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.     

First-trimester aneuploidy screening using serum human chorionic gonadotropin (hCG), free ahCG,and progesterone

Immunochemical serum assays for human chorionic gonadotropin (hCG), the free ohCG subunit, and progesterone (P) were considered separately and in combination for their ability to screen for chromosomally abnormal pregnancies in the first trimester. Maternal serum was collected from 141 women undergoing chorionic villus sampling at 9–12 menstrual weeks. Trisomy 21 pregnancies had significantly higher hCG levels, while trisomy 18 and 13 pregnancies had markedly lower hCG and progesterone levels than those of chromosomally normal pregnancies. However, the discrimination of normal from aneuploid pregnancies was poor with either hCG alone, progesterone alone, or free ahCG alone. Much improved discrimination was obtained by combining hCG, free ahCG, and P into an aneuploidy index [(P/hCG)(free ahCG/hCG)]. This index distinguished 9 out of 17 (53 per cent) of the trisomy 21 pregnancies, while only misidentifying 5 out of 112 (4.5 per cent) of the normal pregnancies. The aneuploidy index thus appears promising as a first-trimester biochemical screen for aneuploid pregnancies.     

Prenatal diagnosis of 5p deletion syndrome following abnormally low maternal serum human chorionic gonadotrophin

Prenatal diagnosis of 5p deletion syndrome, or cri du chat, following an abnormally low measurement of a screening of serum human chorionic gonadotrophin (hCG), is reported. Karyotyping following amniocentesis revealed a terminal deletion in the short arm of one chromosome 5. The pregnancy was electively terminated. 5p deletion syndrome has been described with abnormally high hCG levels and normal hCG levels. This is the first report of its association with abnormally low levels. The association between chromosomal abnormalities and hCG is discussed. Copyright © 2003 John Wiley & Sons, Ltd.     

The effect of thermal instability of intact human chorionic gonadotropin (ihcg) on the application of its free β-subunit (free βhcg) as a serum marker in down syndrome screening

The kinetics of free βhCG concentrations were measured in 30 maternal whole blood samples from second-trimester pregnancies during 72 h incubation at 3, 20, and 30°C. Dissociation of intact hCG (ihCG) was undetectable at 3°C and produced a more than 20 per cent increase of free βhCG at 20°C and a more than 100 per cent increase at 30°C. hCG dissociation at 30°C was not reduced by a protease inhibitor (sodium iodoacetate) and also occurred in purified hCG dissolved in a protease-free incubation medium. These results were reproduced under conditions of sample transport by post at different environmental temperatures. In conclusion, reliable free βhCG assessment requires that the specimen be kept cool from vene puncture until assay or completely other transport strategies have to be considered. Evaluation of free βhCG as an effective marker in prenatal Down syndrome screening must be reconsidered from this aspect.     

Serum hCG assay: A method for detection of contamination of fetal blood samples

Serum human chorionic gonadotrophin (hCG) can be assayed in specimens obtained by percutaneous fetal blood sampling to check for the absence of maternal blood or amniotic fluid contamination. In order to assess the accuracy of this approach, we measured serum hCG in 44 pure fetal blood samples obtained by intracardiac puncture. The mean fetal serum hCG concentration was 52 IU/l, and the ratio of maternal to fetal serum hCG concentration never exceeded 1 · 1 per cent, which represents the smallest contamination rate detectable by this method.     

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).     

The characterization of hCG regulation in cultured human amniotic fluid cells

The media from primary cultures and subcultures of second trimester human amniotic fluid (AF) cells were assayed by radioimmunoassay to quantitate production of human chorionic gonadotropin (hCG). Primary AF cultures produce more hCG per cell than do the corresponding subcultures. Sodium butyrate (2 mM) stimulates AF subcultures to produce 5-13 times more hCG per cell or per mg of cellular protein than do untreated subcultures. This stimulatory effect of sodium butyrate is dose dependent between 0 and 5 mM. Addition of sodium butyrate 24 hours after subculture, while stimulating production of hCG during the subsequent 3 days, also results in fewer cells and less protein per culture. This effect on cell growth is also dose-dependent. Previous investigators have proposed that the stimulation of hCG by sodium butyrate in other types of cell cultures is due to an effect of that agent on culture growth. Therefore, in these studies AF cells are allowed to grow to confluency before sodium butyrate was added. Production of hCG was stimulated by sodium butyrate about four-fold during the next 5 days although no significant changes were observed either in number of cells or amount of cellular protein per culture. These results suggest that stimulation of hCG by sodium butyrate is not dependent on its effect on growth of the cultures.     

