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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4488-4495
Copyright © 2003 by The Endocrine Society

Placental Iodothyronine Deiodinase Expression in Normal and Growth-Restricted Human Pregnancies

S. Chan, S. Kachilele, E. Hobbs, J. N. Bulmer, K. Boelaert, C. J. McCabe, P. M. Driver, A. R. Bradwell, M. Kester, T. J. Visser, J. A. Franklyn and M. D. Kilby

Department of Fetal Medicine, Division of Reproductive and Child Health (S.C., S.K., E.H., M.D.K.), Department of Medical Sciences (K.B., C.J.M., P.M.D., A.R.B., J.A.F.), University of Birmingham, Birmingham, United Kingdom B15 2TG; Department of Pathology, University of Newcastle-upon-Tyne (J.N.B.), Newcastle-upon-Tyne, NE1 4LP United Kingdom; and Department of Internal Medicine III, Erasmus University Medical School (M.K., T.J.V.), 3015 GE Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Prof. Mark Kilby, Department of Fetal Medicine, Division of Reproductive and Child Health, Floor 3, Birmingham Women’s Hospital, University of Birmingham, Edgbaston, Birmingham, United Kingdom B15 2TG. E-mail: m.d.kilby{at}bham.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have described the expression of specific iodothyronine deiodinase mRNAs (using quantitative RT-PCR) and activities in normal human placentas throughout gestation and compared our findings to those in placentas from pregnancies affected by intrauterine growth restriction (IUGR). The predominant deiodinase expressed in placenta was type III (D3); type II (D2) was also present. In general terms, the activities of the enzymes D2 and D3 (and mRNAs encoding these enzymes) were higher earlier in gestation (<28 wk) than at term and displayed an inverse relationship with the duration of gestation (P < 0.05). Comparison of the relative expressions of mRNAs encoding D2 and D3 as well as their activities in placentas associated with IUGR (early and late gestational groups) with findings from normal placentas of similar gestational ages revealed no significant differences. Immunolocalization of D2 and D3 in syncytiotrophoblast (including syncytial sprouts) and cytotrophoblast of human placentas was demonstrated at both early and late gestation. Treatment of primary cultures of term cytotrophoblast cells in vitro with increasing doses of T3 (1, 10, and 100 nM) resulted in increased expression of mRNAs encoding both D2 and D3 at 100-nM concentrations (P < 0.01) compared with control. Experiments with JEG-3 choriocarcinoma cells demonstrated a similar effect on D3 mRNA at 10 and 100 nM T3 (P < 0.01). The demonstrated changes in iodothyronine deiodinase expression in the placenta across pregnancy are likely to contribute to regulation of the thyroid hormone supply to the developing fetus. The lack of difference in deiodinase expression in normal placentas and those found in IUGR argues against placental deiodinases being responsible for the hypothyroxemia in circulating fetal thyroid hormones observed in this condition.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
FETAL GROWTH IS dependent upon a number of endocrine, paracrine, and autocrine events within the fetoplacental unit. Babies born with intrauterine growth restriction (IUGR) are major contributors to both perinatal and neonatal mortality. This pathological process, often associated with malplacentation, also causes significant morbidity, with 10% of low birth weight babies having physical handicap and a further 5% exhibiting chronic neurodevelopment sequalae (1). Thyroid status is one of several factors that have been postulated to play a critical role in the pathogenesis of such morbidity, especially with respect to central nervous system (CNS) development (2, 3). There is increasing support for the view that maternal thyroid hormones cross the placenta into the fetal circulation from early gestation. In thyroid agenesis, the maternal supply of thyroid hormones provides the term fetus with circulating thyroid hormone concentrations that are 25–50% of those in normal infants (4). Furthermore, epidemiological data indicate that subtle maternal hypothyroxinemia in early gestation may have an effect on neurological development (5, 6), and therefore, alterations in transplacental thyroid hormone passage may be of physiological significance. Our own data show that thyroid hormone receptors (TRs) and deiodinases are expressed in the human fetal brain from early gestation, suggesting that the early developing CNS is a thyroid hormone-responsive organ (7).

