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The Journal of Clinical Endocrinology & Metabolism 88(9):4175– 4179
Copyright © 2003 by The Endocrine Society
doi: 10.1210/jc.2003-030522
COMMENT
Establishment of Reference Intervals for Markers of
Fetal Thyroid Status in Amniotic Fluid
PRATIMA K. SINGH, CURTIS A. PARVIN,
AND
ANN M. GRONOWSKI
Washington University School of Medicine, Department of Pathology and Immunology, Division of Laboratory Medicine,
St. Louis, Missouri 63110
Fetal goiter can arise as a result of fetal hyper or hypothyroidism. Although this condition is rare, it can be life threatening. Detection of fetal goiter in utero is possible with the aid
of ultrasound, but proper prenatal treatment depends on
knowledge of hormonal status. Amniotic fluid (AF) sampling
is less technically demanding and poses fewer risks to the
fetus than cordocentesis for fetal serum sampling, but wellestablished reference ranges for AF thyroid studies are not
available in the literature. We have established reference intervals for AF (TSH), total T4 (tT4), and free T4 using stored AF
samples. The reference intervals were: TSH (n ⴝ 127), less than
0.1– 0.5 mU/liter, with a median of 0.1 mU/liter; tT4 (n ⴝ 129),
2.3–3.9 ␮g/dl (30 –50 nmol/liter), with a median of 3.3 ␮g/dl (4
T
HYROID DISORDERS IN the perinatal period are common endocrine disorders (1, 2). Although maternal
thyroid abnormalities can be diagnosed with maternal serum
testing, fetal diagnosis is far more difficult. Proper fetal diagnosis is especially important because thyroid disorders can
lead to growth retardation and possible intrauterine demise.
Approximately 3% of fetal thyroid disorders are associated
with goiter (3), which can cause neck dystocias and respiratory distress.
During pregnancy, the fetus starts to produce its own
thyroid hormones in the first trimester, including TSH, T4,
and T3 (4, 5). Despite the onset of independent fetal thyroid
function, maternal thyroid disorders can affect the fetus
through a variety of physiologic mechanisms. Maternal thyroid-stimulating Igs and anti-TSH receptor antibodies, which
are present in Graves’ and Hashimoto’s diseases, can traverse the placental barrier. These antibodies, if present in
sufficient quantity, can induce fetal hyper or hypothyroidism
(6 – 8). In addition, antithyroid medications used to treat maternal hyperthyroidism, such as propylthiouracil and methimazole, can also cross the placenta and cause fetal hypothyroidism with goiter and possible physical and mental
retardation (9 –11). Fetal hypothyroidism can also be caused
by maternal hypothyroidism in the setting of iodine deficiency because the fetus is normally supplied with iodine by
transplacental passage from the mother (2, 4). This is the most
common cause of fetal hypothyroidism worldwide.
Evaluating thyroid abnormalities in the fetus is clinically
Abbreviations: AF, Amniotic fluid; fT4, free T4; SI, Système Internationale; tT4, total T4.
nmol/liter); and free T4 (n ⴝ 119) less than 0.4 – 0.7 ng/dl (5–9
pmol/liter), with a median of 0.4 ng/dl (5 pmol/liter). These
intervals represent the largest study done to date on third
trimester AF using automated immunoassays. A literature
search of fetal goiter revealed a number of cases of hypothyroidism. Seven cases reported AF TSH concentrations (range,
1.1–28.9 mU/liter) and four reported AF tT4 concentrations
[range, 0.98 –1.25 ␮g/ml (13–16 nmol/liter)], all of which fell
outside our reference intervals. These data support the use of
AF to diagnose fetal hypothyroidism, reducing the need to
resort to a riskier procedure such as cordocentesis. (J Clin
Endocrinol Metab 88: 4175– 4179, 2003)
difficult; yet, because in utero treatment is available, proper
diagnosis is important. Ultrasound examination can be of use
in detecting fetal goiters, but it does not differentiate between
hyper and hypothyroidism. To determine fetal thyroid status, cord blood has been used, but percutaneous umbilical
blood sampling is a technically demanding procedure that
poses a risk of fetal bradycardia, hemorrhage, and possible
fetal demise (5). Although the risk of fetal demise from percutaneous umbilical blood sampling is low, reported as
0.5–1% or less in recent reports (12); amniocentesis is a far
easier and safer procedure.