Second-trimester maternal serum alpha-fetoprotein (MSAFP) iselevated in women with adverse pregnancy outcome associated with inherited thrombophilias

Obstetric complications, such as severe pre-eclampsia, fetal growth restriction, abruptio placentae, or stillbirth are associated with abnormally elevated second-trimester maternal serum alpha-fetoprotein (MSAFP) and β subunit of human chorionic gonadotrophin (βhCG). This has been attributed to placental abnormalities. Women with thrombophilias have been shown to have abnormalities of the placenta resulting in adverse pregnancy outcome in these patients. The purpose of the present study was to evaluate whether women with pregnancy complications and inherited thrombophilias have abnormally elevated second-trimester MSAFP or βhCG. Sixty-two women with pregnancy complications were tested for inherited thrombophilias several months after delivery. The thrombophilia group included 29 women with pregnancy complications and an inherited thrombophilia and the control group included 33 other patients without thrombophilia. Patients in the thrombophilia group had a higher median MoM MSAFP compared to the controls (1.337 vs 1.086, p=0.0516). The incidence of abnormally elevated MSAFP (>2.5 MoM) was also significantly higher in the thrombophilia group compared to controls (21% vs 3%, p=0.04). Neither the median MoM βhCG nor the incidence of abnormally elevated βhCG were significantly different between the groups. We conclude that second trimester MSAFP, but not βhCG, is abnormally elevated in patients with thrombophilia and obstetric complications. Copyright © 2001 John Wiley & Sons, Ltd.     

Screening for fetal Down syndrome with maternal serum hCG and oestriol: A prospective study

We report the preliminary results of a prospective study aimed at evaluating the effectiveness of Down syndrome (DS) screening using second-trimester measurement of maternal serum human chorionic gonadotrophin (hCG) and unconjugated oestriol (uE3) together with maternal age. Reference values for hCG, uE3, and the hCG/uE3 ratio in normal pregnancies were established from more than 3000 normal gestations and found to follow a log-normal statistical distribution. Risk evaluation was made using reference values for affected pregnancies from retrospective studies. Screening of 10 000 women under 38 years resulted in 412 amniocenteses and the prenatal diagosis of six cases of DS, whereas four cases remained undetected until term. In a parallel study, diagnostic amniocentesis was performed in women over 38 years and in women with a previous affected child, and an evaluation of the risk of fetal DS based on serum hCG and uE3 levels was made in all cases. Fourteen cases of DS were detected. Median values for hCG and uE3 in the 24 affected pregnancies were close to the 90th and tenth centiles of the normal reference values, respectively, and thus are in good agreement with the values reported by others in retrospective studies.     

Human chorionic gonadotropin and unconjugated oestriol measurements in insulin-dependent diabetic pregnant women being screened for fetal down syndrome

This prospective study investigates the relationship between insulin-dependent diabetes and maternal serum levels of alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and human chorionic gonadotropin (hCG). It also examines the potential impact on screening for Down syndrome. The population-based cohort included 20 321 pregnant women in Maine who underwent routine serum screening for Down syndrome in the second trimester. The cohort included 52 women with insulin-dependent diabetes. Maternal serum AFP levels are now routinely adjusted for insulin-dependent diabetes. These adjustments, therefore, were made routinely in the diabetic women, but no equivalent adjustments were made for uE3 and hCG values. The initial false-positive rate (using all three markers) among the women with diabetes was not significantly different from that in the non-diabetic population (7·7 and 5·4 per cent, respectively). Prior to adjustment for insulin-dependent diabetes, the median AFP level in the 52 women was 0·73 multiples of the median (MOM); the median levels of uE3 and hCG were 0·93 and 0·98 MOM, respectively. When the uE3 and hCG levels were adjusted, the initial false-positive rate was unchanged. Median serum levels of uE3 were significantly higher in the 33 women whose onset of diabetes was prior to 19 years of age (0·99 MOM) than in the 19 women whose onset of diabetes was at age 19 or older (0·84 MOM). This is the first population-based study to investigate the relationship between diabetes and serum levels of AFP, uE3, and hCG, and confirms earlier observations from a case—control study that found only slightly lower uE3 and hCG levels.     

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.     

Trisomic pregnancies have normal human chorionic gonadotropin bioactivity

Sera from women carrying either chromosomally normal or aneuploid fetuses in the first half of pregnancy were assayed for human chorionic gonadotropin (hCG) bioactivity in order to determine whether differences might provide the basis for a useful antenatal screen for aneuploidy. A mouse uterine weight assay was used to assess hCG bioactivity in sera from 35 patients undergoing chorionic villus sampling (12 normal pregnancies and 23 trisomic pregnancies) and in sera from 18 patients undergoing elective second-trimester abortion (12 presumed normal pregnancies, 3 trisomic pregnancies, and 3 pregnancies with neural tube defects). The hCG bioactivity to immunoactivity (B:I) ratio of normal pregnancies progressively decreased from 7.7±1.3 at 4–5 menstrual weeks, to 4.7±0.4 at 9–12 menstrual weeks, to 3.3±0.5 at 16–20 menstrual weeks. There were no significant differences in the B:I ratios between normal and aneuploid pregnancies in either the first-trimester (4.7±0.4 versus 5.2±0.3) or the second-trimester samples (3.3±0.5 versus 2.6±0.3), despite significantly greater hCG concentrations in the trisomic pregnancies. We conclude that while aneuploid pregnancies display dysfunctional regulation of hCG expression, the bioactivity of their hCG is normal and does not appear to form the basis for a useful screen for aneuploidy.     