The placenta is known to be a rich source of the selenocysteine-containing iodothyronine deiodinases (8). There are three subtypes: type I (D1), type II (D2), and type III (D3) (8). D1, which is nonpropylthiouracil (non-PTU) sensitive, catalyzes monodeiodination of T4 to T3 (responsible for 80% of circulating T3 in humans) and is expressed predominantly in kidney, liver, and thyroid (9). D2, which can be inhibited by PTU, is the primary activating enzyme in tissues and locally catalyzes the monodeiodination of T4 to T3 (10). The highest tissue activities of D2 have been reported in the CNS, the anterior pituitary, and brown fat (11, 12). D3, the deactivating enzyme, catalyzes monodeiodination of T4 to rT3 and of T3 to T2 (13); it is present mainly in placenta and to a lesser extent in the CNS (12, 14). It is postulated that the coordinated regulation of all of the deiodinase subtypes is responsible for the local homeostatic modulation of intracellular T3 concentrations (13).

Despite the relatively high D3 activity in placenta, physiologically important amounts of maternal T4 are transferred to the fetus (15, 16). The fetus is particularly dependent upon maternally derived hormones before the onset of endogenous fetal thyroid hormone production at around 14–16 wk gestation (17). Thyroid hormones can be detected in the fetal circulation and embryonic cavities in the early first trimester (16, 18, 19). In the second and third trimesters, circulating thyroid hormones are of both maternal and fetal origin.

Fetal blood sampling by cordocentesis in pregnancies complicated by IUGR has revealed a significant reduction in circulating free (F) T4 and FT3 and a modest elevation in TSH (20, 21). Umbilical cord blood from very low birth weight babies at delivery also revealed significantly reduced total T4 and T3 concentrations, although this may reflect reduced T4-binding globulin concentrations, as free thyroid hormone concentrations were not markedly lower than those in term neonates (22). In addition, transient hypothyroidism, characterized by low total and FT4 concentrations with or without a rise in TSH, frequently develops in small for gestational age and premature neonates (23, 24). Although treatment of permanent or transient primary hypothyroidism associated with a rise in TSH is considered important in terms of long-term neurological outcome, the significance of physiological hypothyroxinemia of prematurity (with no rise in TSH) (22, 24, 25) and the role of postnatal hormone replacement remain more controversial (25, 26, 27).

Although circulating concentrations of free thyroid hormones are major determinants of the cellular uptake of T4 and T3, other factors modulate thyroid hormone action at the tissue level, including prereceptor modulation by deiodinases and expression of TRs in the target tissue. Our own studies have demonstrated reduced expression of TR {alpha}1, {alpha}2, and ß1 isoforms (3) in the CNS of fetuses affected by IUGR, in contrast to the increased expression of all TR isoforms in placentas of these pregnancies (20). These data suggest specific alterations of thyroid hormone responses within fetal tissues in growth-restricted pregnancies. We postulate that changes in placental modulation of the thyroid hormone supply are reflected in changes in placental TR expression and fetal circulating thyroid hormones in IUGR, and hence in fetal thyroid hormone status.

To investigate this, we quantified deiodinase mRNA expression in placental tissue from normal pregnancies across gestation. The expression of D1, D2, and D3 mRNAs and the activities of these enzymes in normal pregnancy were compared with those in gestationally matched, IUGR pregnancies. To investigate the role of the active TR ligand, T3, in regulating the expression of deiodinase isoforms, we also defined in vitro the effect of T3 on D2 and D3 mRNA in primary cultures of cytotrophoblast cells from term placenta and immortalized choriocarcinoma cell lines.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
A total of 109 placental samples were collected with the approval of the local research ethics committee. Early first trimester (6–8 wk gestation; n = 19), late first trimester (9–13 wk gestation; n = 28), and early second trimester (14–20 wk gestation; n = 15) placentas were collected at surgical termination of pregnancy in accordance with the United Kingdom’s Polkinghorne report (28). Placentas from late second trimester (27–28 wk gestation; n = 3) and early third trimester (29–34 wk gestation; n = 6) pregnancies with appropriately grown fetuses for gestational age were obtained at emergency cesarean section for placenta previa (n = 3), maternal tumors (n = 3), and premature rupture of membranes (with no infection) with a breech presentation (n = 3). Term (37–40 wk gestation; n = 20) placentas from uneventful pregnancies were collected at elective cesarean section. Eighteen cases of IUGR (25–38 wk gestation) were diagnosed prospectively (29), each having at least three of the four following characteristics determined from ultrasound scan examination: 1) fetal abdominal circumference at the third percentile or below for gestational age, 2) abnormal fetal growth velocity (maximum increase in abdominal circumference, <1.5 SD over 14 d), 3) severe oligohydramnios (amniotic fluid index <=10th percentile for gestational age), and 4) absent or reversed velocities in umbilical artery Doppler waveforms. These criteria selected a relatively homogenous, but severe, phenotype, all delivered by cesarean section.

