Assay for fetal thyroid function

Disclosed is an assay for fetal thyroid function involving determining the amount of fetal thyroid function indicator that is present in the maternal blood and maternal urine and then comparing the determined value to a known standard for the gestation age of the fetus. It is defined as below one standard deviation of the normal values. Also disclosed is a method of treating a fetus having hypothyroidism involving determining that the fetus is hypothyroid using the assay of the present invention and thereafter, administering supplemental thyroid hormone (T.sub.4, 500 ug/wk) into the amniotic sac. Also disclosed is a method of identifying euthyroid state using the assay of the present invention in hypothyroid fetuses receiving T.sub.4 therapy.

FIELD OF THE INVENTION 
The present invention relates to methods of detecting inadequate fetal 
thyroid function and to treating a fetus having inadequate fetal thyroid 
function in order to prevent or lessen the associated sequelae. 
BACKGROUND OF THE INVENTION 
Prenatal hypothyroidism can result in a syndrome known as cretinism. This 
syndrome can be manifested at birth by jaundice, umbilical hernia, noisy 
respirations, hypotonia, depressed reflexes and lethargy, and after birth 
by the development of abnormal facial features, retarded bone development, 
enlarged tongue and constipation. 
Most significantly, inadequate thyroid function prior to birth can cause 
abnormal neurologic development including mental retardation, varying 
degrees of deafness and a combination of flexed posture with spasticity 
and rigidity of proximal limb musculature. These nervous system deficits 
can persist throughout life. 
Worldwide, prenatal hypothyroidism most commonly results from a maternal 
iodine deficiency, particularly in some estimated 800 million people in 
geographic areas wherein dietary iodine deficiency is endemic. The 
prevalence of endemic cretinism can reach 10% of the whole population in 
severely affected areas. It can also result from congenital disorders of 
thyroid function. 
Because of the potentially severe consequences, neonates are screened for 
hypothyroidism by measuring serum T.sub.4 or TSH. However, numerous 
reports have shown that some infants have some degree of impaired 
psychological and neuromuscular function at later ages, even when therapy 
has been started early. Ideally, prenatal screening would allow in utero 
intervention to prevent or lessen the effects of prenatal hypothyroidism. 
Among the thyroid hormones, concentrations of hormones resulting from the 
conjugation of phenolic hydroxyl with sulfate, including T.sub.4 sulfate 
(T.sub.4 S), T.sub.3 sulfate (T.sub.3 S), and rT.sub.3 sulfate (rT.sub.3 
S) are markedly elevated in fetal and newborn cord sera. The high levels 
of these sulfated iodothyronines in fetal serum are due to both reduced 
clearance secondary to relatively low type I 5'-monodeiodinase activity in 
fetal tissue and high fetal production rates, particularly of T.sub.4 S. 
The high prevailing levels of sulfated iodothyronines in fetal plasma, 
however, are not associated with increased circulating concentrations of 
T.sub.4 S, T.sub.3 S, or rT.sub.3 S in pregnant women. Thus, measurement 
of these substances in maternal serum cannot be used to detect inadequate 
fetal thyroid function. Similarly, analysis of amniotic fluid 
iodothyronine metabolites or TSH levels also cannot be used to evaluate 
fetal thyroid function, because the concentrations of T.sub.4 or rT.sub.3 
correlate poorly with maternal or fetal thyroid function. Whether amniotic 
fluid TSH reflects fetal or maternal TSH concentrations remains to be 
established. 
Currently, direct sampling of fetal blood levels of fetal thyroid hormone 
or metabolites of fetal thyroid hormone requires an invasive procedure in 
the uterus. This procedure of cordocentesis is expensive, requires highly 
trained personnel and carries a significant risk of injury to the fetus. 
Thus, there remains a need for an effective prenatal test to determine 
fetal thyroid function. In one aspect, the present invention is an 
improved method of testing for prenatal hypothyroidism by screening the 
mother's blood for a novel compound. 