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.     

Abnormal maternal serum chorionic gonadotropin levels in pregnancies with fetal chromosome abnormalities

The alpha subunit of human chorionic gonadotropin (alpha-hCG), human chorionic gonadotropin (hCG) and alpha fetoprotein (AFP) were measured in the serum of 25 women with chromosomally abnormal fetuses between 18 and 25 weeks of gestation and in 74 normal pregnancies. AFP levels less than 0.5 multiples of the median (MoM) or greater than 2.5 MoM were observed in 24 per cent of the abnormal pregnancies and in 6.76 per cent of the normal pregnancies. A low concentration of hCG (< 0.25 MoM) was observed in 8 per cent of abnormals and in 2.7 per cent of normals while an elevated concentration of hCG (>2.5 MoM) was observed in 56 per cent of abnormals and in 1.35 per cent of normals. Elevated hCG-alpha (>2.5 MoM) was observed in 28 per cent of abnormals and in none of the normals. Determination of elevated levels of hCG-alpha or hCG resulted in detection of 68 per cent of pregnancies with chromosomally abnormal fetuses with a false positive rate of 1.35 per cent. Determination of both elevated and depressed gonadotropin levels resulted in detection of 76 per cent of abnormal pregnancies with a false positive rate of 4.05 per cent. Measurement of hCG and hCG-alpha in maternal serum samples can be used as a screening procedure for detecting pregnancies at risk for fetal chromosome abnormalities.     

Human chorionic gonadotropin levels in pregnancies with aneuploid fetuses

Maternal serum human chorionic gonadotropin (hCG) and the free alpha-hCG subunit were evaluated in 249 women from 9 to 11 weeks gestation who subsequently underwent chorionic villus sampling for determination of fetal karyotype and in 20 women of 18 or more weeks gestation who were ascertained to have an aneuploid fetus by genetic amniocentesis. Seven of the first-trimester pregnancies were determined to be aneuploid and six had hCG levels in the normal range (one triploid pregnancy had elevated hCG levels) whereas 12 of the 20 secondtrimester cases had abnormal hCG levels and an additional three had elevated levels of alpha-hCG. This study confirms the previous report of abnormal maternal serum hCG levels in women with an aneuploid fetus at ≥ 18 weeks gestation and demonstrates that hCG evaluation is not useful at 9–11 weeks gestation for selecting pregnancies at risk for fetal aneuploidy.     

First-trimester maternal serum human thyroid-stimulating hormone in chromosomally normal and Down syndrome pregnancies

Maternal serum human thyroid-stimulating hormone (TSH) levels were investigated in chromosomally normal and Down syndrome pregnancies to determine whether TSH can be used as a marker for Down syndrome in the first trimester. Measurements were conducted on stored serum samples collected from 23 Down syndrome pregnancies and 115 unaffected pregnancies before chorionic villus sampling (CVS), between 9 and 11 completed weeks of pregnancy. The samples were matched for gestational age, maternal age, maternal weight and duration of storage of the serum sample. Maternal TSH concentration was slightly decreased in Down syndrome pregnancies, with a median of 0.84 multiples of the median (MoM). Maternal serum human chorionic gonadotropin (hCG) concentration was slightly elevated in Down syndrome pregnancies, with a median of 1.03 MoM. Both differences were not significant applying matched rank analysis (p=0.50 for TSH and p=0.43 for hCG). The association between TSH and hCG in unaffected pregnancies was also measured. The Spearman correlation coefficient between TSH and hCG was −0.21 which was statistically significant (p=0.02, 95% confidence interval −0.38 to −0.03). However, it was concluded that TSH is not a useful marker for distinguishing Down syndrome-affected pregnancies from normal pregnancies in the first trimester. Copyright © 2001 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

The association between alpha-fetoprotein and βhcg levels prior to and following chorionic villus sampling in cases that spontaneously miscarried

Maternal serum alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (βhCG) measurements taken prior to chorionic villus sampling (CVS) in 21 patients who subsequently miscarried were compared with measurements in a control group of 113 patients with uneventful pregnancies. Patients with AFP levels of 10 iu/ml or more prior to the CVS had a 4·3 times greater risk of miscarriage (95 per cent confidence interval 1·3–13·6). AFP levels obtained 1 week after the CVS in the 13 patients with late miscarriages were higher than in the control group (P = 0·06). Patients miscarrying had a greater rise in AFP (P = 0·06) and a greater fall in βhCG levels (P = 0·04) following the CVS procedure, compared with the control subjects. Each 10-unit change in the difference between AFP or βhCG levels prior to and 1 week following the CVS was associated with a significantly increased risk for late miscarriage. Elevated maternal serum AFP levels early in pregnancy and changes in AFP and βhCG levels following CVS may predict an increased risk for subsequent miscarriage.