Studies indicate that from wk 12 of gestation onwards,
there are steady rises in the fetal serum concentrations of T4,
and TSH (5, 13) that correlate with concentrations in amniotic
fluid (14) and are independent of maternal concentrations (5,
14). The presence of small concentrations of T4 in cord blood
of fetuses with thyroid agenesis or organification defects
indicates that there is some transplacental T4 transfer (15);
however, the evidence overall points to a limited maternal
contribution to fetal physiology. Therefore, amniocentesis
provides a safer alternative to cord blood sampling.
This study sought to establish normal amniotic fluid reference intervals for TSH, total T4 (tT4), and free T4 (fT4) using
automated immunoassays.
Patients and Methods
Amniotic fluid samples that had been sent to the Barnes-Jewish Hospital chemistry laboratory (St. Louis, MO) for fetal lung maturity testing
from the preceding 22 months and stored at ⫺20 C were used. Corresponding patient chart review was performed, and samples were excluded if there was a maternal or fetal history of thyroid disorders, fetal
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anomalies, multiple gestations, or if sample collection took place before
the third trimester. Gestational ages ranged from 27–39 wk (median 35).
The AxSym automated immunoassay analyzer (Abbott Laboratories,
Abbott Park, IL) was used for all thyroid hormone measurements. The
AxSym TSH assay is a two-site microparticle enzyme immunoassay with
an analytical sensitivity of 0.1 mU/liter. The AxSym tT4 assay is a
fluorescence polarization immunoassay with an analytical sensitivity of
1.5 ␮g/dl (19 nmol/liter). The AxSym fT4 is also an microparticle enzyme immunoassay assay with an analytical sensitivity of 0.4 ng/dl (5.2
pmol/liter). This assay cannot be diluted and our laboratory has established linearity to 4.5 ng/dl (58 pmol/liter).
All samples were also evaluated for the presence of hemoglobin using
the ABL Radiometer (Copenhagen, Denmark), to eliminate the possibility of maternal blood contamination. None of the samples had detectable concentrations of hemoglobin, with the minimal detectable limit
for the assay being 1.0 ng/dl.
The Washington University Human Studies Committee approved
this study.
Statistical analysis
Central 95% reference intervals for each of the analytes were calculated using nonparametric analysis (16). This process ranks data according to numerical value, and calculates percentiles as a function of
these ranked numbers. The concentrations at the 2.5 and 97.5 percentiles
provide an estimate of the central 95% reference interval for the population based on the given set of data.
Singh et al. • Comments
To examine whether the distributions of amniotic fluid (AF) TSH, fT4,
or tT4 concentrations changed with gestational age during the third
trimester, robust regression analyses with gestational age as the independent variable were performed (17). P ⬍ 0.05 for the slope estimate
was considered statistically significant.
Results
Samples from 132 patients met selection criteria (see Patients and Methods) and were subsequently thawed and assayed immediately for TSH, tT4, and fT4 (not all samples
could have all three assays performed due to insufficient
sample volume). The frequencies of amniotic fluid thyroid
hormone concentrations are shown in Fig. 1. Central 95%
reference intervals, median values, and ranges for each of the
analytes were calculated and are outlined in Table 1. Changes
in AF concentrations of TSH, tT4, and fT4 across gestational
age were also examined (Fig. 2). There was no statistically
significant change in TSH (P ⫽ 0.390) or fT4 (P ⫽ 0.507) with
gestational age during the third trimester. There was a statistically significant decrease in the concentration of tT4 concentrations (Fig. 2B) with gestational age (P ⫽ 0.024; slope ⫽
⫺0.022). However, this difference does not warrant a gesta-
FIG. 1. Frequencies of amniotic fluid thyroid hormone results. TSH (A), tT4 (B), fT4 (C). Conversions to SI units are as follows: TSH, 1 ␮IU/ml ⫽
1 mU/liter; tT4, 1 ␮g/dl ⫽ 12.87 nmol/liter; fT4, 1 ng/dl ⫽ 12.9 pmol/liter.
