reviews

Neonatal screening for congenital adrenal

hyperplasia
Perrin C. White
Abstract | Congenital adrenal hyperplasia (CAH) caused by steroid 21‑hydroxylase deficiency occurs in
1:16,000–1:20,000 births. if not promptly diagnosed and treated, CAH can cause death in early infancy
from shock, hyponatremia and hyperkalemia. Affected girls usually have ambiguous genitalia but boys
appear normal; therefore, newborn babies are commonly screened for CAH in the Us and many other
countries. By identifying babies with severe, salt‑wasting CAH before they develop adrenal crises, screening
reduces morbidity and mortality, particularly among affected boys. Diagnosis is based on elevated levels of
17‑hydroxyprogesterone, the preferred substrate for steroid 21‑hydroxylase. initial testing usually involves
dissociation‑enhanced lanthanide fluorescence immunoassay that has a low positive predictive value
(about 1%), which leads to many follow‑up evaluations that have negative results. The positive predictive
value might be improved by second‑tier screening using DNA‑based methods or liquid chromatography
followed by tandem mass spectrometry, but these methods are not widely adopted. Cost estimates
for such screening range from Us$20,000 to $300,000 per life‑year saved. in babies with markedly
abnormal screen results, levels of serum electrolytes and 17‑hydroxyprogesterone should be immediately
determined, but the most reliable way to diagnose CAH is measurement of levels of steroid precursors
after stimulation with cosyntropin.
white, P. C. Nat. Rev. Endocrinol. 5, 490–498 (2009); doi:10.1038/nrendo.2009.148

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Learning objectives
Upon completion of this activity, participants should be able to:
Department of 1 identify limitations of current first‑tier screening for congenital
Pediatrics, University of adrenal hyperplasia (CAH).
Texas southwestern 2 Describe second‑tier testing for CAH.
Medical Center, Dallas,
TX, UsA.
3 Diagnose CAH in infants effectively.
4 List morbidity outcomes improved with screening programs
for CAH.
Correspondence:
Department of
Pediatrics, University of
Texas southwestern
Medical Center, 5323
Harry Hines Boulevard,
Dallas,
TX 75390‑9063, UsA Competing interests
perrin.white@ The author, the Journal editor v. Heath and the CMe questions
utsouthwestern.edu author C. P. vega declare no competing interests.
Introduction
Congenital adrenal hyperplasia (CAH) refers to a group
of autosomal-recessive, inherited disorders of corti-
sol biosynthesis. More than 90% of cases result from
steroid 21-hydroxylase deficiency caused by muta-
tions in CYP21A2.1–3 (In this Review, CAH refers to
21-hydroxylase deficiency unless stated otherwise.) Steroid
21-hydroxylase (a cytochrome P450 enzyme) converts
17-hydroxyprogesterone (17-OHP) to 11-deoxycortisol,
and progesterone to 11-deoxycorticosterone (Figure 1).
As 11-deoxycortisol and 11-deoxycorticosterone are pre-
cursors for cortisol and aldosterone, respectively, com-
plete loss of 21-hydroxylase activity results in deficiencies
of both of these vital corticosteroids. If this abnormality
is not detected and treated in time, it can cause death in
early infancy owing to shock, hyponatremia and hyper-
kalemia. In addition, accumulated steroid pre cursors
are metabolized to androgens within the adrenal glands
and in other tissues, which causes prenatal virilization in
affected girls and signs of postnatal androgen excess
in both sexes, including rapid linear growth and acceler-
ated skeletal maturation. Disease of this severity is termed
‘classic’ CAH, which is further subdivided into salt-wasting
and simple virilizing forms depending on whether or not
serum electrolyte levels are abnormal. Patients with the
milder ‘nonclassic’ form of CAH are asymptomatic or show
relatively mild signs of postnatal androgen excess.1,2
The incidence of classic CAH in most populations
is approximately 1:16,000–1:20,000, as determined by

