Prenatal screening for open neural tube defect (AFP only), Down

syndrome (AFP, hCG, uE[3], and inhibin A) and trisomy 18 (AFP,

hCG, uE[3])

 

Note:  The use of these markers to screen for Down syndrome is

               not an approved Food and Drug Administration (FDA)

               procedure.

Multiple marker serum screening has become a standard tool

used in obstetrical care to identify pregnancies that may have

an increased risk for certain birth defects, including neural tube

defects (NTDs), Down syndrome, and trisomy 18. The screen is

performed by measuring analytes in maternal serum that are

produced by the fetus and the placenta. The analyte values along

with maternal demographic information such as age, weight,

gestational age, diabetic status, and race are used together in a

mathematical model to derive a risk estimate. The laboratory

establishes a specific cutoff for each condition, which classifies

each screen as either screen-positive or screen-negative. A

screen-positive result indicates that the value obtained exceeds

the established cutoff. A positive screen does not provide a

diagnosis, but indicates that further evaluation should be

considered.

 

Analytes:

Alpha-fetoprotein (AFP):

AFP is a fetal protein that is initially produced in the fetal yolk sac

and liver. A small amount also is produced by the gastrointestinal

tract. By the end of the first trimester, nearly all of the AFP is

produced by the fetal liver. The concentration of AFP peaks in

fetal serum between 10 to 13 weeks. Fetal AFP diffuses across

the placental barrier into the maternal circulation. A small amount

also is transported from the amniotic cavity.

 

The AFP concentration in maternal serum rises throughout

pregnancy, from a nonpregnancy level of 0.2 ng/mL to about

250 ng/mL at 32 weeks gestation. If the fetus has an open NTD, AFP is

thought to leak directly into the amniotic fluid causing unexpectedly high

concentrations of AFP. Subsequently, the AFP reaches the maternal

circulation, thus producing elevated serum levels. Other fetal

abnormalities such as omphalocele, gastroschisis, congenital renal

disease, esophageal atresia, and other fetal distress situations such

as threatened abortion, and fetal demise also may show AFP elevations.

Increased maternal serum AFP values also may be seen in multiple

pregnancies and in unaffected singleton pregnancies in which the

gestational age has been underestimated.

 

Lower values have been associated with an increased risk for

genetic conditions such as trisomy 21 (Down syndrome) and

trisomy 18.

 

Estriol (uE[3])

Estriol, the principal circulatory estrogen hormone in the blood

during pregnancy, is synthesized by the intact feto-placental unit.

Estriol exists in maternal blood as a mixture of the unconjugated

form and a number of conjugates. The half-life of unconjugated

estriol in the maternal blood system is 20 to 30 minutes because the

maternal liver quickly conjugates estriol to make it more water

soluble for urinary excretion. Estriol levels increase during

the course of pregnancy. Decreased unconjugated estriol has

been shown to be a marker for Down syndrome and trisomy 18.

Low levels of estriol also have been associated with

overestimation of gestation, pregnancy loss, Smith-Lemli-Opitz,

and X-linked ichthyosis (placental sulfatase deficiency).

 

 

Human Chorionic Gonadotropin (Total Beta-hCG [ThCG]):

hCG is a glycoprotein consisting of 2 noncovalently bound

subunits. The alpha subunit is identical to that of luteinizing

hormone (LH), follicle-stimulating hormone (FSH), and thyroid-

stimulating hormone (TSH), while the beta subunit has significant

homology to the beta subunit of LH and limited similarity to the

FSH and TSH beta subunits. The beta subunit determines the

unique physiological, biochemical, and immunological properties

of hCG.

 

The CGA gene (glycoprotein hormones, alpha polypeptide)

 is thought to have developed through gene duplication from the

LH gene in a limited number of mammalian species. hCG only

plays an important physiological role in primates (including humans),

where it is synthesized by placental cells, starting very early in

pregnancy, and serves to maintain the corpus luteum, and hence,

progesterone production, during the first trimester. Thereafter,

the concentration of hCG begins to fall as the placenta begins

to produce steroid hormones and the role of the corpus luteum

in maintaining pregnancy diminishes.

 

Increased total hCG levels are associated with Down syndrome,

while decreased levels may be seen in trisomy 18. Elevations

of hCG also can be seen in multiple pregnancies, unaffected

singleton pregnancies in which the gestational age has been

overestimated, triploidy, fetal loss, and hydrops fetalis.  

 

Inhibin A:

Inhibins are a family of heterodimeric glycoproteins, primarily

secreted by ovarian granulosa cells and testicular Sertoli cells,

which consist of disulfide-linked alpha and beta subunits. While

the alpha subunits are identical in all inhibins, the beta subunits

exist in 2 major forms, termed A and B, each of which can occur

in different isoforms. Depending on whether an inhibin heterodimer

contains a beta A or a beta B chain, they are designated as inhibin

A or inhibin B, respectively. Together with the related activins, which

are homodimers or heterodimers of beta A and B chains, the inhibins

are involved in gonadal-pituitary feedback and in paracrine

regulation of germ cell growth and maturation. During pregnancy,

inhibins and activins are produced by the feto-placental unit in

increasing quantities, mirroring fetal growth. Their physiological

role during pregnancy is uncertain. They are secreted into the

coelomic and amniotic fluid, but only inhibin A is found in

appreciable quantities in the maternal circulation during the first

and second trimesters.

 

Maternal inhibin A levels are correlated with maternal hCG levels

and are abnormal in the same conditions that are associated

with abnormal hCG levels (e.g., inhibin A levels are typically higher

in Down syndrome pregnancies). However, despite their similar

behavior, measuring maternal serum inhibin A concentrations in

addition to maternal serum hCG concentrations further improves

the sensitivity and specificity of maternal multiple marker

screening for Down syndrome.