Singh et al. • Comments
J Clin Endocrinol Metab, September 2003, 88(9):4175– 4179 4177
TABLE 1. Reference intervals for TSH, total T4, and f T4 calculated using nonparametric analysis
TSH (mU/liter)
Total T4 (␮g/dl)a
f T4 (ng/dl)
No. of
samples
Median
Range
Central 95%
reference interval
Assay
method
Analytical
sensitivity
127
129
119
0.1
3.3
0.4
⬍0.1– 0.5
1.7– 4.2
⬍0.4–⬎4.5
⬍0.1– 0.4
2.3 –3.9
⬍0.4– 0.7
MPEI
FPIA
MPEI
0.1
1.5
0.4
MPEI, Microparticle enzyme immunoassay; FPIA, fluorescence polarization immunoassay.
Conversion to SI units are as follows: total T4, 1 ␮g/dl ⫽ 12.87 nmol/liter; free T4, 1 ng/dl ⫽ 12.9 pmol/liter.
a
FIG. 2. Concentrations of TSH, tT4, and fT4 during the third trimester of pregnancy. TSH (A), tT4 (B), fT4 (C). Note that in C, the sample with
the fT4 more than 4.5 ng/dl has been excluded. Conversions to SI units are as follows: TSH, 1 ␮IU/ml ⫽ 1 mU/liter; tT4, 1 ␮g/dl ⫽ 12.87 nmol/liter;
fT4, 1 ng/dl ⫽ 12.9 pmol/liter.
tional age-specific reference interval as it takes 1 month for
a 0.1-␮g/dl change to occur.
A review of the literature revealed six reports of fetal
hypothyroidism with measured AF thyroid hormone concentrations (Table 2). No reports of fetal hyperthyroidism
with measured AF thyroid hormone concentrations were
found.
Discussion
This study has established normal amniotic fluid reference
intervals for TSH, tT4, and fT4 (Table 1). To our knowledge,
this is the first study that has established these intervals using
an automated immunoanalyzer, the first study to establish a
range for fT4 and the largest of its kind for third trimester
samples.
This study also examined whether AF TSH, fT4, and tT4
concentrations change during the third trimester (Fig. 2).
Robust regression analysis indicated that TSH concentrations (Fig. 2A) do not change appreciably during wk 27–39.
These findings are in agreement with two previous studies
that have also examined AF TSH concentrations over time.
Chopra and Crandall (18) reported that TSH changed little
between 15 and 42 wk of gestation. A study by Yoshida et al.
(14) suggested that TSH may peak at gestational wk 28 –36
with a mean of 0.218 mU/liter (n ⫽ 5) and drop to a mean
of 0.129 mU/liter (n ⫽ 21) by more than 37 wk. However, the
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Singh et al. • Comments
TABLE 2. Case reports of fetal hypothyroidism with measured amniotic fluid thyroid hormone concentrations
Gestational
age (wk)
b
Reference intervals
Kourides, 1984 (30)c
Perelman, 1990 (28)
Abuhamad, 1995 (29)
Nicolini, 1996 (31)
Bruner, 1997 (26)e
Perrotin, 2001 (26)
a) 26
b) 38
34.5
29
25
a) 29
b) 32
25
TSH (mU/liter)
Total T4 (␮g/dl)a
f T4 (ng/dl)
⬍0.1– 0.4
a) 2.4
b) 2.9
1.1
3.3
28.9
a) 16.6
b) 1.8
1.8
2.3–3.9
a) 0.98
b) 0.5
n/d
n/d
⬍0.1–2
a) ⬍1.25
b) ⬍1.25
n/d
⬍0.4 – 0.7
n/dd
Maternal diagnosis
Euthyroid
n/d
1
n/d
n/d
Euthyroid
Euthyroid
Graves’ disease, on PTU
Graves’ disease, on PTUf
n/d
Euthyroid
Conversions to SI units are as follows: tT4, 1 ␮g/dl ⫽ 12.87 nmol/liter; f T4, 1 ng/dl ⫽ 12.9 pmol/liter.
