Maternal serum free beta hCG screening: Results of studies including 480 cases of down syndrome

The median maternal serum free beta human chorionic gonadotropin (hCG) multiple of the median (MOM) of 480 Down syndrome cases in the second trimester was 2·64, significantly greater than the reported median MOM of intact hCG (p<0·0001). In 234 of these cases from retrospective and prospective studies, the effectiveness of maternal serum free beta hCG was evaluated in combination with alpha-fetoprotein (AFP) and maternal age in second-trimester Down syndrome screening. Down syndrome detection in the gestational age range of 14–16 weeks was 82 per cent. In all gestational weeks (14–22), a 77·7 per cent Down syndrome detection rate was achieved. In prospective screening of 44 272 patients under the age of 35 years, 69 per cent of Down syndrome cases were detected (73 per cent in gestational weeks 14–16). The false-positive rate for the prospective study was 3·8 per cent. The use of free beta hCG combined with maternal serum AFP and maternal age-related risk for Down syndrome in a screening population (i.e., women under 35 years) yields an improved detection efficiency over other protocols.     

Racial differences in maternal serum human chorionic gonadotropin and unconjugated oestriol levels

We assayed maternal serum samples from 134 black and 268 white women from 16 to 18 weeks of gestation for intact human chorionic gonadotropin (hCG), and unconjugated oestriol (uE3). Serum from women with high ( ⩾ 2·5 MOMs) or low (risk for Down syndrome ⩾ 1/365) maternal serum alpha-fetoprotein (MSAFP) levels were excluded. After correcting for maternal weight, we found that median hCG levels were 16 per cent higher in black women but uE3 levels were not significantly different. These results confirm three other studies for hCG and one study for uE3. Corrections are recommended for both maternal serum hCG and AFP before calculating the risk for Down syndrome in black women.     

Second-trimester diurnal variation of maternal serum alpha-fetoprotein,human chorionic gonadotropin,and unconjugated oestriol: Is it present and does it affect the prediction of a patient's risk for fetal down syndrome?

Both a cross-sectional and a longitudinal study were performed to investigate whether or not the collection time should be taken into consideration when generating a patient's risk for fetal Down syndrome with multiple marker screening. Diurnal variations of third-trimester alpha-fetoprotein (AFP) levels and first-trimester human chorionic gonadotropin (hCG) levels have been previously reported. In addition, large episodic fluctuations of conjugated and unconjugated oestriol (uE3) as well as a diurnal variation have also been reported in the third trimester. If the levels of these analytes routinely fluctuate during the day, they could affect a patient's risk calculation for fetal Down syndrome. The longitudinal study evaluated ten non-diabetic women who underwent sequential sampling for AFP, hCG, and uE3. The cross-sectional study evaluated 1953 patients for these three markers whose time of sampling was recorded between 8·00 a.m. and 5·59 p.m. Using either study design, no significant effect was seen in the median MOM levels of the screening analytes as a function of the time of day.     

Biochemical screening for chromosomal disorders and neural tube defects (NTD): is adjustment of maternal alpha-fetoprotein (AFP) still appropriate in insulin-dependent diabetes mellitus (IDDM)?

Maternal serum alpha-fetoprotein (AFP) has been reported to be decreased in insulin-dependent diabetes mellitus (IDDM). The objective of the present study was to reinvestigate this finding in detail. Maternal serum levels of AFP, human chorionic gonadotropin (hCG), and unconjugated estriol (uE3) in 114 diabetic women, of whom 84 had IDDM, were compared to those of 19,251 control pregnancies in the second trimester (15th to 20th gestational weeks). The mean body weight at the date of sampling was 73.7 kg in all diabetic women, 72.7 kg in women with IDDM and 68.3 kg in non-diabetic women, respectively. Body weights were significantly (p<0.001) elevated in all diabetic pregnancies. Using weight-adjusted MoM (multiple of the median) values no statistical difference of serum levels in diabetic and non-diabetic pregnant women was found. Median MoM levels were 1.01 (hCG), 1.01 (uE3), 1.06 (AFP) in all diabetic women, and 0.95 (hCG), 0.96 (uE3), 0.96 (AFP) in women with IDDM, respectively. Ignoring adjustment for maternal weight leads to a reduction of all serum parameters in diabetic pregnancies. However, median MoM values of all three analytes are not statistically different when compared to non-diabetic pregnancies. This finding is contrary to the results of former studies from the 1970s and 1980s. It is concluded that progress in insulin adjustment and blood glucose surveillance of diabetic patients on the whole has balanced out serum levels. Therefore adjustment of serum AFP values for diabetic status no longer seems reasonable. Copyright © 2001 John Wiley & Sons, Ltd.     