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screening.4–6 CAH is suitable for neonatal screening as it
is relatively common and potentially fatal in childhood,
it can be diagnosed by a simple hormonal measurement in
blood, and early recognition and treatment can (in prin-
ciple) prevent serious morbidity and mortality. At present,
49 states in the uS and at least 16 other countries screen
newborn babies for CAH, and 13 additional countries
have pilot or local screening programs.6,7
As the positive predictive value of current screening
methods is relatively low (see below), pediatricians and
pediatric endocrinologists are frequently confronted by
babies who tested positive and require further testing to
eliminate CAH. This population represents a manage-
ment challenge disproportionate to the actual frequency
of the disease. In this Review, we concentrate on the
current status of neonatal screening for CAH.
Initial screening methodology
First-tier screening tests for CAH employ immunoassays
to measure 17-OHP levels in dried blood spots on the
same filter paper cards used for other neonatal screens,
such as the Guthrie test.4–7 Three different methods cur-
rently exist. Radioimmunoassay was the first method to
be developed,8 but this assay, as well as enzyme-linked
immunoassays, have been almost completely supplanted9
(in 46 states and most european countries) by time-
resolved, dissociation-enhanced, lanthanide fluores-
cence immunoassay (DelFIA®, wallac Oy Corporation,
Turku, Finland), which is highly automated.10
Limitations of first-tier screening
Several factors limit the accuracy of these tests. First, levels
of 17-OHP are normally high at birth and decrease rapidly
during the first few postnatal days. By contrast, 17-OHP
levels increase over time in newborn babies who are
affected with CAH. Thus, diagnostic accuracy is poor in the
first 2 days, which can be a problem if newborn babies are
discharged from the hospital within this period unless effi-
cient mechanisms exist to obtain follow-up samples.11 At
least 10 state laboratories in the uS stratify their samples
on the basis of time of collection (whereas at least 26 do
not); no correlation exists, however, between the positive
predictive value of the test and this stratification practice,
according to data from 2007.9
Second, newborn girls have lower mean 17-OHP levels
than newborn boys, which slightly reduces the sensitivity
of neonatal screening for CAH in girls.12 This difference,
however, is not a major problem in practice as almost all
girls with salt-wasting CAH are virilized; neonatal screen-
ing is more necessary for rapid detection of affected boys
than affected girls.
Third, premature, sick or stressed babies tend to have
higher levels of 17-OHP than healthy, term babies do;
therefore, such babies generate many false-positive test
results unless increased normal threshold values are
used. This problem seems to be particularly prominent
with DelFIA®assays, probably because these assays are
usually performed without an organic extraction step that
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reviews
Key points
■ Neonatal screening for congenital adrenal hyperplasia (CAH) caused
by steroid 21‑hydroxylase deficiency has been widely adopted in the Us
and many other countries
■ Neonatal screening for CAH reduces morbidity and mortality, particularly
among boys, by identifying infants with the severe, salt‑wasting form of CAH
before they develop adrenal crises
■ initial testing usually consists of an immunoassay for 17‑hydroxyprogesterone
levels; this assay has a low positive predictive value (approximately 1%), which
results in many follow‑up evaluations that have negative results
■ The positive predictive value might be improved by second‑tier screening
using DNA‑based methods or liquid chromatography followed by tandem mass
spectrometry, but these methods are not widely adopted yet
■ in infants with markedly abnormal test results, clinicians should immediately
determine serum electrolyte and 17‑hydroxyprogesterone levels and start
treatment with hydrocortisone and fludrocortisone pending the results of
hormonal tests
could remove cross-reacting substances but is relatively
laborious.13 no universally accepted standards exist for
stratifying 17-OHP levels in newborn babies, but most
laboratories use a series of birth-weight-adjusted thres-
hold values.11,14,15 Among laboratories in the uS that
report their practice, 12 used two birth weight categories
(generally below and above 2,500 g, respectively); eight
laboratories used three; 10 laboratories used four and
six laboratories used five birth weight categories in 2008.9
In practice, no correlation exists between the number of
birth weight categories that are employed for stratifica-
tion and the recall rate or the positive predictive value
of the test.
The specificity of neonatal screening can be improved
by stratifying test results according to the babies’ actual
gestational age,16,17 because 17-OHP levels correlate much
better with gestational age than with birth weight.18 In
The netherlands, adoption of gestational-age criteria
improved the positive predictive value of CAH screen-
ing tests from 4.5% to 16%.19 elevated 17-OHP levels in
preterm babies have been confirmed by high performance
liquid chromatography and are thus not solely attributable
to cross-reaction of the assay with other steroids. Steroid
profiles in preterm babies suggest a functional deficiency
of several adrenal steroidogenic enzymes, with a nadir
in function at 29 weeks of gestation.20 However, some
17-OHP immunoassays do cross-react with other ste-
roids, including 17-hydroxy pregnenolone sulfate21 and
15β-hydroxylated compounds; the latter are probably
generated by gut bacteria and absorbed via the entero-
hepatic circulation.22 As mentioned above, the speci-
ficity of immunoassays can be improved in some cases
by an organic extraction step to remove cross-reacting
substances, including steroid sulfates.
Fourth, multiple courses of antenatal corticosteroids
administered to mothers at risk of preterm delivery (for
example, two injections of 12 mg betamethasone 12–24 h
apart, administered to the mother at 1 week intervals
until birth or until the baby reaches a gestational age
vOluMe 5 | SePTeMBeR 2009 | 491
 reviews

CH3 CH3
HC CH2 CH2 CH2 CH
CH3
CH3 CH3

HO
Cholesterol
Cholesterol
desmolase 17α-Hydroxylase (CYP17A1) 17,20-Lyase
(CYP11A1)
Pregnenolone 17-OH Pregnenolone Dehydroepiandrosterone
3β-Hydroxysteroid
dehydrogenase
Progesterone 17-OH Progesterone Androstenedione
21-Hydroxylase 17β-Hydroxysteroid
(CYP21A2) dehydrogenase
Deoxycorticosterone 11-Deoxycortisol Testosterone

11β-Hydroxylase 11β-Hydroxylase
(CYP11B2) (CYP11B1) 5α-Reductase
Corticosterone Cortisol Dihydrotestosterone
18-Hydroxylase 21
(CYP11B2) CH2OH OH
CH3
18-Oxidase 18-OH Corticosterone HC O CH3 17
(CYP11B2) HO CH3
11
OH 3
CH3 17
O
Aldosterone 3
21 O
CH2OH
O
18 HC O
HO CH
CH3 11
3
O

Figure 1 | Pathways of steroid biosynthesis in the adrenal cortex. Pathways for the synthesis of progesterone and
mineralocorticoids (aldosterone), glucocorticoids (cortisol), and androgens (testosterone) are shown. enzymes that are
encoded by a single gene are shown in boxes. For activities that are mediated by specific P450 cytochromes (CYP), the
systematic name of the gene that encodes the enzyme is given in parentheses. The enzymes encoded by CYP11B2 and
CYP17A1 have multiple activities. The planar structures of cholesterol, aldosterone, cortisol, and dihydrotestosterone are
shown. Deficient 21‑hydroxylase activity prevents the synthesis of aldosterone and cortisol and shunts precursors, such as
17‑hydroxypregnenolone, into the pathway for androgen biosynthesis. Androstenedione is secreted by the adrenal cortex
and then converted to testosterone in the periphery. Testosterone may be aromatized to estradiol or converted by
5α‑reductase to dihydrotestosterone.