Reference intervals established in the present study for third trimester samples.
In this study, a) and b) are values for the same infant taken at different gestational ages.
d
n/d, Not determined.
e
In this study, a) and b) are values for two separate patients.
f
Both of the mothers in these cases had Graves’ disease treated with propylthiouracil (PTU).
a
b
c
authors point out that there was almost complete overlap
between the two groups. Klein et al. (19) reported that AF tT4
concentrations increased during the first half of pregnancy
reaching peak concentrations at 25–30 wk. The concentrations then decreased for the remainder of the pregnancy. Our
tT4 data also show a statistically significant decrease in concentration during the third trimester (Fig. 2B), but the difference was not great enough to warrant gestational agespecific reference intervals. No one has examined AF fT4
concentrations during gestation before this study, and we
found no statistically significant trend in fT4 concentrations
(Fig. 2C).
We performed a comprehensive literature search of reported cases of fetal hypothyroidism. Our findings are outlined in Table 2. In all cases of fetal hypothyroidism, the AF
TSH concentrations were above 1.1 mU/liter (range 1.1–28.9
mU/liter), which is more than 2.6 times the upper reference
range established in this study. These findings further support the use of AF thyroid hormone measurement for the
diagnosis of primary fetal hypothyroidism.
Fetal hyperthyroidism is currently diagnosed through
findings of fetal goiter, tachycardia, advanced bone maturation, and abnormal cord serum testing. The utility of amniotic fluid thyroid hormone reference intervals for diagnosing hyperthyroidism is unclear. We know of no published
cases of fetal hyperthyroidism where AF thyroid tests were
reported. TSH may not be as useful for diagnosis of fetal
hyperthyroidism, as the normal concentration of TSH in
amniotic fluid is already quite low, and detecting concentrations lower than the reference range exceeds the sensitivity of the second generation TSH assay. It may be possible
using a third generation assay because most of these assays
have analytical sensitivities of 0.01 mU/liter). Reference intervals for tT4 and fT4 may very well be useful in diagnosing
fetal hyperthyroidism, but it is not possible to know until
amniotic fluid thyroid hormones from several cases of fetal
hyperthyroidism are reported.
For the most part, the thyroid concentrations in this study
fell into a relatively tight range for all three analytes measured. However, one fT4 sample had a concentration of more
than 4.5 ng/dl (58 pmol/liter), which is much higher than
expected (⬎3 sd from the mean). The range independent of
this single value was less than 0.4 – 0.7 ng/dl (5–9 pmol/
liter). Patient chart review of the sample with the abnormally
elevated fT4 was significant for abdominal trauma with premature rupture of membranes. The mother had no history of
thyroid disorders, and a healthy baby boy was subsequently
delivered at 32 wk (the amniotic fluid sample was taken at
31 wk). Because the authors could find no reason to exclude
this case from consideration, it is included in the statistical
analysis pertaining to reference intervals. Of note is the fact
that although the fT4 concentration is elevated, this sample
had a tT4 concentration that fell into the normal reference
interval (rank 100 of 129 n).