SUMMARY OF THE INVENTION 
In one aspect, the present invention is an assay for quantifying fetal 
thyroid function of a fetus in a pregnant woman. The assay comprises the 
steps of, first, obtaining a sample of fluid from the pregnant woman, 
wherein the fetus has a gestational age; next, determining the amount of 
Fetal Thyroid Hormone Indicator (FTFI) present in the fluid, wherein the 
FTFI has the characteristics of immunological cross-reactivity with 
L-3,3'-diiodothyronine (T.sub.2) sulfate (T.sub.2 S), presence in cord 
blood at birth, crosses the placenta, presence in maternal blood in a 
concentration versus weeks of gestation having a relative relationship as 
shown by a best fit expressed as y=57-2.9.times.+0.17x.sup.2, wherein y is 
expressed as nanomoles/L or ng/dL T.sub.2 S equivalent and x is weeks of 
gestation, and has a different chromatographic peak than synthetic T.sub.2 
S in high pressure liquid chromatography; and then, comparing the amount 
of determined FTFI to a known normal amount of FTFI for the gestational 
age. A determined FTFI amount lower than about one standard deviation of 
the normal FTFI amount for the gestation age indicates abnormally low 
thyroid function in the fetus. 
In one embodiment the assay is performed using maternal blood. In another, 
the assay is performed using maternal urine. 
In another aspect of the present invention, there is provided a method of 
treating a fetus having inadequate thyroid function, comprising the steps 
of, first, identifying the fetus as having inadequate thyroid function 
according to the assay for quantifying fetal thyroid function of a fetus 
in a pregnant woman of the present invention, and then treating the fetus 
to render the fetus closer to a euthyroid state. 
In one embodiment, the treating step comprises administering thyroid 
hormone to the fetus is an amount and frequency sufficient to render the 
fetus closer to the euthyroid state. In one particular embodiment, the 
thyroid hormone is T.sub.4. 
In another embodiment, the treating step comprises administering thyroid 
hormone to the fetus through the amniotic sac in a dose between about 
200-800 .mu.g weekly. 
In another, the present invention is a substance present in fetal blood, 
comprising a compound that has the following characteristics: 1) 
immunological cross-reactivity with T.sub.2 sulfate (T.sub.2 S), 2) 
presence in cord blood at birth, 3) capability of crossing the placenta, 
4) presence in maternal blood in a concentration versus weeks of gestation 
having relative relationship as shown by a best fit expressed as 
y=57-2.9.times.+0.17x.sup.2, wherein y is expressed as nanomoles/L or 
ng/dL T.sub.2 S equivalent and x is weeks of gestation, 5) presence in 
maternal urine in increasing quantities with progression of pregnancy when 
expressed in ng/gram of urinary creatinine, and 6) a different 
chromatographic peak than synthetic T.sub.2 S in high pressure liquid 
chromatography.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
It is one goal of the present invention to provide an improved method of 
testing for prenatal hypothyroidism which will allow for timely treatment 
of the condition to prevent or lessen the associated sequelae. In one 
embodiment, the present invention related to the discovery of a substance 
hereinafter referred to as "Fetal Thyroid Function Indicator" (FTFI). This 
substance is present in human maternal serum and urine in concentrations 
that increase progressively to peak values before parturition. After 
parturition, FTFI is cleared from the maternal circulation in 7-10 days. 
FTFI was discovered unexpectedly during experiments to determine whether 
3,3'-diiodothyronine sulfate (T.sub.2 S) levels were elevated in pregnant 
women. Serum T.sub.2 S immunoreactivity was measured in non-pregnant women 
and in pregnant women at various gestational ages as well as after 
delivery using the method described below. It is one of the surprising 
discoveries of the present invention that maternal levels of FTFI can be 
used to identify deficient thyroid function in the fetus. 
METHODS AND MATERIALS 
T.sub.2 S Radioimmunoassay 
3,3'-T.sub.2 S and .sup.25 I!T.sub.2 S were prepared by the method of 
Eelkman-Rooda and co-workers (Eelkman-Rooda SJ, Kaptein E, VanLoon MAC, 
Visser TJ. 1988 "Development of a radioimmunoassay for triiodothyronine 
sulfate"), herein incorporated by reference in its entirety, but can be 
prepared by any number of methods known to those with skill in the art. 