of 34 weeks)23 might reduce 17-OHP levels and thus
potentially increase the likelihood of false-negative
CAH screening test results. However, premature babies
are at high risk of respiratory distress syndrome, which is
likely to be associated with increased 17-OHP levels. As
inconsistent effects of antenatal corticosteroid adminis-
tration have been observed in practice,24,25 all babies who
received such treatment and were screened for CAH at
birth should be tested again after several days of life.
Finally, neonatal screening identifies a few babies
with mild, nonclassic CAH (1:130,000 babies in the uS
during 2003–2007).9 As the actual frequency of an auto-
somal recessive disease is approximately one-quarter
of the square of the carrier frequency, the true incidence of
nonclassic CAH is predicted to be approximately 1:2,000
in most populations.26–28 Screening, therefore, identifies
only a small fraction of nonclassic cases (moreover, the
incidence of nonclassic CAH might be much higher than
this predicted value in certain ethnic groups29). Although
we are not aware of any reports of systematic genotyping
of such patients, some studies suggest that patients with
nonclassic CAH who were identified by neonatal screen-
ing tend to be compound heterozygotes for a classic and
a nonclassic CAH allele (that is, they carry one copy of
each allele type, rather than two nonclassic alleles), and/
or carry a Pro30leu mutation in CYP21A2, 30 which com-
promises enzymatic activity more than the val281leu
mutation in the same gene that is most commonly
detected in patients with nonclassic CAH.31 Although ele-
vated 17-OHP levels are obviously not an artifact in such
situations, these babies are generally asymptomatic and
require no treatment unless they subsequently develop
signs of androgen excess.
improving the positive predictive value
To obtain sensitivity close to 100%, most screening
laboratories must use such low cut-off levels for 17-OHP
that about 1% of all test results are reported as positive.
CAH is a rare disease, present in only 1 in 16,000 births,
so the positive predictive value (defined as the propor-
tion of genuinely positive samples within all positive
results) of such tests is obviously very low—approximately

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1%—although their specificity (the proportion of nega-
tive test results among all unaffected individuals) and
sensitivity (the proportion of positive test results among
all affected individuals) are very high (Table 1).
The positive predictive value might be improved by
using a higher threshold 17-OHP level than those cur-
rently employed, with the trade-off of decreased sensi-
tivity.17 Such a decision might depend in part upon the
goals of CAH screening programs. use of relatively high
threshold values may efficiently detect babies with the
severe, salt-wasting form of the disease—those who
have the highest levels of 17-OHP—but might miss as
many as 30% of cases of simple virilizing CAH.32,33 As the
main goal of neonatal screening for CAH is early detec-
tion of babies with the salt-wasting form (as discussed
later), a delay in diagnosis for patients who are relatively
mildly affected might be considered acceptable if the cost
savings are sufficient.
The costs associated with ruling out CAH in patients
with positive screening test results could be greatly
reduced if a second tier of screening was implemented,
using a test that is more specific than the initial one.
Both biochemical and molecular genetic approaches
have been proposed for second-tier screening, but none
has yet been widely implemented.
Second-tier screening
Biochemical second screens
limitations of immunoassays for 17-OHP include the
above-mentioned fact that 17-OHP levels are elevated in
premature babies and in those who are sick or stressed.
Testing of serial filter paper blood samples from such
patients might improve the positive predictive value. 17
Some immunoassays for 17-OHP have low specificity,
which can be increased by organic solvent extraction, as
mentioned previously. A second-tier screen using this
approach is currently mandated in four uS states.
Direct biochemical analysis of steroid levels by liquid
chromatography then tandem mass spectrometry
(lC–MS/MS) addresses these issues more effectively
than do organic solvent extraction immunoassays.34,35
The run times for individual samples in lC–MS/MS
assays are 6–12 min, which would be too long for a first-
tier screen, but are suitable for a second-tier screen using
the original, dried blood samples.34,36 Of note, approxi-
mately 40% of samples that test positive in first-tier
immunoassay screening actually have normal 17-OHP
levels as measured by lC–MS/MS, which is consistent
with the suboptimal specificity of antibodies used in
these immunoassays. Measurement of steroid ratios
further improves the screening specificity of lC–MS/MS.
One approach assessed the ratio of the sum of 17-OHP
and androstenedione levels relative to the cortisol level.37
when this strategy was used in second-tier CAH screen-
ing in a laboratory in Minnesota for 3 years (204,000
births), the positive predictive value improved to 7.3%,
whereas the primary immunoassay screen used in
the same laboratory had a positive predictive value of
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Table 1 | sensitivity, specificity, and predictive value of neonatal CAH screening
Test result CAH Predictive value
Present Not present
Positive True positive False positive Positive
n = 860 n = 79,731 860/80,591 = 1.07%
Negative False negative True negative Negative
n = 10 n = 18,257,881 18,257,881/
18,257,891 = 100%
Test value sensitivity specificity –
860/870 = 98.9% 18,257,881/
18,337,612 = 99.6%
Aggregated data are from the entire Us in 2003–2007;9 these values have not been verified with
individual laboratories and should be considered approximate. Note that the positive predictive value of
the screening test is only approximately 1%, although its sensitivity and specificity are very high.
Abbreviation: CAH, congenital adrenal hyperplasia.
0.64%.38 Androstenedione, however, is not a potential
substrate for steroid 21-hydroxylase, as its C20–21 segment
has been removed by the 17,20-lyase activity of steroid
17α-hydroxylase (Figure 1). Consequently, this steroid is
only indirectly relevant for screening purposes.
By contrast, 21-deoxycortisol (produced by 11β-
hydroxylation of 17-OHP) is not normally secreted in
large amounts even in preterm babies; thus, elevated levels
of this steroid are highly specific for 21-hydroxylase defi-
ciency. One modified lC–MS/MS protocol has utilized
the ratio of the sum of 17-OHP and 21-deoxycortisol
levels relative to the cortisol level. when 1,609 samples
that tested positive at primary screening in a German
program (out of a total of 242,500 samples) were tested
prospectively, this protocol correctly identified all 16
affected children and had no false-positive results—a
positive predictive value of 100%.36
Molecular genetic second-tier screens
CYP21A2 mutations can be detected in DnA samples
extracted from the same dried blood spots used for hor-
monal screening. Detection methods include dot-blotting
protocols,39 ligation-detection assays,26,40 real-time, quan-
titative polymerase chain reaction (PCR),28,41 full sequenc-
ing 42 and minisequencing.43 More than 90% of mutant
alleles involve one or more recombination events (dele-
tion of CYP21A2 or gene conversions that result in trans-
fer of deleterious mutations from the nearby CYP21A1P
pseudogene to CYP21A2).1,2 Therefore, samples that carry
none of these mutations might be presumed with >99%
confidence to be from unaffected individuals. If at least
one of these mutations is detected, the patient requires
additional evaluation.
The positive predictive value of this strategy depends
on the carrier rate for classic CAH in the general popula-
tion. whereas the observed disease frequency of about
1:16,000 leads to an estimated carrier rate of 1.6%, direct
ascertainment of mutation frequencies in a total of 1,100
normal individuals in three independent studies from
new Zealand and europe suggests that the carrier fre-
quency is 3.5%; this difference is greater than can be
expected by chance (P = 0.006).26–28 The explanation for
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First neonatal screen (17-OHP levels)