Immediately post delivery, full-thickness placental biopsies (in triplicate) were excised from the central (periumbilical) region, which has previously been demonstrated to be representative of the entire placenta. They were washed thoroughly in ice-cold 0.9% saline and fixed in neutral-buffered 10% formal saline or were cut into small pieces (<1 g) and snap-frozen in liquid nitrogen before storage at -80 C.

RNA preparation and RT

Total RNA was extracted from approximately 100 mg tissue after homogenization, using a single-step acid guanidinium phenol-chloroform extraction method (Tri-Reagent, Sigma-Aldrich, Poole, UK) following the manufacturer’s guidelines. One microgram of RNA was reverse transcribed in a total reaction volume of 20 µl, containing 0.25 µg oligo(deoxythymidine), 2 µl 10x avian myeloblastosis virus reverse transcriptase buffer, 10 pmol deoxy-NTP, 10 U ribonuclease inhibitor (RNasin), and 7.5 U avian myeloblastosis virus reverse transcriptase (all reagents from Promega, Madison, WI).

Quantitative PCR

Relative expression of deiodinase subtypes was calculated using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA), which employs TaqMan chemistry (30, 31). Target genes were labeled with the reporter dye FAM. The housekeeping gene 18S was assessed as an internal reference to allow for differences in RT efficiency and was labeled with VIC. Details of the methods and the quantitative primer and probe sequences have been previously reported (7). D1 reactions were single-plexed to avoid potential interference with 18S reactions indicated by preliminary experiments, whereas D2 and D3 were multiplexed. This also controlled for contamination with genomic DNA in RT-PCR of D2 and D3 mRNA.

According to the manufacturer’s guidelines, values were expressed as Ct values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine {Delta}Ct values [{Delta}Ct = Ct of the target gene (e.g. D1) minus Ct of the housekeeping gene], upon which all statistical tests were performed (30, 31).

Iodothyronine deiodinase (D1, D2, and D3) activity assays

The activities of specific deiodinase subtypes were estimated (in a representative group of 89 normal and 14 IUGR samples) using methods previously described (9, 14, 32, 33). The placental samples were homogenized in 10 vol 0.1 M phosphate (pH 7.2), 2 mM EDTA and 1 mM dithiothreitol (P100E2D1 buffer). D1 and D2 activities were determined by measurement of the release of radioiodide from the outer ring-labeled rT3 and T4, respectively, and D3 activity was assayed by HPLC analysis of the formation of radioactive T2 and 3'-iodothyronine from outer ring-labeled T3. Deiodination in the presence of placental homogenate (~1 mg protein/ml) was corrected for nonenzymatic deiodination in the absence of homogenate. The assays are briefly described below (14, 33, 34).

D1 activity assay. Incubations were performed for 30 min at 37 C with 0.1 µM (105 cpm) [3',5'-125I]rT3 in the absence or presence of 0.1 mM PTU, a specific D1 inhibitor, in 0.1 ml P100E2D10 buffer. Deiodinase activity was ascribed to D1 if inhibited by PTU.

D2 activity assay. Incubations were carried out for 120 min at 37 with 1 nM (105 cpm) [3',5'-125I]T4 in the presence of 1 µM T3 to block D3 and in the absence or presence of 100 nM T4 to saturate D2 in 0.1 ml P100E2D25 buffer. Deiodinase activity was ascribed to D2 if inhibited by excess unlabeled T4. HPLC analysis demonstrated that in homogenates with significant D2 activity, labeled T4 was converted under these conditions to equivalent amounts of radioactive T3 and iodide.