L-3,3'-Diiodothyronine (L-T.sub.2) is purchased from Henning Berlin 
(Berlin, Germany). The radioactive .sup.125 I! T.sub.2 (S.A. 300-500 
mCi/mg) was prepared by iodination of 3,3'-T.sub.2 (exchange reaction) 
using methods described in Nakamura Y, Chopra I J, Solomon DH. 1977 
"Preparation of high specific activity radioactive iodothyronines and 
their analogues." Journal of Nuclear Medicine. 18:1112-1115, herein 
incorporated by reference in its entirety, but can be prepared by any of a 
number of methods known to those with skill in the art. In 10.times.75 mm 
disposable glass culture tubes, 10 .mu.l of methanol containing 10.sup.-9 
moles of the substrate (T.sub.2) were mixed with 25 .mu.l of 0.4 M 
phosphate buffer (pH 6.2) containing approximately 1 mCi of I-125. The 
radioiodination reaction was started by adding 10 .mu.l of 0.05M phosphate 
buffer (pH 6.2) containing 4 .mu.g of chloramine-T. Two minutes later, the 
reaction was stopped by addition of 20 .mu.l of a solution prepared by 
1:10 dilution of saturated sodium metabisulfite in 0.05 M phosphate buffer 
(pH 6.2). The reaction mixture was next transferred to a column made from 
a 3-mL disposable plastic syringe filled with a suspension of Sephadex 
LH-20 up to 1.2 mL; the reaction vessel was washed once with 50 .mu.l of 
0.05 M phosphate buffer (pH 6.2), and washings were also transferred to 
the column. Unreacted radioiodine was removed by washing of the column 
with 1 mL of 0.05M phosphate buffer (pH 6.2) followed by 4 mL of water and 
0.6 mL 99:1 (methanol: 2N NH.sub.4 OH). The radioactive compounds were 
then eluted with 4 mL of 99:1 (methanol: 2N NH.sub.4 OH) and were dried 
under a thin stream of air. The residue was dissolved in 100 .mu.l of 
methanol (2N NH.sub.4 OH) and applied to a paper strip to separate the 
radioiodinated iodothyronines. 
Descending paper chromatography was performed using a 1:5:6 hexane: 
tertiary amyl alcohol: 2N NH.sub.4 OH system and 3 MM Whatman 
chromatographic paper. Chromatography was allowed to proceed for 20 hr. 
The radioactive spots were identified by a X-ray film overlaying the paper 
chromatographic strip for 1 min. The radioactive spots were cut and eluted 
with 5-10 mL of 99:1 methanol-2N NH.sub.4 OH. The eluate was dried under 
air and the residue dissolved in 1-3 mL of 50% propylene glycol for 
storage at 4.degree. C. The .sup.125 I!-T.sub.2 spot was identified by 
specific T.sub.2 antibody by RIA. 
Radioactive tracer amounts of 125-I-T.sub.2 S or relatively small 
quantities (ng to .mu.g) of T.sub.2 S were synthesized as following. 
Reactions involving cholorosulfonic acid were started by the slow addition 
of 200 .mu.l chlorosulfonic acid (CISO.sub.3 H, 15 M, Aldrich Chemical 
Co., Milwaukee, Wis.) to 800 .mu.l N,N-dimethylformamide (DMF) at 
0.degree. C. In another tube, a solution of (usually) 100 pmol unlabeled 
plus .sup.125 I!-T.sub.2 in ammonia ethanol was evaporated under a stream 
of nitrogen. To the residue of the latter tube, 200 .mu.l of the 
CISO.sub.3 H solution in DMF was added at 0.degree. C. Subsequently, the 
mixtures were brought to room temperature, and, in general, reactions were 
continued overnight. After dilution with 800 .mu.l ice-cold water, 
reaction products were analyzed by Sephadex LH-20 chromatography. Products 
were applied to a small 1-mL Sephadex LH-20 column. Serial elution was 
performed with 4.times.1-mL fractions of 0.1N HCl, 7.times.1-mL water, and 
4.times.1-mL of 1N ammonia in ethanol. The sulfated T.sub.2 was eluted 
with water. 
Larger (mg) quantities of T.sub.2 S were similarly synthesized. Five to 20 
mg anhydrous T.sub.2 (Sigma) and 1 .mu.Ci .sup.125 I-T.sub.2 was added to 
0.5 mL of a mixture of CISO.sub.3 H and DMF (1/4, v/v) at 0.degree. C. 
After reacting for 40 hours at room temperature, T.sub.2 S was 
precipitated by addition of this mixture to 5 mL H.sub.2 O at 0.degree. C. 