12% of all individuals may carry such chromosomes.44
These chimeric loci are not actual CAH alleles but could
complicate efforts to interpret the results of a molecular
Normal 17-OHP level Abnormal 17-OHP level screening program.
(>99th percentile for birth weight or gestational age) If we assume that the apparent carrier frequency is
3.5% and that 1% of all individuals will test positive for
CAH in first-tier screening, then a second-tier screen
No further Second 17-OHP assay DNA screen for
action CYP21A2 mutations using molecular genetic techniques would be expected
to identify 0.035% (1:2,900) of all infants as positive for
CAH. As the actual frequency of CAH is about 0.006%
Normal 17-OHP level Abnormal 17-OHP level No One or more (1:16,000), the positive predictive value of this approach
mutations mutations should be approximately 18% (0.006:0.035).
For several reasons, DnA analysis of a single sample
No further Other symptoms No other No further cannot actually be used to diagnose CAH. First, an indivi-
action (e.g. abnormal symptoms action
electrolyte levels) dual who is heterozygous for a known mutation associ-
ated with classic 21-hydroxylase deficiency could have a
novel mutation within the other allele that might not be
Treatment with hydrocortisone Cosyntropin stimulation test detected. Second, alleles that carry a common mutation
and fludrocortisone
in intron 2 may be preferentially amplified under some
experimental conditions, which would result in heterozy-
gous carriers being incorrectly classified as homozygous
17-OHP level 17-OHP level 17-OHP level 17-OHP level
<300 ng/dl 300–1,500 ng/dl 1,500–10,000 ng/dl >10,000 ng/dl and affected.45 Finally, many CAH alleles include more
(<9 nmol/l) (9–45 nmol/l) (45–300 nmol/l) (>300 nmol/l) than one deleterious mutation, depending on the size of
the recombination that generated each one; determining
Unaffected Heterozygote Nonclassic CAH Classic CAH whether two mutations are in different alleles or within
the same one is impossible without genotyping at least
one parent.
No further action No symptoms Symptoms present Several studies in which samples from screening pro-
grams were genotyped have suggested that this approach
is a potentially useful adjunct to hormonal measure-
Follow-up Treatment with hydrocortisone ments,26,28,39,42 but, to the best of our knowledge, no large-
and fludrocortisone
scale study has assessed the efficacy of genotyping as a
second-tier screen.
Genotyping remains more costly and time-consuming
Adjust therapy on the
basis of response than lC–MS/MS per sample. Although the equipment
for lC–MS/MS is expensive, most neonatal screening
Figure 2 | Algorithm for screening newborn babies for CAH. Protocols vary among programs already have it available for use in other tests. If
clinical centers. The first screen is performed using dried capillary blood. A 17‑OHP
the previously reported success of second-tier screening
level above the 99th percentile for birth weight prompts second‑tier screening: either
another 17‑OHP assay (repeat immunoassay or liquid chromatography followed by with lC–MS/MS can be replicated in other programs,
tandem mass spectrometry) or a genetic test for common mutations in CYP21A2, it should become the method of choice for confirming
which encodes 21‑hydroxylase. if the results of either test are abnormal, the baby is positive CAH screening results.
referred for a cosyntropin stimulation test or, if the risk of CAH is very high (for
example, on the basis of abnormal serum electrolyte levels), treatment might be evaluation after positive tests
started immediately after the second 17‑OHP test, without a stimulation test. If a baby tests positive for CAH, the decision whether to
Depending on the results, the baby is classified as unaffected by CAH, a probable
inform only the primary-care physician or a pediatric
heterozygous carrier, having nonclassic disease, or having classic disease. The
need for further management is determined by these results. Abbreviations:
endocrinologist as well depends on the availability of
CAH, congenital adrenal hyperplasia; 17‑OHP, 17‑hydroxyprogesterone. subspecialists in that particular geographic area. In Texas,
both the infant’s primary-care physician and a pediatric
endocrinologist are notified of all positive CAH screening
this discrepancy remains uncertain. An adverse effect of results.32 Babies with moderately elevated 17-OHP levels
these mutations on fetal survival cannot be ruled out, but are followed up by obtaining a second filter-paper blood
no distortion of the Mendelian ratio has been reported specimen. Infants whose 17-OHP levels remain high are
(that is, the number of affected babies is approximately evaluated by measurements of their electrolyte and serum
one-quarter that of the number of pregnant women who 17-OHP levels; if these values are also abnormal, the baby
are at risk of having such babies). Many of the ‘excess’ is referred to a pediatric endocrinologist (Figure 2).
mutations detected might actually occur in chromo- The gold standard for diagnosis of CAH is a cosyn-
somes that contain a third, chimeric locus in addition tropin stimulation test (Figure 3).46 This test employs a
to the usual CYP21A1P and CYP21A2 genes; up to pharmacologic dose of 0.125–0.25 mg cosyntropin, which