Adjustment formulae for maternal serum alpha-fetoprotein,human chorionic gonadotropin,and unconjugated oestriol to maternal weight and smoking

Serum levels of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and un-conjugated oestriol (uE3) were measured in serum samples of 4131 non-smoking and 1018 smoking women during the second trimester of pregnancy. The levels of all three analytes decreased with increasing body weight. The AFP median was significantly increased in smokers in a dose-response association; hCG decreased by 21 per cent and uE3 decreased by 3 per cent in smokers in a non-dose-related fashion. Regression functions for adjustment of serum levels for weight and smoking should be considered in risk estimation for Down syndrome in order to give a woman's individual risk more precisely.     

Maternal serum human chorionic gonadotrophin and pregnancy-associated plasma protein a,markers for fetal down syndrome at 8–14 weeks

Maternal serum levels of human chorionic gonadotrophin and its subunits (intact, α, and free βhCG) and pregnancy-associated plasma protein A (PAPP-A) were measured in 279 women between 8 and 14 weeks' gestation. This group included 23 pregnancies in which the fetus had Down syndrome (DS), diagnosed either at birth or during the second trimester (n=17) or from chorionic villus sampling (CVS) (n=6). Normal medians were determined from the 258 apparently normal pregnancies. The median levels of intact hCG (1·4 MOM) and free βhCG (2·1 MOM) were significantly raised, whereas the median level of PAPP-A (0·39 MOM) was significantly lower in the DS pregnancies when compared with the control group. Levels of αhCG were similar in both the control and the DS pregnancies. Analysis of samples taken prior to 14 weeks' gestation demonstrated that only PAPP-A (0·34 MOM) was significantly altered in DS pregnancies. However, after the exclusion of DS cases diagnosed at CVS, the median intact hCG (1·56 MOM), free βhCG (2·27 MOM), and αhCG (1·8 MOM) were all raised in DS pregnancies. This emphasizes the problem of the interpretation of biochemical markers when DS cases are diagnosed at CVS.     

An association between fetal abdominal wall defects and elevated levels of human chorionic gonadotropin in mid-trimester

We analysed maternal serum human chorionic gonadotropin (hCG) in 16 pregnancies with fetal abdominal wall defects previously identified prenatally by elevated maternal serum alpha-fetoprotein (AFP) or at birth. The AFP levels had a mean of 6·38 MOM (range 0·34–15·65), as expected with these defects. The hCG levels had a mean of 1·82 MOM (range 0·23–4·11). The hCG levels in five pregnancies (31·25 per cent) were above 2·30 MOM. Elevated levels of hCG may be associated with fetal abdominal wall defects.     

Risk-based prenatal screening for trisomy 18 using alpha-fetoprotein,unconjugated oestriol and human chorionic gonadotropin

Nine centres collaborated to examine the feasibility of a screening method for trisomy 18 that was based on assigning individual risk, using a combination of maternal age and measurements of alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and human chorionic gonadotropin (hCG). Second-trimester measurements of these analytes were obtained from 94 trisomy 18 pregnancies. In the 89 pregnancies without an associated open defect, the median levels for AFP, uE3, and hCG were 0.65, 0.43 and 0.36 multiples of the unaffected population median, respectively. The strongest individual predictor of risk for trisomy 18 was uE3, followed by hCG, AFP, and maternal age, in that order. Using a method of individual risk estimation that is based on the three markers and maternal age, 60 per cent of pregnancies associated with trisomy 18 would be detected at a risk cut-off level of 1:100, with a false-positive rate of about 0.2 per cent. One in nine pregnancies identified as being at increased risk for trisomy 18 would be expected to have an affected pregnancy. This risk-based screening method is more efficient than an existing method that is based on fixed analyte cut-off levels. Even though the birth prevalence of trisomy 18 is low, prenatal screening can be justified when performed in conjunction with Down syndrome screening and when a high proportion of women offered amniocentesis have an affected fetus.     