The pellet was dissolved in 1 mL 2N NH.sub.4 OH and reprecipitated with 5 
mL1 N HCl. The pellet was further washed by repeated suspension in 3 mL 
0.1 N HCl. 
Identification and further purification of T.sub.2 S had been achieved by 
HPLC. Reverse phase HPLC was performed on a Radial PAK uBondapak C.sub.18 
column using a model 6000A solvent delivery system, and absorbance was 
monitored at 254 nm with a variable wavelength detector (Waters, Milford, 
Mass.). Products were eluted isocratically with a mixture of acetonitrile 
and 0.02M ammonium acetate, pH 4 (22:78, v/v), at a solvent flow of 2 
mL/min. Absorbance was recorded at 254 nm. A semipreparative column 
(Blochtom 1010 ODS, Regis) was used for separation and quantitative 
recovery of other pure iodothyronine sulfate conjugates including T.sub.2 
S. 
The T.sub.2 S (approx. 1-5 mg, with tracer amount of 125-I-T.sub.2 S) was 
dissolved in 5 mL dimethylformamide and added to a solution of 100 mg 
bovine albumin (BSA) in 20 mL H.sub.2 O. After adjusting the mixture to pH 
5 with 0.1 N NaOH, 50 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide 
(Sigma Co., St. Louis, Mo.) was added in 5 mL H.sub.2 O. This was repeated 
after 3 hr, and the mixture was further stirred for 16 hr at room 
temperature. The product was dialyzed at 4.degree. C. against successively 
2.times.2 liter H.sub.2 O, 2 liter 0.1N NaOH and 3.times.2 liter H.sub.2 
O, changed twice daily. Analysis of the remaining radioactivity indicated 
a low degree of T.sub.2 S incorporation (&lt;5%), corresponding to &lt;5 mol 
T.sub.2 S/mol BSA. The immunogen was stored at -20.degree. C. in a 
concentration of 1.5 mg protein/mL. Three New Zealand white rabbits were 
immunized by S.C. injections of 1 mL conjugates mixed with complete 
Freund's adjuvant at multiple sites in the back. This was repeated after 
one month and subsequently at bimonthly intervals. 
The RIA employed an anti-L-T.sub.2 S (WO213) obtained from rabbits 
immunized with L-T.sub.2 S BSA conjugate. In a final dilution of 1:15,000, 
anti-T.sub.2 S antibody WO213 bound 35-45% of a tracer amount (.about.5 pg 
or 6.8 fmol) of .sup.125 I!T.sub.2 S in 0.075 mol/L barbitol buffer (pH 
8.6; containing 0.15% sodium azide and 0.125% normal rabbit serum) and 19% 
ethanol. 
The T.sub.2 S RIA procedure was performed using serum or urine samples that 
were extracted with 2 volumes 95% ethanol (final concentration, 63%) 
before assay. Preliminary experiments showed that the extraction 
efficiency of T.sub.2 S in serum exceeded 96%. Final T.sub.2 S 
concentrations were not corrected for recovery. The lower limit of 
detection of the assay was 8.3 fmol (5 pg) or 82.6 pmol/L in a 300-.mu.l 
ethanol extract of serum. 
The cross-reactivities of various thyroid hormone analogs were determined 
using this assay. Only T.sub.4 S, rT.sub.3 S and T.sub.3 S cross-reacted 
significantly (3.2%, 1.4%, and 0.02%, respective). Other thyroid hormone 
analogs and sulfated steroids (dehydroepiandrosterone sulfate, estradiol 
sulfate and estrone sulfate) cross-reacted less than 0.0001%. 
Identification of FTFI 
FTFI was identified during determination of T.sub.2 S in the following 
manner. T.sub.2 S immunoreactivity was determined in maternal serum or 
urine extracts by acid hydrolysis, HPLC, and RIA. Ten to 20 mL maternal 
serum or urine were extracted with 2 volumes 95% ethanol and subsequently 
lyophilized. The dried extracts were dissolved in 1-2 mL H.sub.2 O and 
purified with an LH-20 column. One milliliter of the solubilized extract 
was applied to a small LH-20 column (bed volume, 1.2 mL) equilibrated in 
0.1 mol/L HCl. 
Next, the column was rinsed with 4 mL 0.1 mol/L HCl. The ensuing 7 mL 
H.sub.2 O eluent fractions were collected and lyophilized. The dried 
fractions were dissolved in 500 .mu.l 0.025 N NaOH. 