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maximally stimulates the adrenal cortex. This diagnostic
test is different from the low-dose cosyntropin stimula-
tion test, which is often used to evaluate the integrity of
the hypothalamic–pituitary–adrenal axis.47
Girls with ambiguous genitalia are usually assessed
shortly after birth, before the results of neonatal blood
screening are available. Approaches used to assess chil-
dren with ambiguous genitalia are reviewed elsewhere.48,49
In brief, the key steps are a prompt determination of the
child’s genetic sex and internal anatomy, which is best
accomplished by a thorough physical examination, pelvic
ultrasonography (if a competent operator is available)
and counting of sex chromosomes within interphase
nuclei by fluorescence in situ hybridization. These assess-
ments can generally be completed within one working
day. CAH due to 21-hydroxylase deficiency should be
suspected in any girl with two X chromosomes and
normal internal Müllerian structures (that is, a uterus
detected by ultrasonography).
when CAH is suspected on the basis of ambiguous
genitalia, the cosyntropin stimulation test should be
deferred until after the first 24 h of life, because this test
has a high incidence of both false-positive and false-
negative results in samples that are obtained immediately
after birth.
During stimulation testing, clinicians should keep in
mind that 17-OHP levels might be elevated as a result
of other enzymatic defects, particularly 11β-hydroxylase
deficiency and, more rarely, 3β-hydroxysteroid dehydro-
genase deficiency and P450 oxidoreductase deficiency
(Antley–Bixler syndrome).50 To differentiate between the
various enzymatic defects that could potentially cause
CAH, the clinician should ideally measure levels of
17-OHP, cortisol, deoxycorticosterone, 11-deoxycortisol
and 17-OH-pregnenolone at 0 min and 60 min of
stimulation and perform at least one measurement each
of dehydroepiandrosterone and androstenedione. In
low-birth-weight babies who have a small blood volume,
only one sample is collected at 60 min. Determination of
precursor:product ratios is particularly useful to distin-
guish between the different enzymatic defects. If second-
tier screening with lC–MS/MS is employed, all relevant
steroids can be measured at the same time, which greatly
facilitates the diagnoses of other forms of CAH.51,52
If a diagnosis of CAH is highly probable (on the
basis of ambiguous genitalia in girls, markedly elevated
17-OHP level at neonatal screening, and/or electrolyte
abnormalities in either sex), treatment should be insti-
tuted immediately without waiting for the results of a
cosyntropin stimulation test. The decision whether or
not to conduct stimulation testing at all under these
circumstances will depend on the local situation. large
pediatric referral hospitals with an on-call pediatric
endocrinologist might be able to conduct such tests
quickly. However, in many institutions, the risks posed by
delaying treatment to perform a cosyntropin stimulation
test may outweigh its benefits, particularly in sick babies
with abnormal electrolyte levels, who presumably already
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4,000 –
17-OHP level after stimulation (nmol/l)
Classic
CAH
400 –
Nonclassic
CAH
40 –
Carrier
Normal
4–
0_
0 4 40 400 4,000
Basal 17-OHP level (nmol/l)
Figure 3 | Nomogram for comparing 17‑OHP levels before
and 60 min after an intravenous bolus of cosyntropin in
individuals with or without 21‑hydroxylase deficiency.46
Note that values for normal individuals and heterozygous
carriers of a CAH allele overlap. Abbreviation: 17‑OHP,
17‑hydroxyprogesterone.
have markedly elevated adrenocorticotropic hormone
levels even without pharmacologic stimulation.
If the baby does not seem to be ill and electrolyte abnor-
malities are not yet present or are mild (for example, the
serum sodium level is 130–135 mmol/l, and the potas-
sium level is 5.5–6.5 mmol/l), initial treatment should
include hydrocortisone at approximately 20 mg/m 2
daily, divided into three equal oral doses, and fludro-
cortisone (a synthetic mineralocorticoid) 0.1 mg daily. If
further evaluation rules out CAH within a few days, these
medications can be immediately discontinued. Severely
ill infants might initially require intravenous fluids with
0.9% naCl and high doses (100 mg/m2 daily in divided
doses) of intravenous hydrocortisone followed by oral
hydrocortisone, a higher dose (0.1 mg twice daily) of
fludrocortisone, and oral naCl at 4–8 mmol/kg daily.
Cost-effectiveness
Screening markedly reduces the time to diagnosis of
infants with CAH. 32,53–55 The main putative benefit
of early diagnosis is reduced morbidity and mortality,
particularly among babies with the salt-wasting form of
this disease. CAH might, however, remain undiagnosed
in babies who die suddenly, with the consequence that a
benefit of screening is difficult to demonstrate by direct
comparison of CAH-related mortality in unscreened and
screened populations. Indeed, a retrospective analysis
of neonatal blood samples from cases of unexplained
sudden infant death in the Czech Republic and Austria
identified 3 genotype-proven cases of classic CAH among
242 samples screened.56 Furthermore, as boys with salt-
wasting CAH are more likely than girls to be diagnosed
after a delay, a relative paucity of boys with salt-wasting
CAH in a given population may be taken as indirect evi-
dence of unreported deaths from salt-wasting crises. In
fact, girls do outnumber boys in some57,58 although not
vOluMe 5 | SePTeMBeR 2009 | 495
 reviews

