Prospective prenatal serum screening for Down syndrome in Venezuela

1. Introduction 

Prenatal screening for Down syndrome has evolved from a risk assessment based on maternal age to an approach that also uses maternal serum concentrations of specific analytes. Abnormal maternal serum levels of alpha fetoprotein (AFP), human chorionic gonadotropin (hCG or, its fraction-free subunit β-hCG), and unconjugated estriol (uE3) during the second trimester of pregnancy have been shown to be associated with Down syndrome [1], [2]. For each pregnancy, the separate probabilities derived from the serum levels of each of the 3 markers and the risk associated with maternal age generate a compound probability for a fetus to be affected with Down syndrome. If the screening result is positive, the pregnant woman is offered an amniocentesis [3]. The screening strategy requires strict quality control and audit.

There have been tremendous advances in prenatal diagnosis over the past decades, and triple-marker maternal serum screening for Down syndrome has become an established part of obstetric practice in developed countries. However, data from Latin American countries are insufficient to achieve consensus regarding implementation of prenatal screening for Down syndrome. The objective of this study was to determine whether triple-marker maternal serum screening for Down syndrome would be useful in Venezuela pregnant women.

3. Results 

Of 28 cases of chromosomal aberrations detected by amniocentesis there were 13 cases of trisomy 21, 11 resulting from meiotic nondisjunction and 2 from Robertsonian translocation, one homologous and the other heterologous (Table 1). The median MoM values were 0.69 (95% CI, 0.66 to 0.74; range 0.61–0.83) for AFP; 2.10 (95% CI, 1.85 to 2.35; range 1.58–3.32) for β-hCG and 0.67 (95% CI, 0.59 to 0.75; range 0.34–0.87) for uE3 in the study group. They were 1.03 for AFP, 1.02 for β-hCG, and 0.97 for uE3 in the control group, and the differences were significant for each marker (P<0.001 by the Mann-Whitney test in affected pregnancies with Down syndrome and normal pregnancies.

Table 1. Values for each marker and risks for trisomy 21

	Karyotype	Gestational age, wk	Maternal age, y	AFP, MoM	β-hCG, MoM	uE3, MoM	Estimated risk
	47,XY+21	17	29	0.78	2.05	0.87	1:372
	47,XY+21	18	29	0.71	1.88	0.73	1:308
	47,XX+21	15	30	0.83	1.98	0.69	1:300
	48,XYY+21	17	31	2.15	1.96	0.34	1:270
	47,XY+21	17	35	0.71	3.32	0.75	1:56
	46,XX+t(14;21)	18	38	2.31	1.87	0.84	1:300
	47,XX+21	18	38	0.71	2.32	0.67	1:31
	46,XX+t(21;21)	18	39	0.67	2.15	0.62	1:21
	47,XY+21	19	39	0.61	2.01	0.67	1:30
	47,XX+21	17	40	0.67	2.30	0.66	1:20
	47,XY+21	15	41	0.68	1.58	0.64	1:30
	47,XX+21	16	42	0.69	1.97	0.74	1:16
	47,XY+21	15	43	0.63	1.97	0.53	1:6

Abbreviation: AFP, alpha fetoprotein; β-hCG, beta human chorionic gonadotropin; MoM, multiples of the median (MoM) values for the same gestational age in the control group; uE3, unconjugated estriol.

The distributions of the AFP, β-hCG, and uE3 MoM values on the logarithmic transformation fit a normal Gaussian distribution in both groups, and the Kolmogorov-Smirnov test showed no deviation from linearity at the 0.01 probability level. The means and standard deviations of Gaussian distributions of the logarithmic MoM values and the Pearson correlation coefficient for each marker are shown in Table 2. No correlations were found between the logarithmic distributions of AFP MoM and β-hCG MoM, AFP MoM and uE3 MoM, or of β-hCG MoM and uE3 MoM in either group. No significant correlations were found between maternal age and AFP, β-hCG, or uE3 either in the study group (r=−0.22, P=0.47; r=−0.06, P=0.85; or r=−0.11, P=0.71, respectively or in the control group (r=0.03, P=0.70; r=−0.03, P=0.69, and r=−0.02, P=0.79).

Table 2. Gaussian distribution of the logarithmic MoM values and Pearson correlation coefficients for each marker in pregnancies unaffected and affected with trisomy 21

	Marker	Unaffected Pregnancies		Affected Pregnancies
	MOM values
		Mean	Standard deviation	Mean	Standard deviation
	AFP	0.01053	0.05762	–0.1664	0.03101
	β-hCG	0.002529	0.01466	0.3142	0.07972
	uE3	0.003733	0.03005	–0.1844	0.1121
	Pearson correlation coefficients
		r	P value	r	P value
	AFP plus β-hCG	0.33	<0.05	0.35	0.29
	β-hCG plus uE3	0.03	<0.05	0.30	0.32
	AFP plus uE3	0.06	<0.05	0.60	0.05

Abbreviation: AFP, alpha fetoprotein; β-hCG, beta human chorionic gonadotropin; MoM, multiples of the median (MoM) values for the same gestational age in the control group; uE3, unconjugated estriol.

