusion— These results suggest that the abnormal maternal serum levels of uE3 and hCG in affected pregnancies are due mainly to abnormal feto-placental synthesis, rather than feto-maternal transfer.

Maternal serum free alpha-human chorionic gonadotrophin (free alpha-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 alpha-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 alpha-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.

Objective: To investigate the value of the measurement of free beta human chorionic gonadotrophin (hCG) as a serum marker of Down's syndrome in the second trimester of pregnancy. Design: A prospective observational study using stored antenatal serum samples. Setting: Serum samples collected from women receiving routine antenatal care in Oxford. Subjects: Seventy-five singleton pregnancies with fetal Down's syndrome and 367 unaffected singleton pregnancies. Each affected pregnancy was matched with five control pregnancies for maternal age, gestational age, and duration of storage of the serum sample. None of the pregnancies were associated with neural tube defects. Main study measures: Maternal serum free beta-hCG levels. These were compared with total hCG levels in the same pregnancies. The performance of screening using free beta-hCG was compared with that using the principal markers, namely alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and total hCG together with maternal age. Results: The median free beta-hCG level in the affected pregnancies was 2.22 multiples of the normal median (MoM), significantly higher than in the unaffected pregnancies (95% confidence interval, 1.84-2.68 MoM). The discriminatory performances of free beta-hCG and total hCG, each considered separately, were similar; with a cut-off level of 2.5 MoM the detection rate was 43% and 5.7 of unaffected pregnancies had raised free beta-hCG levels (likelihood ratio of 7.5 (43/5.7)), somewhat better discrimination than the 32% and 4.6% respectively using total hCG (likelihood ratio of 7.0 (32/4.6)). With a higher cut-off level of 3.5 MoM, the rates were 19% and 2.7% respectively (likelihood ratio of 7.0), using free beta-hCG, worse than the 19% and 1.4% using total hCG (likelihood ratio of 13.6). Screening using maternal age, AFP, uE3 and free beta-hCG (instead of total hCG) yielded a detection rate of 62% (instead of 58%) at a screening risk cut-off level corresponding to a 5% false-positive rated). Conclusion: The main advantage in using free beta-hCG instead of total hCG is that there is a small increase in the detection rate (4%) for a given false-positive rate when used with maternal age, AFP and uE3. The main disadvantage is that there is less practical experience with free beta-hCG measurement and insufficient data to screen in certain categories of pregnancy (e.g. twins). The best practical advice is to use total hCG for the present but consider changing to free beta-hCG either (i) after further data are available that will permit the interpretation of screening results in the same way as is currently available with total hCG, or (ii) if its use with another marker confers a worthwhile increase in the detection rate for a given false-positive rate.

The risk of having a Down's syndrome term pregnancy was estimated using maternal age and the results of two maternal serum alpha-fetoprotein (AFP) tests carried out between 14 and 20 weeks gestation. The estimates of risk were derived from published data and the AFP results from nine affected and 145 unaffected pregnancies in which repeat testing had been carried out. Repeat AFP testing is unjustified in antenatal screening programmes for Down's syndrome, but if a second test happens to have been done, the appropriate estimate of risk that would be applicable when counselling individual patients depends on both results. For example a woman aged 35 years and 6 months with a first AFP level of 0.50 multiples of the normal median (MoM) was estimated to have a risk of 1:172. If a second test were done on a fresh sample and the AFP level were 0.80 MoM then the estimated risk would be 1:216 which is higher than the estimated risk of 1:353 obtained if the second test were regarded as the only result. Estimates of risk are given for maternal serum AFP results in first and second tests ranging from 0.40 to 2.50 MoM.

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/l, 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 non-pregnant women who had had pregnancies associated with Down's syndrome.

The median maternal serum unconjugated oestriol level between 13 and 27 weeks gestation in 77 pregnancies associated with Down's syndrome was lower than the median level in 385 unaffected control pregnancies matched for maternal age, gestational age, and duration of serum sample storage (P less than 0.001). The median level for the affected pregnancies was 73% of that in the controls. Low unconjugated oestriol levels can be used to detect fetal Down's syndrome; at cut-off levels selected to detect at least 35% of affected pregnancies, unconjugated serum oestriol was a better screening test than either maternal age or serum alpha-fetoprotein (AFP). The use of all three variables in combination to select women with a 1:250 or greater risk of a Down's syndrome term pregnancy would yield a 45% detection rate with a false-positive rate of 5.2%. The same detection rate using maternal age alone or using age and serum AFP in combination would yield higher false-positive rates, 15% and 9.8% respectively. The addition of unconjugated oestriol to a Down's syndrome screening programme would therefore be more efficient than the use of age and AFP alone; for a given detection rate fewer women would need an amniocentesis or, for a given percentage of women having an amniocentesis, more pregnancies with Down's syndrome would be detected.