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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, Syste`me 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.
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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-
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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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