NEURAL TUBE DEFECTS

An AFP multiple of the median (MoM) < 2.5 is reported as screen

negative. AFP MoMs > or =2.5 (singleton pregnancies) and > or =5.33

(twin gestation) are reported as screen positive. 

DOWN SYNDROME

Calculated screen risks <1/270 are reported as screen negative,

risks 1/270 are reported as screen positive. 

TRISOMY 18

Calculated screen risks <1/100 are reported as screen negative,

risks 1/100 are reported as screen positive.

 

An interpretive report will be provided.

Screen-positive results are called back to ordering client.

NTD:  

Screen Negative-a screen-negative result indicates that the calculated

AFP multiple of the median (MoM) falls below the established cutoff

of 2.50 MoM. A negative screen does not guarantee the absence of NTD.

 

Screen Positive-a screen-positive result indicates that the calculated

AFP MoM is > or =2.50 MoM, and may indicate an increased risk

for open NTD. The actual risk depends on the level of AFP and the

individual's pretest risk of having a child with NTD based on family history,

geographical location, maternal conditions such as diabetes and

epilepsy, and use of folate prior to conception. A screen-positive result

does not infer a definitive diagnosis of NTD, but indicates that further

evaluation should be considered. Approximately 80% of pregnancies

affected with NTD have elevated AFP, MoM values >2.5.

 

Down syndrome and Trisomy 18:

Screen Negative-a screen-negative result indicates that the

calculated screen risk is below the established cutoff of 1/270 for

Down syndrome and 1/100 for trisomy 18. A negative screen

does not guarantee the absence of trisomy 18 or Down syndrome.

 

When a Down syndrome second trimester risk cutoff of 1/270

is used for follow-up, the combination of maternal age, AFP,

estriol, hCG, and inhibin A has an overall detection rate of

approximately 77% to 81% with a false-positive rate of 6% to 7%. In

practice, both the detection rate and false-positive rate increase

with age. The detection rate ranges from 66% (early teens) to

99% (late 40s), with false-positive rates of between 3% to 62%,

respectively. The detection rate for trisomy 18 is 60% to 80% using

a second trimester cutoff of 1/100.

 

Follow-up:

Upon receiving maternal serum screening results, all information used

in the risk calculation should be reviewed for accuracy (maternal date

of birth, gestational dating, etc). If any information is incorrect, the laboratory

should be contacted for a recalculation of the estimated risks.

 

Screen-negative results typically do not warrant further evaluation.

 

Ultrasound is recommended to confirm dates for NTD

or trisomy 21 screen-positive results. Many pregnancies affected

with trisomy 18 are small for gestational age. Recalculations that

lower the gestational age may decrease the detection rate for

trisomy 18. If ultrasound yields new dates that differ by at least 7

days, a recalculation should be considered. If dates are confirmed,

high-resolution ultrasound and amniocentesis (including amniotic

fluid AFP and acetylcholinesterase measurements for NTD) are

typically offered.

Variables Affecting Marker Levels:

Race, weight, multiple fetus pregnancy, insulin-dependent

diabetes (IDD), and in vitro fertilization (IVF) may affect marker

concentrations. Black mothers tend to have higher AFP levels

but lower risk of NTD and are assigned to a separate AFP median

set. All MoMs are adjusted for maternal weight (to account for

dilution effects in heavier mothers). The AFP, uE(3), and inhibin

MoMs are adjusted upward in IDD to account for lower values in

diabetic pregnancies. hCG levels are higher and uE(3) levels are

lower in pregnancies conceived by IVF, MoMs are adjusted

accordingly to account for the alterations.

 

The estimated risk calculations and screen results are

dependent on accurate information for gestation, maternal age,

race, IDD, and weight. Inaccurate information can lead to significant

alterations in the estimated risk. In particular, erroneous

assessment of gestational age can result in false-positive or false-

negative screen results. Because of its increased accuracy, we

therefore recommend determination of gestational age by

ultrasound, rather than by maternal dates alone, when possible.

 

A screen-negative result does not guarantee the absence of fetal

defects. A screen-positive result does not provide a diagnosis, but

indicates that further diagnostic testing should be considered (an

unaffected fetus may have screen-positive result for unknown

reasons).

 

Valid measurements of AFP in maternal serum cannot be made

after amniocentesis.

 

Triplet and higher multiple pregnancies cannot be interpreted. Twin

pregnancies in insulin-dependent mothers cannot be interpreted.

 

Each center offering maternal serum screening to patients should

establish a standard screening protocol, which provides pre- and

post screening education and appropriate follow-up for screen-

positive results.

1.   Christensen RL, Rea MR, Kessler G, et al:  Implementation of a

      screening program for diagnosing open neural tube defects:

      selection, evaluation, and utilization of alpha-fetoprotein

      methodology. Clin Chem 1986;32:1812-1817

 

2.   Wald NJ, Densem JW, Smith D, Klee GG:  Four marker serum

      screening for Down's syndrome. Prenat Diagn 1994;14:707-716

 

3.   Florio P, Cobellis L, Luisi S, et al:  Changes in inhibins and

      activin secretion in healthy and pathological pregnancies.

      Mol Cell Endocrinol 2001;180:123-130

 

4.   Benn PA:  Advances in prenatal screening for Down syndrome:

      I. General principles and second trimester testing. Clin Chim

      Acta 2002;324:1-11

 

5.   Wald NJ, Cuckle HS, Densem JW, et al:  Maternal serum unconjugated

      oestriol and human chorionic gonadotrophin levels in pregnancies with

      insulin-dependent diabetes: implications for screening for Down's

      syndrome. Br J Obstet Gynaecol 1992;99:51-53