D3 activity assay. Incubations were carried out for 60 min at 37 C with 1 nM (2 x 105 cpm) [3'-125I]T3 in the absence or presence of 100 nM T3 to saturate D3 in 0.1 ml P100D2D50 buffer. Deiodinase activity was ascribed to D3 if inhibited by excess unlabeled T3.

All placental samples were assayed in two batches, each time showing similar results. The inter- and intraobserver coefficients of variation using these assays were less than 4% and 2%, respectively.

Immunohistochemistry

The methods have been published previously (35), but are briefly outlined below. Three-micrometer sections of first and third trimester placentas (n = 4 for each trimester) were dewaxed and rehydrated in xylene. Antigen retrieval was performed by pressure-cooking slides for 1 min in citrate buffer, pH 6.0. Sections were then treated with 1% (vol/vol) H2O2 in methanol to block endogenous peroxidase activity. After washing in 0.05 M Tris-buffered 0.15 M saline, pH 7.6 (TBS), the slides were overlain with 10% normal rabbit serum to block nonspecific binding sites, and then incubated for 60 min at room temperature with specific sheep polyclonal antibodies of human D2 and D3, respectively (The Binding Site Bioreagents, Birmingham, UK). Both primary antibodies were diluted 1:200 (vol/vol) in 10% normal human serum. Negative controls were performed for each tissue by replacing the primary antibody with nonimmune serum. Western immunoblotting using these antibodies demonstrated the specificity of the polyclonal antibodies for D2 and D3 protein extracted from trophoblast. Immunoblots demonstrated a single peptide band at 31 kDa (for D2) and 36 kDa (for D3) in accord with previous reports (8, 10, 11, 12).

After three washes in TBS, the sections were incubated for 40 min with peroxidase-conjugated rabbit antisheep immunoglobulins (DAKO, Ely, UK) diluted 1:100 (vol/vol) in TBS. After three additional TBS washes, the reaction was developed in 1 mg/ml 3,3'-diaminobenzidine containing 0.02% H2O2. The reaction was stopped after 5–10 min by immersing sections in tap water. Sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted in DPX (Raymond Lamb, London, UK). Immunostaining of specific placental cell types was assessed qualitatively as the presence or absence of brown reactivity, and D2 and D3 in either nucleus or cytoplasm were recorded. Immunolocalization of D1 was not examined, as no measurable enzyme activity was noted.

Cell culture experiments

The effects of increasing concentrations of T3 on D2 and D3 mRNA expression in primary (term) cytotrophoblast cell cultures and a choriocarcinoma cell line (JEG-3) were studied. Term placentas (38–41 wk) obtained from women undergoing elective cesarean section were collected, and primary placental cytotrophoblast cells were isolated using previously described methods (35, 36, 37). These cells were plated in six-well plates at a density of 4 x 106 cells/well and cultured as previously described (35). In similar experiments choriocarcinoma JEG-3 cells were plated at a density of 3 x 105 cells/well in standard six-well plates and cultured at 37 C and 5% CO2. Primary cytotrophoblast cells were plated at a higher density than the choriocarcinoma cell line to compensate for a greater rate of cell mortality and lower proliferation. JEG-3 cells were passaged in DMEM (Life Technologies, Inc., Paisley, UK). All media contained antibiotics and were supplemented with 10% fetal bovine serum (Life Technologies, Inc.). For 6 h immediately before experimentation, cells were treated with medium supplemented with charcoal-stripped fetal bovine serum (First Link UK Ltd., Midlands, UK), then treated with T3 (0, 1 , 10, or 100 nM; Sigma-Aldrich) for 16 h before total RNA was extracted using the Tri-Reagent extraction method and reversed transcribed using random hexamer primers. RT-PCR was performed as described for the placental tissues (30).

Statistical methods

Data from the real-time RT-PCR were expressed as the mean {Delta}Ct value ± SE for quantification of mRNA within a gestation range for each encoded gene. Data for all variables were noted to not be normally distributed, and nonparametric statistics were used. The differences in mRNA expression and activities between gestations were investigated by the Kruskal-Wallis one-way ANOVA test with post hoc multiple comparisons. Where two sample groups were compared, then unpaired Mann-Whitney U test was used. In addition, Spearman’s rank correlation coefficient ({rho}) was obtained with 95% confidence intervals (CI) for {rho} using Fisher’s z transformation test. Statistical significance for rejection of the null hypothesis was taken at P = 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Normal first, second, and third trimester placental tissue

mRNA expression. Relative expression of mRNA encoding deiodinase subtypes across gestation was calculated with the term group as a reference (arbitrary value of 1).