Second-trimester prenatal screening markers for Down syndrome in women with insulin-dependent diabetes mellitus

Published studies have shown that some serum markers used in screening for Down syndrome tend to be lower among women with insulin-dependent diabetes mellitus (IDDM). On this basis, many screening programmes adjust the marker levels to take account of this difference. Recent studies suggested that the marker levels were not different, and so adjustment may no longer be needed, possibly because of better diabetic control. Data from a prenatal screening programme for Down syndrome were examined to see whether the median values of second-trimester screening markers were still reduced in pregnant women with IDDM. A total of 366 women with IDDM singleton pregnancies without Down syndrome were identified from the screening programme at Barts from 1989 to 2002. After allowing for maternal weight, the median multiples of the median (MoM) for IDDM-unaffected singleton pregnancies were as follows: 0.88 (95% confidence interval 0.84–0.93) for alphafetoprotein (AFP), 0.95 (0.91–0.99) for unconjugated oestriol (uE₃), 0.90 (0.80–1.01) for total human chorionic gonadotrophin (total hCG), 0.98 (0.88–1.08) for free β-hCG, and 0.99 (0.89–1.10) for inhibin-A. The median levels for AFP and uE₃ were statistically significantly lower in pregnant women with IDDM. The other markers were not significantly different in women with and without IDDM. There remains a case for adjusting AFP and uE₃ levels in women with IDDM in prenatal screening programmes for Down syndrome. Copyright © 2004 John Wiley & Sons, Ltd.     

Prenatal detection of X-linked ichthyosis by maternal serum screening for down syndrome

Maternal serum unconjugated oestriol (uE3) was measured in 15 375 pregnancies during 2 years of second-trimester risk assessment for Down syndrome using biochemical markers. Very low levels of uE3 (<0·1 MOM) were detected in 22 serum samples (0·14 per cent). Very low uE3 was associated with an adverse outcome in 13 pregnancies including fetal death and miscarriage (N=11), anencephaly (N=1), and Meckel—Gruber syndrome (N=1). Dry scales on the skin appeared in the first year of life in four boys. From dermatological diagnosis, prenatal uE3 levels, and pedigree analysis, it is concluded that at least 5 in approximately 7500 male births in the study population are affected by steroid sulphatase deficiency, which is the biochemical defect in X-linked ichthyosis. Very low uE3 levels in the second trimester are indicative of this disease in pregnancies with normal ultrasound findings.     

First-trimester maternal serum biochemical indicators in Down syndrome

A set of 21 early maternal serum samples (19 first-trimester and two at 14 weeks) from pregnancies resulting in a child with Down syndrome was matched for gestation and length of storage with 63 samples from unaffected pregnancies. The concentrations of alpha-fetoprotein (AFP), unconjugated oestriol (uE₃), human chorionic gonadotrophin (hCG), pregnancy-specific β₁–glycoprotein (SP₁), and placental alkaline phosphatase (PALP) were measured. The ratios of the medians for Down syndrome pregnancies compared with the medians for controls were AFP 0·71, uE₃ 0·67, hCG 1·43, SP₁ 0·79, and PALP 0·92. Although the differences between the medians for affected and unaffected pregnancies were not significant, the trends for AFP, uE₃, and hCG confirm earlier findings on first-trimester samples.     

First-trimester free beta (hCG) screening for Down syndrome

Maternal serum free beta (hCG) levels are elevated (median 2·20 MOM) in the first trimester of pregnancy in 38 Down syndrome cases as compared with appropriate controls. This observation may form the basis for its use as a marker in screening for Down syndrome in the first trimester. Altered levels of the free beta analyte are observed in pregnancy conditions or complications other than Down syndrome.     

Second-trimester levels of maternal serum unconjugated oestriol and human chorionic gonadotropin in pregnancies affected by fetal anencephaly and open spina bifida

Unconjugated oestriol (uE3) and human chorionic gonadotropin (hCG) levels were determined in second-trimester maternal serum (MS) samples from 21 pregnancies associated with fetal anencephaly and 15 pregnancies associated with fetal open spina bifida. Each measurement was expressed as a multiple of the median (MoM) for unaffected pregnancies for each completed week of gestation. In pregnancies associated with anencephaly, the median value for MSuE3 was very low (0–17 MoM, range <0·12–0·33 MoM), suggesting a functional defect in the fetal adrenal prior to 20 weeks' gestation; the median value for MShCG was also low (0–73 MoM), although not to the same extent as for MSuE3. A biological explanation for the hCG result is not apparent. In pregnancies associated with open spina bifida, the MSuE3 and MShCG values were unremarkable, consistent with a lack of involvement of these open fetal defects in the synthesis and secretion of uE3 and hCG.     