After application of 200 .mu.l sample to a HPLC uBondapak C.sub.18 column, 
the serum extract was eluted isocratically with a mixture of acetonitriles 
and 0.02 mol/L ammonium acetate, pH 4.0 (22:78, vol/vol) at a flow rate of 
2 mL/minutes. Aliquots of eluent of 1-mL fractions were collected, and 100 
.mu.l of these aliquots were subjected to T.sub.2 S RIA. Radioactive or 
non-radioactive T.sub.2 S-supplemented serum extracts from non-pregnant 
women were used to identify the T.sub.2 S peak. 
One milliliter of 63% ethanol serum extract was mixed with 1 mL 1.0N HCl, 
followed by heating at 80.degree. C. for 1 hour to hydrolyze sulfated 
iodothyronines. After the addition of 1 mL 1N NaOH, the concentration of 
3,3'-T.sub.2 was measured in duplicate 300-.mu.l aliquots of the resulting 
mixture by specific RIA. 
Sources of Specimens 
Serum samples were obtained from 200 pregnant women with estimated 
gestational ages ranging from 3-40 weeks (18-39 years of age). Serum 
samples were also obtained from 25 women (20-39 years of age) at the time 
of delivery and from paired newborn cord blood samples. Additionally, 67 
serum samples were obtained from 35 women 12-184 hours postpartum. Control 
samples were obtained from 14 normal non-pregnant women, 19-35 years of 
age (NP control). Urine samples were also obtained from 85 pregnant women 
with estimated gestational ages ranging from 4-40 weeks. 
Statistical Analysis 
Student's unpaired t test was used to assess between-group differences. 
Analysis of variance was used to test multigroup comparisons. If 
significant differences were present, Dunnett's multicomparison test was 
used to compare the control or baseline mean and the mean values of other 
groups. 
Significance was defined as P&lt;0.05. Results are reported as the 
mean.+-.SEM. In addition, simple linear and multiple regression analyses 
of the serum FTFI concentrations of pregnant women of different 
gestational ages were also performed. 
RESULTS: 
Identification of FTFI in Serum of Pregnant Women 
Referring now to FIG. 1, there is presented the identification of T.sub.2 S 
and FTFI by RIA in third trimester women (filled triangles) and newborn 
cord serum extracts (open circles) on HPLC. Serum extracts from 
non-pregnant women with (open squares) or without (filled circles)! 
supplementation of T.sub.2 S were used to identify T.sub.2 S peaks. 
Serum extracts were eluted from HPLC (uBondapak C.sub.18 column) 
isocratically with a mixture of acetonitrile and 0.02 mol/L ammonium 
acetate, pH 4.0 (22:78, vol/vol), at a flow rate of 2 mL/minutes. Aliquots 
of eluents in 1-mL fractions were collected, and 100 .mu.L of each were 
studied in the T.sub.2 S RIA. 
Note that, in cord serum, part of the T.sub.2 S immunoreactivity 
cochromatographed with synthetic T.sub.2 S (with a retention time of 7.5 
minutes), but there was an additional peak at 10 minutes. This additional 
peak represents FTFI in the maternal serum and urine sample. 
In the serum of pregnant women, all of the T.sub.2 S immunoreactivity was 
found in the FTFI peak. Neither T.sub.2 S nor FTFI activity was 
identifiable in the serum of non-pregnant women. 
Upon acid hydrolysis, 36.+-.2.6% of the T.sub.2 S immunoreactivity in cord 
serum extracts was recovered as 3,3'-T.sub.2. 3,3'-T.sub.2 was 
undetectable (&lt;10 ng/dL) in the acid hydrolysates of serum from pregnant 
women. The recovery of FTFI after hot acid hydrolysis of pregnant women 
serum extract was 98.2.+-.4.8% (n=10). 
FTFI Concentrations in Human Maternal and Fetal Sera 
A total of 200 maternal serum samples were assayed for FTFI levels. The 
results are shown below in Table 1. Concentrations are given in ng/dL 
(T.sub.28 -equivalent).+-.SD(100 ng/dL=1.65 nmol/L). 
Following delivery (158.+-.11, N=25) serum levels decreased to 89.+-.7 
(N=15) at 1 day, 54.+-.4 (N=15) at 3 days, and 20.+-.2 (N=8) at 7 days. 
The peak levels remain significantly lower than the 3,3'-diiodothyronine 
sulfate (T.sub.2 S) concentrations in cord serum at birth. 
TABLE 1 
______________________________________ 
Weeks of Pregnancy 
14- NB 
Pt. 3-7 8-13 19 20-26 27-33 34-40 (cord) 
NP 
______________________________________ 
(N) (38) (33) (38) (18) (51) (22) (45) (15) 
Ser- 55# 51 70 98 145* 217* 310* 11* 
um 
Conc. 