D2 mRNA expression was significantly higher in the 6–8 wk placentas (20-fold; P < 0.0001), the 9–12 wk placentas (5.7-fold; P < 0.001), and the 13–20 wk placentas (6.2-fold, P < 0.01) compared with term samples (P < 0.001; Fig. 1AGo). However, after 27 wk gestation there was no significant difference in D2 mRNA expression compared with term. When considering each placental sample separately, a significant negative correlation between D2 mRNA expression and gestation was noted ({rho} = 0.53; 95% CI, 0.37 to 0.67; P < 0.0001).



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  		FIG. 1. The relative expression of mRNA encoding iodothyronine deiodinase type II (D2) (A) and type III (D3) (B) in normal human placentae from 6–34 wk gestation compared to term, given an arbitary value of 1. The number of samples expressing mRNA and the mean {Delta}CT values and SEM obtained using quantitative RT-PCR for each group are given in the corresponding columns in the table below each graph. The mean (±SE) D2 (C) and D3 (D) enzyme activity in normal human placentae from 6–34 wk gestation and at term. Statistically significant differences were *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, compared to term (K-W ANOVA D2 mRNA P < 0.0001; D2 activity P < 0.0001; D3 mRNA P < 0.0001, and D3 activity P < 0.05).

 
D3 mRNA expression was significantly increased throughout gestation compared with term samples, being maximally expressed in the 9–12 wk placentas (8.4-fold; P < 0.0001) and the 13–20 wk placentas (7.5-fold; P < 0.0001) before declining toward term values in the third trimester (Fig. 1BGo), when considering each placental sample separately. There was also a significant negative correlation between D3 mRNA expression with gestation ({rho} = 0.33; 95% CI, 0.13–0.50; P < 0.01).

D1 mRNA expression was at the limits of detection, being apparent in only 29% of 6–20 wk, 17% of 29–34 wk placentas, and none of the late second trimester and term placentas. We have therefore not attempted quantification.

Deiodinase activity. Measurable D2 and D3 enzyme activities were noted throughout gestation. Throughout gestation, D3 activity levels were significantly higher than those of D2. In the first trimester the mean D2 and D3 activities were 0.83 and 155.06 fmol/min/mg, respectively, and at term they were 0.14 and 58.60 fmol/min/mg. Assuming equal efficiency of the activity assays for D2 and D3, these values suggest that D3 activity is 187 and 419 times higher than D2 activity in first trimester and term placentas, respectively.

When considering each placental sample separately, D2 activity was significantly positively correlated with D2 mRNA expression ({rho} = -0.41; 95% CI, -0.57 to -0.22; P < 0.0001). In common with mRNA expression, D2 activity showed a significant negative correlation with gestation ({rho} = -0.49; 95% CI, -0.64 to -0.32; P < 0.0001). Compared with term placental samples there were significant increases in D2 activity in the 6–8 wk (P < 0.0001), 9–12 wk (P < 0.001), and 13–20 wk (P < 0.01) placental cohorts (Fig. 1CGo). After this time, no significant difference was noted.

There was a wide variation in D3 activity within each gestational age group. Although there was a significant negative association between D3 activity and gestation ({rho}= -0.24; 95% CI, -0.43 to -0.04; P < 0.05), a significant increase in activity was observed only at 9–12 wk (P < 0.05) and 13–20 wk (P < 0.05) gestation compared with term samples (Fig. 1DGo). Unlike D2, D3 mRNA expression did not significantly correlate with D3 activity ({rho}= -0.016; 95% CI, -0.19 to -0.22).

In all placental samples, D1 activity was below the threshold for assay detection.