all59 retrospective studies in which CAH was diagnosed
clinically. Moreover, the preponderance of girls is par-
ticularly prominent among patients with genotypes that
predict a complete absence of 21-hydroxylase enzymatic
activity and thus severe salt-wasting.58 By contrast, babies
with salt-wasting CAH who are identified through neo-
natal screening programs are at least as likely to be boys
as girls.53–55 Mortality from salt-wasting CAH in popula-
tions that do not undergo neonatal screening has been
assumed to be around 10%,60 but was recently estimated
to be 4% in contemporary, developed economies.61
Screening reduces morbidity from CAH for several
reasons. Babies who are identified through screening
have less-severe hyponatremia than babies who are
not screened (mean serum sodium levels at diagnosis
134 mmol/l versus 124 mmol/l, respectively)19,54 and tend
to be hospitalized for shorter periods of time (although
the difference falls short of statistical significance).55
learning disabilities have been reported in patients who
have had salt-wasting crises;62,63 however, whether neo-
natal screening reduces the frequency and severity of
such abnormalities is not known. Although boys with
salt-wasting CAH would seem to derive the greatest
benefit from neonatal screening, such programs also
markedly reduce the delay in correct sex assignment for
severely virilized girls.54,64 Moreover, in the absence of
neonatal screening, boys with simple virilizing disease
might not be diagnosed until rapid growth and acceler-
ated skeletal maturation are detected later in childhood,
at which time their final height may already be adversely
affected. However, whether this last benefit itself justi-
fies the costs of a screening program is debatable.
Furthermore, the early identification of a small number
of patients with nonclassic CAH has little, if any, benefit,
given that such patients are generally not treated unless
they develop signs of androgen excess.
Several reviews have performed cost–benefit analyses
of neonatal screening for CAH. Such estimates generally
assume that the only adverse outcome of late diagno-
sis of CAH is death, particularly in boys, and thus that
the benefit of screening is best quantified in ‘life-years’
(calculated as the number of babies who are saved by
prompt diagnosis, multiplied by their life expectancy in
years). Calculations of costs per life-year saved are sensi-
tive to assumptions about mortality, and recent estimates
have ranged widely from uS$20,000 65 to $250,000–
300,000.66 A conventional assumption is that screening for
a particular disease is cost-effective at less than $50,000
per life-year (or per quality-adjusted life-year, if quality
of life is considered).65
The downstream costs of following up false-positive
screening results are difficult to estimate, as follow-
up might entail a large amount of physician time for
evaluation and counseling, plus nursing time if a cosyn-
tropin test is undertaken, in addition to the labora-
tory’s costs. Moreover, parents of babies with positive
screening results may suffer substantial psychological
distress at the prospect of their child having a poten-
tially life-threatening, chronic disease,67 although their
distress generally resolves if CAH is subsequently ruled
out.68 These problems can be markedly reduced by the
adoption of screening methods with improved positive
predictive values.
Conclusions
neonatal screening for CAH has been widely adopted in
the uS and many other countries. By identifying babies
with the severe, salt-wasting form of the disease before
they develop adrenal crises, neonatal screening reduces
morbidity and mortality. The positive predictive value of
existing first-tier immunoassay-based screening is gener-
ally very low, which leads to many follow-up evaluations
of children with positive first-tier screen results who
do not have CAH. Positive predictive values might be
improved by second-tier screening using lC–MS/MS or
DnA-based methods.
Review criteria
we searched PubMed using the search terms “neonatal”,
“newborn”, “screening”, “CAH” and “adrenal”; we
limited the search to english‑language literature. we
also searched the reference lists of identified articles for
additional papers.

1. white, P. C. & speiser, P. w. Congenital adrenal
hyperplasia due to 21‑hydroxylase deficiency.
Endocr. Rev. 21, 245–291 (2000).
2. speiser, P. w. & white, P. C. Congenital adrenal
hyperplasia. N. Engl. J. Med. 349, 776–788
(2003).
3. Joint LwPes/esPe CAH working Group.
Consensus statement on 21‑hydroxylase
deficiency from the Lawson wilkins Pediatric
endocrine society and the european society for
Paediatric endocrinology. J. Clin. Endocrinol.
Metab. 87, 4048–4053 (2002).
4. Pang, s. & shook, M. K. Current status of
neonatal screening for congenital adrenal
hyperplasia. Curr. Opin. Pediatr. 9, 419–423
(1997).
5. Therrell, B. L. Newborn screening for congenital
adrenal hyperplasia. Endocrinol. Metab. Clin.
North Am. 30, 15–30 (2001).
6. van der Kamp, H. J. & wit, J. M. Neonatal
screening for congenital adrenal hyperplasia.
Eur. J. Endocrinol. 151 (Suppl. 3), U71–U75
(2004).
7. Loeber, J. G. Neonatal screening in europe; the
situation in 2004. J. Inherit. Metab. Dis. 30,
430–438 (2007).
8. Pang, s., Hotchkiss, J., Drash, A. L., Levine,
L. s. & New, M. i. Microfilter paper method for
17α‑hydroxyprogesterone radioimmunoassay:
its application for rapid screening for congenital
adrenal hyperplasia. J. Clin. Endocrinol. Metab.
45, 1003–1008 (1977).
9. National Newborn screening information
system 2009. Disorder Report for Congenital
Adrenal Hyperplasia (CAH) [online] http://
www2.uthscsa.edu/nnsis/ (2009).
10. Gonzalez, r. r., Mäentausta, O., solyom, J.
& vihko, r. Direct solid‑phase time‑resolved
fluoroimmunoassay of 17α‑hydroxyprogesterone
in serum and dried blood spots on filter paper.
Clin. Chem. 36, 1667–1672 (1990).
11. Olgemoller, B., roscher, A. A., Liebl, B. &
Fingerhut, r. screening for congenital adrenal
hyperplasia: adjustment of
17‑hydroxyprogesterone cut‑off values to both
age and birth weight markedly improves the
predictive value. J. Clin. Endocrinol. Metab. 88,
5790–5794 (2003).
12. varness, T. s., Allen, D. B. & Hoffman, G. L.
Newborn screening for congenital adrenal
hyperplasia has reduced sensitivity in girls.
J. Pediatr. 147, 493–498 (2005).
13. al saedi, s., Dean, H., Dent, w., stockl, e. &
Cronin, C. screening for congenital adrenal
hyperplasia: the Delfia screening test
overestimates serum 17‑hydroxyprogesterone in
preterm infants. Pediatrics 97, 100–102 (1996).