.+-.25 .+-.23 .+-.35 
.+-.47 
.+-.50 
.+-.108 
.+-.94 
.+-.4 
(ng/ 
dL) 
______________________________________ 
#in mean .+-. SD T.sub.2 Sequivalent ng/dL; 
*p &lt; 0.05 cf., 3-7 wk pregnancy; 
NP = nonpregnant women, 
NB = new born infant 
Referring now to FIG. 2, 119 pregnant women were studied at gestational 
ages ranging from 3-40 weeks. Serum FTFI concentrations (expressed both as 
nanogram/dL and as nanomoles/L T.sub.2 S equivalent; mean.+-.SE) were 
elevated in the first trimester (3-13 weeks pregnancy, n=41) and found to 
be 0.73.+-.0.04 nmol/L (p&lt;0.05). 
The mean FTFI level increased moderately during the second trimester 
(1.4-26 weeks pregnancy, 1.14.+-.0.09 nmol/L; n=43; p&lt;0.05 vs. first 
trimester) and the first two-thirds of the third trimester (27-35 weeks, 
1.67.+-.0.11 nmol/L; n=21; p&lt;0.01 cf. first trimester). A rather sharp 
increase was noted near term (36-40 weeks, 3.49.+-.0.49 nmol/L; n=14; 
p&lt;0.01 vs. first trimester). 
In FIG. 2, the filled triangles indicate concentrations obtained from 
maternal serum samples during first trimester pregnancies (3-13 weeks 
gestation; n=41). The open squares indicate concentrations obtained during 
second trimester pregnancies (14-26 weeks gestation; n=43). The circles 
indicate concentrations obtained during third trimester pregnancies (27-40 
weeks gestation; n=35). Finally, the filled circles indicate 
concentrations obtained from fourteen patients approximately 1 month 
before delivery at term. 
The straight fines labeled "A", "B", and "C" represent linear regression 
lines for T.sub.2 S concentrations in samples from the first, second, and 
third trimesters (y=ax+b linear representation). The slopes of these lines 
were 3.69 and 10.76 for the second and third trimesters, respectively 
(p&lt;0.05). Overall FTFI values in all 118 samples were best fitted by a 
curvilinear line (y=57-2.9.times.0.17x.sup.2 , correlation =0.7036; 
p&lt;0.001). 
Referring now to FIG. 3, there is presented the concentrations of T.sub.2 S 
and FTFI in cord serum of newborns and FTFI levels in maternal serum 
samples at the time of delivery (D). The connected lines represent serial 
measurements in the same patients (n=18). T.sub.2 S concentrations also 
were measured in 14 nonpregnant women (NP) for comparison. 
The decrease in serum FTFI concentrations after parturition is depicted in 
the semilog plot in the inset. The closed circles in vertical bars 
represent the mean (.+-.SEM) and (n) represents total number of samples 
studied at each time period in a total of 35 patients. 
As can be seen in FIG. 3, the serum levels of FTFI in pregnant women are 
shown at parturition (2.61.+-.0.18 nmol/L; n=25). Immunoreactivities were 
measured in the paired cord sera obtained at birth (4.40.+-.0.21 nmol/L; 
T.sub.2 S and FTFI mixture; n=25). After parturition, maternal serum FTFI 
levels decreased from 2.61.+-.0.18 nmol/L (n=25) to 1.47.+-.0.12 nmol/L 
(n=18) at 1 day (decay t.sub.1/2 =1.2 days) and then to 0.89.+-.0.07 
nmol/L (n=15) at 3 days and 0.33+0.03 nmol/L (n=8) at 7 days (decay 
t.sub.1/2 =2.9 days; FIG. 3). 
A 6.2-fold increase in the concentration of serum T.sub.2 S-cross-reactive 
material was observed in six non-pregnant women who received hCG treatment 
(9 days post-hCG, 1.12.+-.0.18 nmol/L; pre-hCG baseline concentration, 
0.18.+-.0.02 nmol/L; p&lt;0.01). The T.sub.2 S-cross-reactivities remained 
unchanged after hot acid hydrolysis of post-hCG serum extracts. 