Placental tissue from pregnancies complicated by IUGR

For analysis, the IUGR samples were divided into an early and a late gestation group. The early IUGR group (n = 14; 25–32 wk) had a mean birth weight 786 ± 87 g, all complicated by oligohydramnios. and 12 of 14 had absent end-diastolic flow velocity in the umbilical artery. These samples were compared with late second and early third trimester placentas from normal pregnancies (n = 9; 27–34 wk). The late IUGR group (n = 4; 37–38 wk) had a mean birth weight of 2101 ± 139 g, with four of four having both absent end-diastolic flow velocity in the umbilical artery and oligohydramnios. Samples from this cohort were compared with the normal term placentas (n = 20; 37–40 wk).

There were no significant differences in either D2 or D3 mRNAs (Fig. 2Go, A and B) and enzyme activities (Fig. 2Go, C and D) in early or late IUGR placentas compared with normal samples. D2 and D3 mRNAs expression and activities were also similar when the early IUGR group was compared with the late IUGR group.



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  		FIG. 2. The relative expression mRNA encoding iodothyronine deiodinase D2 (A) and D3 (B) in IUGR human placentae (early and late gestational groups) compared to normal placentae of similar gestational ages, given an arbitrary value of 1. The number of samples expressing mRNA and the mean {Delta}CT values and SE obtained using quantitative RT-PCR for each group are given in the corresponding columns in the table below each graph. The mean (±SE) D2 (C) and D3 (D) enzyme activity in the early IUGR human placentae (25–32 wk, n = 14) compared to normal placentae of simlar gestational ages (27–34 wk, n = 8). There were no statistically significant differences observed.

 
D1 mRNA expression and activity were undetectable in all IUGR samples (as in the corresponding normal samples).

Immunohistochemistry

Both D2 and D3 protein were localized to villous syncytiotrophoblast and villous cytotrophoblast layers in the first and third trimesters of pregnancy (Fig. 3Go). D2 was consistently expressed by villous cytotrophoblast in the first trimester, but expression by villous syncytiotrophoblast was variable, weak, and often focal. Cytotrophoblast columns also showed consistent moderate to strong immunostaining (Fig. 3AGo). In contrast, in the first trimester D3 was strongly expressed by villous syncytiotrophoblast and by syncytial sprouts, but the reactivity of villous cytotrophoblast was focal and weak (Fig. 3BGo). Cytotrophoblast columns showed moderate D3 immunostaining, which was less intense than that observed for D2. In third trimester placentas both D2 and D3 immunostaining were expressed by villous syncytiotrophoblast. The reactivity of villous cytotrophoblast for D2 was stronger than that observed for D3 in the third trimester (Fig. 3Go, C and D).



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  		FIG. 3. Immunoreactivity for D2 (A and C) and D3 (B and D) in first trimester (A and B) and term (C and D) placenta. A, In first trimester placenta, D2 shows variable reactivity with syncytiotrophoblast, including syncytial sprouts with more intense cytotrophoblast reactivity. B, Immunoreactivity for D3 is intense and consistent in syncytiotrophoblast, including syncytial sprouts, with weaker reactivity of cytotrophoblast and variable reactivity of the cytotrophoblast cell column. C, At term D2 reactivity in syncytiotrophoblast remains variable, and cytotrophoblast immunostaining persists. D, Syncytiotrophoblast reactivity for D3 persists at term with more variable immunostaining of the residual cytotrophoblast (arrows, syncytiotrophoblast; arrowheads, cytotrophoblast).

 
Expression of iodothyronine deiodinase subtypes in primary cytotrophoblast cultures of and choriocarcinoma JEG-3 cells: effect of T3 treatment

D2 and D3 mRNA were expressed in primary cytotrophoblast cell cultures and JEG-3 choriocarcinoma cells investigated under basal conditions. Significantly higher D2 mRNA expression (170- to 250-fold change) was noted in the primary cytotrophoblast cell cultures compared with the JEG-3 choriocarcinoma cells (P < 0.01). A similar difference in mRNA encoding D3 was also noted (P < 0.0001).

Primary cytotrophoblast cell cultures demonstrated a significant increase in D2 mRNA after treatment with 100 nmol/liter T3 (Fig. 4AGo; P < 0.01). The JEG-3 cell line showed no response in D2 expression with increasing T3 concentrations (Fig. 4BGo).