A caveat to our study is that our samples were obtained
from a population of women receiving amniocentesis for
fetal lung maturity analysis and does not necessarily represent a “normal healthy population.” Although this is important, we feel that our reference intervals are still useful as
supported by previously published case reports of fetal hypothyroidism (Table 2).
Apart from the lack of adequate reference intervals, amniocentesis for the diagnosis of thyroid disorders has not
been widely used for several other reasons. First, there has
been uncertainty as to whether AF thyroid hormone concentrations derive from fetal or maternal sources. If AF thyroid testing reflects contamination from maternal physiology, it would not be a useful method. It is generally accepted
that TSH does not cross the placenta (8, 20, 21), and therefore,
AF TSH concentrations originate solely from the fetus. However, maternal-fetal transfer of T4 has a more complex physiology. Detectable, albeit low concentrations of T4 have been
reported in fetuses with thyroid agenesis or total organification defects (15). On the other hand, some infants are born
with hypothyroidism despite normal maternal serum thyroid hormone concentrations (22), indicating insufficient
contribution of maternal T4 to alter fetal physiology. Also, as
was previously stated, AF thyroid hormone concentrations
rise throughout gestation independently of maternal concentrations (14). Together, these findings suggest that the
majority of AF T4 concentrations are fetal derived.
A second reason that AF thyroid hormone concentrations
have not been used is that previously published reports
clamed a lack of utility in this method. One of the most
widely cited is that by Hollingsworth and Alexander (23),
whereby the authors were unable to correlate AF concen-
Singh et al. • Comments
J Clin Endocrinol Metab, September 2003, 88(9):4175– 4179 4179
trations of TSH, T4, and T3 to clinical outcome. Interestingly,
the reference intervals for AF TSH that Hollingsworth’s
study provided are considerably wider than the reference
intervals that have been reported in the literature since that
time (14, 24), including the current study. A difference in
assay techniques may explain part of the apparent discrepancy. Furthermore, Hollingsworth and Alexander report that
their TSH assay had cross-reactivity with human chorionic
gonadotropin and their amniotic fluid contributed a positive
bias for TSH, making their TSH results unreliable. They
further state that their AF TSH results “should not be taken
as absolute values.” Therefore, their assay should not have
been used to define a reference interval. Several of the case
reports in Hollingsworth’s paper do not confirm AF thyroid
hormone concentrations with neonatal serum, which may
account for the failure of AF concentrations to correlate with
clinical diagnosis. Finally, it is worth noting that a number
of reports since Hollingsworth and Alexander’s study have
successfully used AF thyroid hormones to diagnose fetal
thyroid disorders (25–27).
The utility of amniotic fluid thyroid hormone measurement lies not only in the diagnosis of thyroid disorders but
also in management. Many authors (26 –29) have treated
cases of fetal hypothyroid goiter with intrauterine T4, using
ultrasound examination to show regression of fetal goiter as
well as amniotic fluid to show decreases in AF TSH and/or
increases in AF T4. Application of our amniotic fluid reference intervals may provide a more relevant endpoint for
therapy than ultrasound monitoring, since the regression of
goiter does not necessarily confer an euthyroid state. In addition, amniotic fluid is relatively simple to obtain in the
setting of intrauterine T4 administration, as it is readily accessible when the injections are performed.
In conclusion, we have established reference intervals for
TSH, fT4, and tT4 in third trimester amniotic fluid. We believe
that these reference intervals can be of significant use in the
diagnosis and management of fetal hypothyroidism, reducing the need for umbilical blood sampling in these patients.
Acknowledgments
Received March 26, 2003. Accepted May 29, 2003.
Address all correspondence and requests for reprints to: Ann
Gronowski, Ph.D., Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid, Box 8118, St.
Louis, Missouri 63110. E-mail: [email protected].
This study has been presented in part at the Academy of Clinical
Laboratory Physicians and Scientists Annual Meeting, New York, NY,
June 2002. This research was sponsored in part by Abbott Laboratories
by supplying reagents.
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