Serum PAPP-A and free β-hCG are first-trimester screening markers for down syndrome

Serum measurements of pregnancy-associated plasma protein A (PAPP-A) and the free β-human chorionic gonadotrophin (hCG) subunit were made in 13 women with Down syndrome (DS) pregnancies and six other women with fetal aneuploidy ascertained at chorionic villus sampling (CVS), as well as 89 women with contemporaneous normal control pregnancies. Median serum PAPP-A measurements (0·31 MOM, 95 per cent confidence interval (CI) 0·22–0·65 vs. normal 1·06, 95 per cent CI 0·89–1·20) were lower and free β-hCG subunit measurements (1·13 MOM, 95 per cent CI 0·93–2·63 vs. normal 0·91, 95 per cent CI 0·79–1·03) were higher at statistically significant levels. Receiver operator characteristic (ROC) curves showed that the highest sensitivity for detection, 71·2 per cent (95 per cent CI 54·7–87·6 per cent), was for depressed PAPP-A levels; the combination of low serum PAPP-A levels, maternal age, and elevated free β-hCG levels yielded a detection rate of 78·9 per cent (95 per cent CI 64·9–92·8 per cent) of the affected pregnancies at 8–12 weeks' gestation.     

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.     

Abnormal amniotic fluid levels of CA 125 in second-trimester Down syndrome pregnancies

The aim of this study was to evaluate the concentration of CA 125 in second trimester amniotic fluid from Down syndrome pregnancies. CA 125 was measured in stored amniotic fluid samples from pregnancies of 14–19 weeks' gestation with and without Down syndrome fetuses. CA 125 levels were expressed in multiples of the median (MOM) for normal pregnancies of the same gestational age. Twenty-one pregnancies with Down syndrome fetuses and 63 unaffected controls matched for maternal age, gestational age, and duration of storage were studied. The median MOM values of the affected pregnancies were significantly higher than those of the controls (1·41 MOM versus 0·99 MOM). These findings show that there is an increased concentration of CA 125 in second-trimester amniotic fluid from Down syndrome pregnancies.     

Second-trimester levels of maternal urinary gonadotropin peptide in down syndrome pregnancy

Urinary gonadotropin peptide (UGP; β-core fragment), a major metabolite of human chorionic gonadotropin (hCG), was shown recently to be markedly elevated in Down syndrome pregnancy between 19 and 22 weeks of gestation. To confirm and extend this finding, we obtained maternal urine and matching maternal serum samples from 14 cases of Down syndrome and six other aneuploidies between 17 and 21 weeks of gestation. UGP was measured in all these samples and in 91 singleton control urines. Results were corrected for urinary creatinine level and expressed as multiples of the control median (MOM). hCG levels were assayed in all serum samples from the cases and compared with previously established reference values. The median UGP level in Down syndrome cases was 5.34 MOM (range 2.71–12.57); 88 per cent of the values were above the 95th centile of control levels after modelling. The median maternal serum hCG level for the same cases was 2.20 MOM (range 0.84–3.40); 36 per cent of the values were above the 95th centile. The level of UGP in every case including all other aneuploidies was higher than the comparable maternal serum hCG level. Elevated UGP measurements are strongly associated with fetal Down syndrome during the second trimester and could contribute to improved Down syndrome screening protocols that are more accessible and less expensive than are currently available.     

Second-trimester unconjugated oestriol levels in maternal serum from chromosomally abnormal pregnancies using an optimized assay

Second-trimester unconjugated oestriol (UE3) levels were measured retrospectively in maternal serum from 78 chromosomally abnormal pregnancies and 390 matched controls using a radioimmunoassay kit (Amersham AMERLEX-M) optimized for use in the second trimester. Reduced levels of UE3 were found in a group of 49 Down's syndrome pregnancies with a median UE3 level of 0·79 multiples of the median (MOM) of the controls. Four trisomy 18 pregnancies had UE3 levels less than 0·7 MOM. There was a highly significant level of correlation between alpha-fetoprotein (AFP) and UE3 levels in the controls (r = 0·25, P <0·01), the Down's syndrome pregnancies (r = 0·44, p 0·01), and the other chromosome abnormalities (r = 0·61, p0·01). When used as an additional marker to AFP and human chorionic gonadotrophin in screening for Down's syndrome, UE3 does not appear to add to the sensitivity of such screening.     

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.     

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.