Enhanced Second Trimester Maternal Serum Screening for Down Syndrome: Addition of Dimeric Inhibin A

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Learning Objectives
On successful completion of this activity, the participant will be able to:

  1. Recognize the major source of dimeric inhibin A in pregnant women.
  2. Differentiate screening tests from diagnostic tests in two respects.
  3. Recall the objectives of maternal serum Down syndrome screening programs.
  4. Distinguish the levels of maternal serum dimeric inhibin A in Down syndrome from those found in unaffected pregnancies.
  5. Appraise the strengths and weaknesses associated with the different ways that dimeric inhibin A may be incorporated into a maternal serum screening program.
  6. Identify the means for equating results between different clinical laboratories.
  7. Select the optimal gestational age for four-marker screening.
  8. Identify detection rates of common screening tests for trisomy 21.

Down syndrome (DS), also called trisomy 21, occurs in one out of every 800 live births, making it the most common genetic cause of moderate mental retardation.1 In addition to mental retardation, children with DS have a high incidence of congenital heart defects, connective tissue abnormalities and childhood leukemia.

Prenatal diagnosis is available by examining fetal cells obtained by chorionic villus sampling (CVS) or amniocentesis. These procedures are not appropriate for every pregnant woman because they are invasive and carry an increased risk of miscarriage. There are no ultrasound findings specific to DS. The disorder usually is not inherited; it is caused by an error in cell division in meiosis I or II, such that children with DS have an extra chromosome 21. A patient's family history, therefore, usually does nothing to determine her risk to have a baby with DS. The challenge to the obstetrics community is to identify those pregnancies at increased risk for DS that might benefit from prenatal diagnosis.

Maternal age is associated with errors in meiosis I and II. As maternal age increases, so does the risk for conceiving a child with DS or other chromosome problems.2 It is common practice to offer CVS or amniocentesis to pregnant women aged 35 years or older in order to diagnose these potential chromosome problems. The risk of miscarriage associated with amniocentesis is 0.5 percent (1/200).3

Age 35 is chosen as the threshold at which invasive testing is offered because at this maternal age the risk of having a child with DS approaches the risk of miscarriage from the diagnostic procedure. Unfortunately, no matter what her age, every woman has some risk of having a child with DS. Moreover, because the majority of the reproductive population is younger than age 35, most babies with DS are born to younger women, even though individually younger women have a lower risk.

Since the mid 1980s, screening tests that measure specific proteins in maternal serum have been developed to assist obstetricians in identifying at-risk pregnancies in women under age 35.

Screening tests are different from diagnostic tests in that they do not identify affected individuals. Rather, they identify a population of at-risk individuals who may be referred for diagnostic testing. Screening tests necessarily have a high number of false-positive and false-negative results. The detection rate and false-positive rate of any screening program can be modulated to some extent by the lab's choice of abnormal cut-off values. The challenge for the laboratory is to achieve the highest detection rate possible while maintaining an acceptable false-positive rate. In the case of DS screening, this means defining an acceptable number of women who will be offered amniocentesis even though the majority will not have affected fetuses.

Until recently, maternal serum screening panels consisted of either two markers (alpha-fetoprotein [AFP] and human chorionic gonadotropin [hCG]) or three markers (AFP, hCG and unconjugated estriol [uE3]). A fourth marker, dimeric inhibin A (DIA), is emerging as an excellent adjunctive serum analyte to screen for DS. When DIA information is combined with patient specific data and other serum marker results, 75 percent to 80 percent of DS pregnancies can be identified with less than a 5 percent false-positive rate.4

Historical Perspective
Since the 1970s, maternal serum AFP has been used to screen for fetuses with open neural tube defects such as spina bifida and anencephaly.5 Identifying pregnant women with elevations of maternal serum AFP detects more than 80 percent of open neural tube defects.5 In 1984, an association was reported between low levels of maternal serum AFP in the second trimester and DS.6 Maternal serum AFP values identified 20 percent of fetal DS in women less than 35 years old. This finding started the era of noninvasive screening to identify at-risk pregnancies. Women whose maternal serum AFP was screen-positive were referred for genetic counseling and offered amniocentesis for prenatal diagnosis.