Relation of Fetal Thyroid Function Indicator to Fetal Thyroid Hormone 
The preterm increase in fetal serum FTFI appears to coincide with the known 
prenatal surge of serum T.sub.3 in fetuses. The simultaneous peaking of 
fetal serum T.sub.3 and maternal serum FTFI levels makes fetal serum 
T.sub.3 a candidate for a precursor for the perinatal increases in 
maternal T.sub.2 S levels. 
In pregnant sheep near term, significant quantities of authentic .sup.125 
I!-T.sub.2 S are present in the maternal compartment after the infusion of 
ovine fetuses with .sup.125 I!-T.sub.3. More recently it was found that 
fetal T.sub.3 infusion rapidly increased T.sub.2 S concentrations in 
maternal urine and serum in sheep. These results are consistent with the 
view that at least part of the circulating FTFI, a T.sub.2 S-like 
compound, may be fetal in origin. 
An increase in FTFI levels, similar to that found in pregnant women, was 
found in serum of non-pregnant women who received acute injections of hCG. 
Thus, placental hCG may be involved in a mechanism to increase FTFI in 
early pregnancy. Note, however, that serum hCG concentrations peak during 
the first trimester and decrease progressively thereafter. Therefore, hCG 
cannot fully account for the continuing increase in FTFI concentrations in 
maternal serum during the second and third trimesters. Rather, the third 
trimester increase in maternal T.sub.2 S immunoreactivity may be 
associated with the onset and progressive maturation of fetal thyroid 
function. 
The marked rise of serum FTFI in near-term pregnant women occurs while hCG 
levels are relatively low and fetal serum T.sub.3 levels are increasing. 
Thus, transplacental fetal to maternal transfer of T.sub.2 S or metabolite 
may be involved. 
FTFI Concentrations in Human Maternal Urine 
FTFI and creatinine levels were measured in 85 maternal urine samples. The 
results are presented in Table 2, below. 
Note that maternal urine FTFI concentration, expressed in ng (T.sub.2 
S-equivalent)/gm creatinine, rises above baseline during the second 
trimester and continues to rise throughout the remainder of gestation. 
Therefore, maternal urine FTFI concentration can also be used to test for 
fetal hypothyroidism. In addition, urinary FTFI, expressed as ng/gram 
urinary creatinine, can be used as a complimentary test to serum FTFI 
values. It is possible that in certain patients' serum, FTFI values alone 
may be misleading as a result of more rapid or slow than normal clearance 
of serum FTFI (see case #2 below). 
TABLE 2 
______________________________________ 
Weeks of Pregnancy 
14- 
Pt. 3-7 8-13 19 20-26 27-33 34-36 37-40 NP 
______________________________________ 
(N) (19) (9) (9) (8) (11) (11) (18) (6) 
Urine 
601# 697 814 1463* 2189* 2430* 4323* 338 
Conc. 
.+-.155 
.+-.202 
.+-.231 
.+-.682 
.+-.1419 
.+-.979 
.+-.2057 
.+-.97 
______________________________________ 
#in mean .+-. SD T.sub.2 Sequivalent ng/gm; 
NP = nonpregnant; 
*p &lt; 0.05 cf., 3-7 wk pregnancy. 
Thus, whereas hCG stimulation may account for some increase in maternal 
serum concentrations of FTFI in the first trimester, the more rapid 
increase in maternal serum FTFI concentrations during the late third 
trimester are probably related to changes that occur in fetal thyroid 
hormone economy. Further, placental transfer and transformation of fetal 
T.sub.3 may be related to the rise in the level of FTFI in the serum of 
pregnant women. 
EXAMPLE #1, DETECTION OF HYPOTHYROID FETUS AND INTRAUTERINE TREATMENT 
THEREOF 
Case #1 
A fetus was identified with a goiter on ultrasound examination. 
Cordocentesis was carried out to obtain fetal serum sample at 28 weeks of 
gestation. This serum sample showed a low free thyroxine value 0.6 ng/dL; 
(normal 0.8-2.8 ng/dL)! and elevated TSH 126 .mu.U/mL (normal&lt;20 
.mu.U/mL)!. The FTFI concentration in maternal serum was also reduced, 93 
ng/dL (normal mean .+-.SD is 145.+-.50 ng/dL or 95-195 ng/dL). A diagnosis 
of potential fetal hypothyroidism was made. 
The pregnant woman received an intraamniotic injection of thyroxine 
(levothyroxine sodium, USP, 500 .mu.g) weekly for three consecutive weeks. 