496 | SEPTEMBER 2009 | voluME 5 www.nature.com/nrendo

© 2009 Macmillan Publishers Limited. All rights reserved


14. Allen, D. B. et al. improved precision of
newborn screening for congenital adrenal
hyperplasia using weight‑adjusted criteria for
17‑hydroxyprogesterone levels. J. Pediatr. 130,
128–133 (1997).
15. Gruñeiro‑Papendieck, L. et al. Neonatal
screening program for congenital adrenal
hyperplasia: adjustments to the recall protocol.
Horm. Res. 55, 271–277 (2001).
16. Ohkubo, s., shimozawa, K., Matsumoto, M. &
Kitagawa, T. Analysis of blood spot
17α-hydroxyprogesterone concentration in
premature infants—proposal for cutoff limits in
screening congenital adrenal hyperplasia. Acta
Paediatr. Jpn 34, 126–133 (1992).
17. steigert, M., schoenle, e. J., Biason‑Lauber, A.
& Torresani, T. High reliability of neonatal
screening for congenital adrenal hyperplasia in
switzerland. J. Clin. Endocrinol. Metab. 87,
4106–4110 (2002).
18. van der Kamp, H. J. et al. Cutoff levels of
17‑α‑hydroxyprogesterone in neonatal
screening for congenital adrenal hyperplasia
should be based on gestational age rather than
on birth weight. J. Clin. Endocrinol. Metab. 90,
3904–3907 (2005).
19. van der Kamp, H. J. et al. Newborn screening
for congenital adrenal hyperplasia in The
Netherlands. Pediatrics 108, 1320–1324
(2001).
20. Nomura, s. immature adrenal steroidogenesis
in preterm infants. Early Hum. Dev. 49,
225–233 (1997).
21. wong, T., shackleton, C. H., Covey, T. r. &
ellis, G. identification of the steroids in
neonatal plasma that interfere with
17α‑hydroxyprogesterone radioimmunoassays.
Clin. Chem. 38, 1830–1837 (1992).
22. Lange‑Kubini, K., Zachmann, M., Kempken, B. &
Torresani, T. 15‑β‑hydroxylated steroids may be
diagnostically misleading in confirming
congenital adrenal hyperplasia suspected by a
newborn screening programme. Eur. J. Pediatr.
155, 928–931 (1996).
23. Crowther, C. A. & Harding, J. e. repeat doses of
prenatal corticosteroids for women at risk of
preterm birth for preventing neonatal
respiratory disease. Cochrane Database of
Systematic Reviews issue 3. Art. No.:
CD003935. doi:10.1002/14651858.
CD003935.pub2 (2007).
24. Gatelais, F. et al. effect of single and multiple
courses of prenatal corticosteroids on
17‑hydroxyprogesterone levels: implication for
neonatal screening of congenital adrenal
hyperplasia. Pediatr. Res. 56, 701–705
(2004).
25. King, J. L. et al. Antenatal corticosteroids and
newborn screening for congenital adrenal
hyperplasia. Arch. Pediatr. Adolesc. Med. 155,
1038–1042 (2001).
26. Fitness, J. et al. Genotyping of CYP21, linked
chromosome 6p markers, and a sex‑specific
gene in neonatal screening for congenital
adrenal hyperplasia. J. Clin. Endocrinol. Metab.
84, 960–966 (1999).
27. Baumgartner‑Parzer, s. M., Nowotny, P.,
Heinze, G., waldhäusl, w. & vierhapper, H.
Carrier frequency of congenital adrenal
hyperplasia (21‑hydroxylase deficiency) in a
middle european population. J. Clin. Endocrinol.
Metab. 90, 775–778 (2005).
28. Kosel, s. et al. rapid second‑tier molecular
genetic analysis for congenital adrenal
hyperplasia attributable to steroid
nATuRe RevIewS | eNdoCRiNoLogy
© 2009 Macmillan Publishers Limited. All rights reserved
21‑hydroxylase deficiency. Clin. Chem. 51,
298–304 (2005).
29. speiser, P. w. et al. High frequency of
nonclassical steroid 21‑hydroxylase deficiency.
Am. J. Hum. Genet. 37, 650–667 (1985).
30. Tajima, T., Fujieda, K., Nakae, J., Mikami, A.
& Cutler, G. B. Jr. Mutations of the CYP21 gene
in nonclassical steroid 21‑hydroxylase
deficiency in Japan. Endocr. J. 45, 493–497
(1998).
31. Tusie‑Luna, M. T., speiser, P. w., Dumic, M.,
New, M. i. & white, P. C. A mutation (Pro30 to
Leu) in CYP21 represents a potential
nonclassic steroid 21‑hydroxylase deficiency
allele. Mol. Endocrinol. 5, 685–692 (1991).
32. Therrell, B. L. Jr et al. results of screening
1.9 million Texas newborns for
21‑hydroxylase‑deficient congenital adrenal
hyperplasia. Pediatrics 101, 583–590 (1998).
33. votava, F. et al. estimation of the false‑negative
rate in newborn screening for congenital
adrenal hyperplasia. Eur. J. Endocrinol. 152,
869–874 (2005).
34. Lacey, J. M. et al. improved specificity of
newborn screening for congenital adrenal
hyperplasia by second‑tier steroid profiling
using tandem mass spectrometry. Clin. Chem.
50, 621–625 (2004).
35. rauh, M., Gröschl, M., rascher, w. & Dörr, H. G.
Automated, fast and sensitive quantification of
17 α‑hydroxy‑progesterone, androstenedione
and testosterone by tandem mass
spectrometry with on‑line extraction. Steroids
71, 450–458 (2006).
36. Janzen, N. et al. Newborn screening for
congenital adrenal hyperplasia: additional
steroid profile using liquid chromatography–
tandem mass spectrometry. J. Clin. Endocrinol.
Metab. 92, 2581–2589 (2007).
37. Minutti, C. Z. et al. steroid profiling by tandem
mass spectrometry improves the positive
predictive value of newborn screening for
congenital adrenal hyperplasia. J. Clin.
Endocrinol. Metab. 89, 3687–3693 (2004).
38. Matern, D., Tortorelli, s., Oglesbee, D.,
Gavrilov, D. & rinaldo, P. reduction of the false‑
positive rate in newborn screening by
implementation of Ms/Ms‑based second‑tier
tests: the Mayo Clinic experience (2004–
2007). J. Inherit. Metab. Dis. 30, 585–592
(2007).
39. Yang, Y. P., Corley, N. & Garcia‑Heras, J. reverse
dot‑blot hybridization as an improved tool for
the molecular diagnosis of point mutations in
congenital adrenal hyperplasia caused by
21‑hydroxylase deficiency. Mol. Diagn. 6,
193–199 (2001).
40. sorensen, K. M. et al. Multiplex ligation‑
dependent probe amplification technique for
copy number analysis on small amounts of DNA