In 1988, a pattern of low AFP, low uE3 and high hCG in maternal serum was associated with fetal DS.7 The combination of maternal age and these three markers, commonly referred to as the "triple screen," was shown to detect more than 60 percent of DS with a false-positive rate of 5 percent in women under 35 years of age.7

The triple screen has been considered standard of care for several years in many parts of the world. In addition to screening for DS, three-marker screening provides risk estimates for other fetal anomalies. The AFP component of the triple screen continues to offer information about fetal open neural tube defects. Low levels of AFP, uE3 and hCG indicate a higher risk for fetal trisomy 18 with a detection rate of 60 percent.8

Dimeric Inhibin A and Fetal Down Syndrome
DIA is produced by the ovaries (in nonpregnant women) and by the placenta.9,10 It is thought to suppress follicle-stimulating hormone (FSH).11 Maternal serum DIA levels increase during the first trimester until 10 weeks of gestation, remain stable until approximately 25 weeks, then rise again to reach their peak levels at term.12

Several studies have shown that DIA levels in the maternal sera of pregnancies affected with DS are approximately twice as high as in unaffected pregnancies.4,13,14 This significant separation of DIA values between affected and unaffected pregnancies can identify 41 percent of fetal DS in the second trimester.4

To increase the value of DIA as a DS screening tool, other serum analytes that were previously identified as markers for DS screening have been examined in various combinations with DIA. Statistical modeling of different combinations of AFP, uE3, hCG and DIA indicates that the highest detection rates and lowest false-positive rates are always achieved when using all four markers and maternal age.4 Screening with AFP, hCG and DIA appears to be nearly as effective. But uE3 levels are useful for other screening goals; they provide risk estimates for fetal trisomy 18 and can be a prenatal marker for steroid sulfatase deficiency and Smith-Lemli-Opitz syndrome.15,16 DIA provides no information regarding fetal trisomy 18 risk; therefore, four-marker screening that includes DIA appears to be the most informative combination (Table).

DIA Correlations With Other Variables
To determine the efficacy of DIA as a screening tool, researchers examined a number of clinical and demographic variables for associations that might confound interpretation of results. Since fetal DS is more likely to occur in older women, the correlation between DIA and maternal age was examined. No association was found.4,17 Race of the pregnant woman has been identified as a factor for maternal serum AFP measurements, and some studies suggest that race could be a factor for hCG testing as well.18 Black women, on average, have higher levels of maternal serum AFP than Caucasian, Asian or Hispanic women; therefore, an adjustment must be made in order not to lower the detection rate of DS among blacks.

To date, DIA levels have been examined in Asian women, and no statistical difference between Asians and Caucasians was noted.19 At this time there are no published data suggesting that adjustments are necessary for maternal serum DIA levels in black or Hispanic women.

Maternal insulin-dependent diabetes prior to pregnancy has been associated with lower maternal AFP levels; however, studies have demonstrated that no adjustment of DIA was necessary for women taking insulin.12 Maternal weight is the only factor studied to date that has a significant effect on DIA levels.17 As for all maternal serum markers currently used, there is an inverse relationship between marker level and maternal weight. Little correlation is found between the levels of AFP, uE3 and DIA, but a significant correlation is noted between DIA and hCG.17

How Four-Marker Screening Results Are Determined
Although DIA levels are relatively stable throughout the second trimester, AFP, hCG and uE3 values fluctuate; therefore, the timing of maternal serum screening will affect the DS detection rate. It is optimal to obtain a maternal serum specimen between 16 and 18 weeks of gestation, and an accurate gestational age must be provided to the lab.7 As discussed above, markers are influenced by patient-specific variables, so weight, race, insulin status and number of fetuses must also be provided. The analytes are measured by radioimmunoassay or enzyme immunoassay, and the raw lab values are converted to a unit known as a multiple of the median (MoM).