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  		FIG. 4. The relative expression of mRNA D2 (A and C) and D3 (B and D) in response to increasing doses of T3 (1, 10, 100 mM) in cultures of primary term cytotrophoblast (A and B) and the JEG-3 choriocarcinoma cell line (C and D) compared to 0 nM of T3, given an arbitrary value of 1. Statistically significant differences were **, P < 0.01, ***, P < 0.001 compared to 0 nM of T3 for simplicity. The number of samples expressing mRNA and the mean {Delta}CT values and SE obtained using quantitative RT-PCR for each group are given in the corresponding columns in the table below each graph (K-W ANOVA for D2 in PCT P < 0.001 and JEG-3 P > 0.1. In D3 for PCT P < 0.01 and JEG-3 P < 0.01).

 
A significant increase in D3 mRNA expression was also observed in primary cytotrophoblast cultures at 100 nM T3 compared with no T3 (Fig. 4CGo; P < 0.001) Also, in primary cytotrophoblast cultures there was an increase in mRNA encoding D3 between 1 and 10 nM T3 (P < 0.05). Likewise, increasing concentrations of T3 significantly increased D3 mRNA expression in JEG-3 cells at both 10 and 100 nM T3 (Fig. 4DGo; compared with 0 nMT3, P < 0.01; compared with 1 nM T3, P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study we have described the expression of specific deiodinase mRNAs and activity in normal human placenta throughout gestation and compared these findings to placentas from pregnancies affected by IUGR. We demonstrated that the predominant subtype expressed in human placenta is D3, but D2 is also present. In general, the decreasing expression of D2 and D3 mRNA with increasing gestation is reflected in enzyme activity. The association between D2 mRNA expression and D2 enzyme activity suggests a similar degree of posttranscriptional regulation of this enzyme among placentas throughout gestation. The lack of such an association with D3 expression implies more variation in the degree of posttranscriptional or posttranslational regulation in the synthesis of functional D3 protein in the human placenta. The observation that D2 and D3 enzyme activities decreased with gestation confirms previously published findings (14, 34, 38). Koopdonk-Kool et al. (14) also reported significantly higher overall placental D3 than D2 activities at all gestations. The magnitude of difference between levels of D2 and D3 activity noted in the present study (~200-fold in the first trimester and 400-fold at term) is comparable with the 300-fold difference described previously (14).

Both D2 and D3 protein were localized by immunohistochemistry to the villous syncytiotrophoblast and cytotrophoblast layers in first and third trimester human placentas. The villous syncytiotrophoblast layer, which expressed D3 consistently throughout pregnancy, is in direct contact with maternal blood in the intervillous space and is ideally placed to protect the fetus from excessive maternal thyroid hormone transfer. On the other hand, the relatively undifferentiated villous cytotrophoblast layer (the trophoblast stem cell line) expressed D2 protein more consistently with focal, weak immunoreactivity of syncytiotrophoblast in the first trimester, suggesting a role in supply for the fetoplacental unit.

The observed changes in deiodinase mRNA and activity throughout gestation are likely to influence thyroid hormone delivery to the developing fetus and may reflect changing thyroid hormone requirements. The relatively higher D2 activity in the first half of pregnancy may be important for generating sufficient local (intraplacental) T3 to allow trophoblast development and differentiation (39). In vitro studies have demonstrated that T3 is required to enhance hormone (human chorionic gonadotropin-ß and estradiol) and cytokine production (epidermal growth factor) associated with trophoblast differentiation in first trimester, but not in third trimester, primary cytotrophoblast cell cultures (40). High placental D2 activity at earlier periods of gestation may also have a role in enhancing the supply of T3 to developing fetal thyroid-responsive organs, especially the CNS.

D3 is believed to play a role in protecting the developing fetus from excessive maternal transfer of thyroid hormones as well as being responsible for the additional release of iodide into the fetal circulation for in utero thyroid hormone synthesis (15, 41, 42). As the total placental surface area available for fetomaternal exchange is much lower in the first than the third trimester, a higher level of D3 activity per milligram of protein in early gestation may be required for effective maternal T3 (in)activation to protect the fetus. However, as trophoblast mass increases with gestation, the net (mass) placental D3 activity will be highest in the third trimester (14). This increase in total D3 activity may sustain the increasing fetal iodide requirements for endogenous thyroid hormone production, especially in the final trimester of pregnancy (18, 19), independently of maternal iodide availability (14). Relatively high D3 activity in human placenta also accounts for the high circulating levels of rT3 in the fetus (41, 42). rT3 does not bind to TRs, and a specific role for rT3 in human fetal life has yet to be defined (42).