The FTFI concentration in maternal serum one day after the 3rd treatment 
was 260 ng/dL, which was well within the normal range for 32 weeks 
gestational women. The treatment of thyroxine was continued weekly until 
delivery. The infant was delivered at 37 weeks of gestation with normal 
labor, small goiter, normal cord blood TSH and free T.sub.4. 
Case #2 
A pregnant woman, who had a hypothyroid baby discovered at birth in her 
last pregnancy, had three consecutive maternal serum samples at 
gestational ages of 20 weeks, 30 weeks and 33 weeks drawn and analyzed for 
FTFI concentration. All samples had FTFI levels below 1 SD of normal (30 
ng/dL, 49 ng/dL, 56 ng/dL). 
A diagnosis of potential fetal hypothyroidism was made. The patient began 
receiving weekly intraaminiotic thyroxine injections (200 .mu.g 
levothyroxine sodium) on Apr. 29, 1994. The FTFI concentration in maternal 
serum 6 days following the 3rd treatment was 103 .mu.g/dL (still below 1 
SD). However, the urinary FTFI was within normal range 3636 ng/gm 
creatinine; normal, 2266-6380 ng/gm creatinine)!. The infant was delivered 
at 38 weeks of gestation; cord blood TSH and free T.sub.4 were within 
normal limits. This case illustrates the usefulness of urinary FTFI in 
patients who may have a rapid clearance of FTFI from maternal circulation. 
Case #3 
A pregnant woman had been treated with propylthiouracil (PTU) for Graves' 
disease in daily doses of 300-500 mg. The PTU dose was reduced to 250 
mg/day at the 32nd week of gestation. Ultrasound examination demonstrated 
fetal goiter. The maternal serum showed a normal FT.sub.4 of 0.81 ng/dL 
(normal 0.5-1.6 ng/dL) and low FTFI 67 ng/dL, (normal 95-195 ng/dL 
T.sub.2 S-equivalent)! at 33 weeks of gestation. A diagnosis of potential 
fetal hypothyroidism was made. 
The pregnant woman started to receive weekly thyroxine (levothyroxine 
sodium, USP, 250 .mu.g) therapy intraamniotically. The ensuing serum FTFI 
tests were persistently low (69, 70, and 75 ng/dL) between 33 and 35 weeks 
of gestation despite thyroxine therapy. The infant was delivered at 36 
weeks of gestation with elevated TSH (80 .mu.U/mL) and decreased FT.sub.4 
(0.6 ng/dL) in cord blood. 
EXAMPLE #2, PROTOCOL FOR THE DETECTION OF HYPOTHYROID FETUSES AND 
INTRAUTERINE TREATMENT THEREOF 
In order to detect fetal hypothyroidism, fluid samples are obtained from 
pregnant women according to one of the following protocols: a) monthly 
serum (2 mL) and urine (10 mL) samples at the beginning of the 2nd 
trimester (week 14 of gestation); b) one or two consecutive serum and 
urine samples after 20 weeks of pregnancy; c) one or two consecutive serum 
and urine samples after 30 weeks of pregnancy. 
From these samples, the serum and urine FTFI concentrations and urinary 
creatinine concentrations are measured. If serum FTFI concentrations 
(ng/dL) and/or urinary FTF| concentrations (ng/gm creatinine) are below 
one standard deviation (SD), other ancillary tests are performed. These 
tests can include fetal serum thyroid hormone concentrations by 
cordocentesis, ultrasound to detect the presence of fetal goiter or 
retesting FTFI concentrations in maternal serum and/or urine samples. 
Once fetal hypothyroidism is confirmed using maternal serum FTFI 
concentrations or maternal urine concentrations in conjunction with the 
other tests, treatment with intraamniotic administration of higher doses 
of thyroxine (500 .mu.g/wk) should be started after obtaining the 
patient's consent. In one case (case #3), the dose of 250 .mu.g/wk may be 
inadequate to correct fetal hypothyroidism. FTFI concentrations should be 
determined in maternal serum samples one to three weeks following the 
initiation of T.sub.4 therapy. The treatment should be continued until the 
baby is delivered. 
The present invention can be embodied in other specific forms without 
departing from its spirit or essential characteristics. The described 
embodiments are to be considered in all respects only as illustrative and 
not restrictive. The scope of the invention was, therefore, indicated by 
the appended claims rather than the foregoing description. All changes 
which come within the meaning and range of equivalency of the claims are 
to be embraced within their scope.