MoM values normalize results to provide an equivalent measure between laboratories. Each laboratory uses its own internal data to establish median values for each analyte at every gestational age. These medians are used to convert results to MoMs. The MoM values are adjusted according to the patient's gestational age, weight, race, insulin-dependent diabetes status and number of fetuses. The AFP, hCG, uE3 and DIA MoMs are then compared to the known data for affected and unaffected pregnancies and a likelihood ratio is calculated for each marker. The likelihood ratios are then combined with the patient's age-related risk for DS. The resulting number is the patient's specific risk for DS. Because the four-marker results are combined with patient-specific data and each other, there are no reference intervals for each analyte.

How Screen Results are Interpreted
The choice of a screen-positive cut-off level determines the detection rate and false-positive rate achieved by the screening program. Maternal serum marker levels are used to modify a pregnant woman's age-related DS risk (a priori risk) to calculate a patient-specific risk that she is carrying a fetus with DS. A common practice is to call a test positive if a woman's fetal DS risk by maternal serum screening is the same or greater than the fetal DS risk of a 35-year-old woman.

At 35 years of age, the risk that a woman is carrying a child with DS during the second trimester is 1/270 (1/350 at term).4 Using a risk of 1/270 as a cut-off level, four-marker screening will achieve a DS detection rate of 75 percent to 80 percent.4 This increase of 15 percent to 20 percent over traditional three-marker screening is achieved with approximately the same false-positive rate as with three markers. If the cut-off level is raised to a second trimester fetal DS risk of 1/150 (1/200 at term), the false-positive rate drops to about 3 percent with approximately the same DS detection rate, 60 percent, found in three-marker screening.

At this point, the goal of prenatal DS screening must be addressed. Choosing a cut-off level that reduces the false-positive rate will mean less patient anxiety and fewer amniocenteses performed on women with unaffected pregnancies at a DS detection rate of 60 percent, a rate that is already accepted by the obstetric and genetics communities.

A detection rate of 60 percent means that 40 percent of DS pregnancies will be missed by maternal serum screening. Increasing the detection rate to 75 percent to 80 percent will give many more couples reproductive choices at the cost of a false-positive rate that is the same or slightly lower than the 5 percent rate currently accepted with three-marker screening. Many commercial laboratories in the United States that offer four-marker screening have chosen to increase their DS detection rates and maintain a false-positive rate similar to that of their three-marker screening programs.

The Future of Maternal Serum Screening
Other maternal serum markers continue to be investigated to try to raise the DS detection rate and lower the false-positive rate even further. Current interest is focused on markers that may be used to predict DS risk in the first trimester, such as pregnancy-associated plasma protein A and free ß-hCG.20 Major clinical trials are being performed in the United States and the United Kingdom to compare first trimester and second trimester DS screening directly. Some authors have suggested that the most effective DS screening program will use a combination of both first- and second-trimester maternal serum markers.21

Maternal serum screening during the second trimester of pregnancy is a standard practice for identifying pregnancies at higher risk for several birth defects, including DS. DIA is a protein produced primarily by the placenta. High levels of inhibin A are correlated with DS pregnancies. When incorporated into a maternal serum DS screening program, it can improve the efficiency of the screening program dramatically, either by significantly raising the DS detection rate or by lowering the false-positive rate, depending on the goals of the program. *

Dr. McDowell is director of biochemical genetics at the LabCorp Center for Molecular Biology and Pathology, Research Triangle Park, NC. Correspondence regarding this article may be addressed to Dr. McDowell via e-mail at mcdoweg@labcorp.com.

Trisha Brown is director of genetics services; Dr. Papenhausen is national director of genetics; and Dr. Anderson is vice president, chief scientific officer for molecular diagnostics.


1. Jones KL. Smith's Recognizable Patterns of Human Malformation. 4th ed. Philadelphia: W.B. Saunders Company 1988:10­15.

2. Cuckle HS, Wald NJ, Thompson SG. Estimating a woman's risk of having a pregnancy associated with Down's syndrome using her age and serum alpha-fetoprotein level. Brit J Obstet Gynaecol 1987;94:387­402.