Our in vitro data indicate that primary cultures of cytotrophoblast cells display increases in mRNA encoding D2 and D3 after treatment with increasing T3 concentrations, but marked changes were observed only at the relatively high T3 concentration of 100 nM. Increases in mRNA encoding D3 was more in keeping with a dose-response to increasing T3 concentrations in JEG-3 cells. It is important to note that these are local T3 concentrations, and local cellular concentrations of T3 may not correlate with circulating FT3 concentrations in the fetus in vivo. We did not measure D2 or D3 enzyme activities in these in vitro data. Our T3 dose-response results generally contrast with those reported by Hidal et al. (43), who found up-regulation of D2 activity during deprivation of T3 and a down-regulation with thyroid hormone repletion in dispersed cells prepared from human placental chorionic-decidual membrane (43). This difference may, however, imply a deiodinase response to T3 at a posttranscriptional level. The lack of correlation between deiodinase mRNA expression and activity is not unusual and has been observed in other tissues, such as the fetal cerebral cortex (7).

In severe IUGR, fetal FT4 and FT3 concentrations are significantly lower than in appropriately grown fetuses, with a modest increase in TSH (20). The early IUGR group has a very high perinatal mortality (57%). This cohort of IUGR pregnancies is likely to represent a relatively homogenous group because of gestational age at diagnosis and the ultrasound features described for diagnosis. Although the fetus is often hypoxic, the placenta is hyperoxemic due to an inability of the fetus to effectively extract oxygen from the maternal/placental circulation (44, 45). It has been postulated that the fetal hypothyroxinemia associated with IUGR may be secondary to the effects of fetal hypoxia on endogenous thyroid hormone secretion or to changes in placental (in)activation of T3 and T4 (46). The effects of oxygen tension on trophoblast deiodinase expression are unknown. However, our data suggest no significant difference in placental mRNA encoding D3 or D2, and there is no difference in enzyme activities to account for the observed fetal hypothyroxinemia associated with IUGR. This is perhaps not surprising, as the in vitro studies demonstrate little change in D2 or D3 mRNA expression with alterations in T3 concentrations in primary cultures of cytotrophoblast. This is further in keeping with the finding that total placental D3 activity in hypothyroid fetuses (lower serum T4, but comparable serum T3) does not differ significantly from that in euthyroid fetuses, suggesting that changes in circulating fetal T4 are not responsible for regulating D3 placental activity (14). Interestingly, maternal T4, in both hypothyroid and hyperthyroid states also does not regulate placental D3 activity (32, 34).

Our previous finding of up-regulation of placental TR {alpha}1, {alpha}2, and ß1 expression in pregnancies complicated by IUGR (20) suggests that there may be an alternative mechanism by which the trophoblast compensates for reduced fetal circulating thyroid hormone concentrations. This may be a homeostatic mechanism that optimizes the action of T3 in the trophoblast. In contrast, in thyroid-responsive tissues such as brain (at least in rodents), fetal hypothyroidism may result in up-regulation of local D2 activity but down-regulation of D3 activity in an attempt to increase local T3 concentrations (47). Furthermore, in the human adult brain, increased D3 activity is associated with decreased local T3 concentrations, suggesting a homeostatic role for D3 in local T3 regulation in this thyroid-responsive tissue (48).


   Acknowledgments
 
We thank Ellen Kaptein and Asha Mangnoesing (Erasmus University Medical School). We also thank Mrs. Barbara Innes (University of Newcastle) for her technical expertise with the immunocytochemistry.


   Footnotes
 
This work was supported by Medical Research Council, United Kingdom; the Wellcome Trust; and the Endowment Fund of the former United Birmingham Hospitals and the Masons Trust.

Abbreviations: CI, Confidence intervals; CNS, central nervous system; D1, deiodinase type I; D2, deiodinase type II; D3, deiodinase type III; FT3, free T3; FT4, free T4; IUGR, intrauterine growth restriction; PTU, propylthiouracil; TBS, Tris-buffered saline; TR, thyroid hormone receptor.

Received February 12, 2003.

Accepted June 16, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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