The detection and false-positive rates were 69.2% (9 of 13) and 5.8% (173 of 2992), respectively. The specificity was 94.2% (2819 of 2992) and the likelihood ratio (ie, the odds of being affected given a positive result) was 1:19.

4. Discussion 

Triple-marker serum screening was developed to identify pregnant women whose risk of having a fetus with trisomy 21 is sufficiently high to be offered fetal karyotyping. It is now part of routine prenatal care in developed countries, but to the best of our knowledge the present serum screening program is the first reported study in Latin-American pregnant women.

For accurate interpretation of the results, reliable and precise estimates of the maternal serum markers are needed for the design and implementation of prenatal biochemical screening programs. Median values were monitored throughout the study and updated when necessary. In our study with Venezuelan women, the serum levels of the 3 markers fitted a Gaussian distribution similar to the distributions seen for white or Asian women both in normal pregnancies and in pregnancies affected by Down syndrome [4], [5], [6], [7]. Nine of 13 (69.23%) cases of fetal Down syndrome were identified using a cut-off risk for fetal Down syndrome of 1:270, with a false-positive rate of 5.8%.

The detection and false-positive rates varied with maternal age in our study (Table 3). The detection rate was about 25% for pregnant women younger than 35 years, with a false-positive rate of 2.98%. For pregnant women older than 35 years, the detection and false-positive rates were of 88.88% and 14.16%, respectively. Thus, the sensitivity of second-trimester maternal serum screening was much higher for women of older maternal age in our population, a finding that has also been reported by others [1], [8], [9], [10]. Although the screening's main target are pregnant women younger than 35 years, the screening is also of great importance for pregnant women 35 years or older. Still, the proportion of women older than 35 years was significantly higher in our study group than in the general population of pregnant Venezuelan women. This effect could be due to an ascertainment bias in our population.

Table 3. False-positive and detection rates of triple-marker screening for Down syndrome according to maternal age a

	Maternal age, y	All screened pregnant women	Pregnancies with a risk≥1:270	All pregnancies with trisomy 21	Pregnancies with a risk≥1:270 and trisomy 21	Percentage of detection
	<30	1403 (46.68)	37 (2.63)	2 (0.14)	0	0
	≥30–34	776 (25.81)	28 (3.61)	2 (0.26)	1	50
	≥35–37	471 (15.67)	52 (11.04)	1 (0.21)	1	100
	≥38	355 (11.84)	65 (18.31)	8 (2.25)	7	85.70
	Total	3005	182 (6.05)	13 (0.43)	9	69.23

a Values are given as number (percentage) unless otherwise indicated.

Two fetuses with Down syndrome had de novo Robertsonian translocations. Serum concentrations of β-hCG and uE3, expressed as MoM, were similar in these pregnancies and other pregnancies with trisomy 21 (P>0.05 by the Mann-Whitney test). The MoM value for AFP was higher in one case, however. This fetus (46,XX + t[21;21]) also had omphalocele, esophageal atresia, and a tracheo-esophageal fistula, which could have caused the increase in the AFP level, affecting its detection by marker serum screening. Saller et al. [11] found AFP, β-hCG, and uE3 MoMs to be similar in pregnancies with fetal trisomy 21 resulting from meiotic nondisjunction and those resulting from Robertsonian translocation, which suggests that Down syndrome from both causes will be detected in a similar percentage of cases.

Of the 3 markers usually used in maternal serum screening programs, hCG has clearly been shown to provide the best predictive value for fetal Down syndrome. A retrospective analysis demonstrated the usefulness of assessing free β-hCG levels in the identification of pregnancies with Down syndrome [12]. Assessing β-hCG is preferred to assessing intact or total hCG because, statistically, the Mahalanobis distance (ie, the number of standard deviations between the mean levels in affected and unaffected pregnancies) is more discriminatory [13]. Although the β-hCG MoM value was the best discriminating marker between unaffected and affected pregnancies in this study, the dual-marker combination β-hCG/AFP was also a good discriminator. However, in one fetus, trisomy 21 could be not ascertained by bivariate function (β-hCG and AFP), but was detected by trivariate function (β-hCG, AFP, and uE3). Since the false-positive rate did not increase when uE3 was added to calculate the risk of fetal Down syndrome, we recommend using the triple-marker rather than the double-marker screening.

In conclusion, the detection rate, false-positive rate, and specificity in this report are consistent with those reported in other studies, thus confirming the triple-marker serum screening as a suitable and efficient part of prenatal care. Governments and Health Agencies should recommend and support its implementation in routine prenatal care in Latin America.