3. Obstetric and Gynecologic Procedures. In Bechman CRB, Ling FW. Obstetrics and Gynecology for Medical Students. Baltimore, MD: Williams and Wilkins; 1992:19.

4. Haddow JE, Palomaki GE, Knight GJ, Foster DL, Neveux LM. Second trimester screening for Down's syndrome using maternal serum dimeric inhibin A. J Med Screen 1998;5:115­119.

5. Maternal serum-alpha-fetoprotein measurement in antenatal screening for anencephaly and spina bifida in early pregnancy: Report of UK collaborative study on alpha-fetoprotein in relation to neural-tube defects. Lancet 1977;2:1323­1332.

6. Merkatz IR, Nitowsky HM, Macri JN, Johnson WE. An association between low maternal serum a-fetoprotein and fetal chromosome abnormalities. Am J Obstet Gynecol 1984;148:886­894.

7. Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum screening for Down's syndrome in early pregnancy. Br Med J 1988;297:883­887.

8. Canick JA, Palomaki GE, Osathanondh R. Prenatal screening for trisomy 18 in the second trimester. Prenat Diagn 1990;10:546­548. [letter]

9. Illingworth PJ, Reddi K, Smith KB, Baird DT. The source of inhibin secretion during the human menstrual cycle. J Clin Endocrinol Metab 1991;73(3):667­673.

10. Qu J, Thomas K. Inhibin and activin production in human placenta. Endocrine Rev 1995;16(4):485­507.

11. Ying SY. Inhibins, activins, and follistatins: Gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocrine Rev 1988; 9(2):267­293.

12. Wald NJ, Watt HC, George L. Maternal serum inhibin-A in pregnancies with insulin-dependent diabetes mellitus: Implications for screening for Down's syndrome. Prenat Diagn 1996;16:923­926.

13. Lambert-Messerlian GM, Canick JA, Palomaki GE, Schneyer AL. Second trimester levels of maternal serum inhibin A, total inhibin, alpha inhibin precursor, and activin in Down's syndrome pregnancy. J Med Screen 1996;3(2):58­62.

14. Wenstrom KD, Owen J, Chu DC, Boots L. a-Fetoprotein, free b-human chorionic gonadotropin, and dimeric inhibin A produce the best results in a three-analyte, multiple-marker screening test for fetal Down syndrome. Am J Obstet Gynecol 1997; 177(5):987­991.

15. Glass IA, Lam RC, Chang T, Roitman E, Shapiro LJ, Shackleton CH. Steroid sulphatase deficiency is the major cause of extremely low estriol production at mid-pregnancy: A urinary steroid assay for the discrimination of steroid sulphatase deficiency from other causes. Prenat Diagn 1998;18:789­800.

16. Bick DP, McCorkle D, Stanley WS, et al. Prenatal diagnosis of Smith-Lemli-Opitz syndrome in a pregnancy with low maternal serum oestriol and a sex-reversed fetus. Prenat Diagn 1999;19:68­71.

17. Spencer K, Wallace EM, Ritoe S. Second-trimester dimeric inhibin-A in Down's syndrome screening. Prenat Diagn 1996;16:1101­1110.

18. Crandall BF, Lebherz TB, Schroth PC, et al. Alpha-fetoprotein concentrations in maternal serum: Relation to race and body weight. Clin Chem 1983;29(3):531­535.

19. Lam YH, Tang MHY. Second-trimester maternal serum inhibin-A screening for fetal Down syndrome in Asian women. Prenat Diagn 1999;19:463­467.

20. De Biasio P, Siccardi M, Volpe G, Famularo L, Santi F, Canini S. First trimester screening for Down's syndrome using nuchal translucency measurement with free beta-hCG and PAPP-A between 10 and 13 weeks of pregnancy: The combined test. Prenat Diagn 1999;19:360­363.

21. Wald NJ, Watt HC, Hackshaw AK. Integrated screening for Down's syndrome based on tests performed during the first and second trimesters. N Engl J Med 1999;341(7):461­467.

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