material. Anal. Chem. doi:10.1021/ac801688c.
41. Olney, r. C., Mougey, e. B., wang, J.,
shulman, D. i. & sylvester, J. e. Using real‑time,
quantitative PCr for rapid genotyping of the
steroid 21‑hydroxylase gene in a north Florida
population. J. Clin. Endocrinol. Metab. 87,
735–741 (2002).
42. Nordenstrom, A., Thilén, A., Hagenfeldt, L.,
Larsson, A. & wedell, A. Genotyping is a
valuable diagnostic complement to neonatal
screening for congenital adrenal hyperplasia
due to steroid 21‑hydroxylase deficiency. J. Clin.
Endocrinol. Metab. 84, 1505–1509 (1999).
43. Krone, N. et al. Multiplex minisequencing of the
21‑hydroxylase gene as a rapid strategy to
reviews
confirm congenital adrenal hyperplasia. Clin.
Chem. 48, 818–825 (2002).
44. wu, Y. L. et al. Phenotypes, genotypes and
disease susceptibility associated with gene
copy number variations: complement C4 CNvs
in european American healthy subjects and
those with systemic lupus erythematosus.
Cytogenet. Genome Res. 123, 131–141
(2008).
45. Day, D. J. et al. identification of non‑amplifying
CYP21 genes when using PCr‑based diagnosis
of 21‑hydroxylase deficiency in congenital
adrenal hyperplasia (CAH) affected pedigrees.
Hum. Mol. Genet. 5, 2039–2048 (1996).
46. New, M. i. et al. Genotyping steroid
21‑hydroxylase deficiency: hormonal reference
data. J. Clin. Endocrinol. Metab. 57, 320–326
(1983).
47. Abdu, T. A., elhadd, T. A., Neary, r. &
Clayton, r. N. Comparison of the low dose
short synacthen test (1 μg), the conventional
dose short synacthen test (250 μg), and the
insulin tolerance test for assessment of the
hypothalamo–pituitary–adrenal axis in patients
with pituitary disease. J. Clin. Endocrinol.
Metab. 84, 838–843 (1999).
48. white, P. C. The endocrinologist’s approach to
the intersex patient. Adv. Exp. Med. Biol. 511,
107–119 (2002).
49. Lee, P. A. et al. Consensus statement on
management of intersex disorders.
international Consensus Conference on
intersex. Pediatrics 118, e488–e500 (2006).
50. Flück, C. e. et al. Mutant P450 oxidoreductase
causes disordered steroidogenesis with and
without Antley–Bixler syndrome. Nat. Genet. 36,
228–230 (2004).
51. Peter, M. et al. A case of 11β‑hydroxylase
deficiency detected in a newborn screening
program by second‑tier LC–Ms/Ms. Horm. Res.
69, 253–256 (2008).
52. Kushnir, M. M. et al. Development and
performance evaluation of a tandem mass
spectrometry assay for 4 adrenal steroids. Clin.
Chem. 52, 1559–1567 (2006).
53. Balsamo, A. et al. Congenital adrenal
hyperplasia: neonatal mass screening
compared with clinical diagnosis only in the
emilia‑romagna region of italy, 1980–1995.
Pediatrics 98, 362–367 (1996).
54. Thil’en, A. et al. Benefits of neonatal screening
for congenital adrenal hyperplasia
(21‑hydroxylase deficiency) in sweden.
Pediatrics 101, e11 (1998).
55. Brosnan, P. G. et al. effect of newborn
screening for congenital adrenal hyperplasia.
Arch. Pediatr. Adolesc. Med. 153, 1272–1278
(1999).
56. strnadová, K. A. et al. Prevalence of congenital
adrenal hyperplasia among sudden infant
death in the Czech republic and Austria. Eur. J.
Pediatr. 166, 1–4 (2007).
57. Thompson, r., seargeant, L. & winter, J. s.
screening for congenital adrenal hyperplasia:
distribution of 17α‑hydroxyprogesterone
concentrations in neonatal blood spot
specimens. J. Pediatr. 114, 400–404 (1989).
58. Nordenström, A. et al. Female preponderance in
congenital adrenal hyperplasia due to CYP21
deficiency in england: implications for neonatal
screening. Horm. Res. 63, 22–28 (2005).
59. Thilen, A. & Larsson, A. Congenital adrenal
hyperplasia in sweden 1969–1986:
prevalence, symptoms and age at diagnosis.
Acta Paediatr. Scand. 79, 168–175 (1990).
vOluMe 5 | SePTeMBeR 2009 | 497
 reviews
60. watson, M. s., Lloyd‑Puryear, M. A., Mann,
M. Y., rinaldo, P. & Howell, r. r. (eds) Newborn
Screening: Toward a Uniform Screening Panel
and System. [online] http://www.acmg.net/
resources/policies/NBs/NBs‑sections.htm
(2009).
61. Grosse, s. D. & van, v. G. How many deaths can
be prevented by newborn screening for
congenital adrenal hyperplasia? Horm. Res. 67,
284–291 (2007).
62. Nass, r. & Baker, s. Learning disabilities in
children with congenital adrenal hyperplasia.
J. Child Neurol. 6, 306–312 (1991).
63. Donaldson, M. D. et al. Presentation, acute
illness, and learning difficulties in salt‑wasting
498 | SEPTEMBER 2009 | voluME 5
21‑hydroxylase deficiency. Arch. Dis. Child 70,
214–218 (1994).
64. Pang, s. Y. et al. worldwide experience in
newborn screening for classical congenital
adrenal hyperplasia due to 21‑hydroxylase
deficiency. Pediatrics 81, 866–874 (1988).
65. Carroll, A. e. & Downs, s. M. Comprehensive
cost‑utility analysis of newborn screening
strategies. Pediatrics 117, s287–s295 (2006).
66. Yoo, B. K. & Grosse, s. D. The cost effectiveness
of screening newborns for congenital adrenal
hyperplasia. Public Health Genomics 12, 67–72
(2009).
67. Gurian, e. A., Kinnamon, D. D., Henry, J. J. &
waisbren, s. e. expanded newborn screening for
© 2009 Macmillan Publishers Limited. All rights reserved
biochemical disorders: the effect of a false‑
positive result. Pediatrics 117, 1915–1921
(2006).
68. Prosser, L. A., Ladapo, J. A., rusinak, D. &
waisbren, s. e. Parental tolerance of false‑
positive newborn screening results. Arch.
Pediatr. Adolesc. Med. 162, 870–876 (2008).
Acknowledgments
Charles P. vega, University of California, irvine, CA, is
the author of and is solely responsible for the content
of the learning objectives, questions and answers of
the MedscapeCMe‑accredited continuing medical
education activity associated with this article.
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