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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 11 4148-4155
Copyright © 1998 by The Endocrine Society

Original Studies

Ontogeny of Endogenous Secretion of Immunoreactive-Thyrotropin Releasing Hormone by the Human Placenta

Rekha Bajoria and Michael Babawale

Imperial College of Science, Technology and Medicine, Division of Paediatrics, Obstetrics and Gynaecology, Institute of Obstetrics & Gynaecology, Hammersmith Hospital, Du Cane Road, London, United Kingdom

Address correspondence and requests for reprints to: Dr. Rekha Bajoria, Department of Obstetrics & Gynaecology, Research Floor, St. Mary’s Hospital, Whitworth Park, Manchester M13 0JH, United Kingdom. E-mail: rekha.bajoria{at}man.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied endogenous production of immunologically active TRH (ir-TRH) by human placenta throughout gestation. Fragments (20 g) of placentae obtained between 7 and 41 weeks’ gestation were incubated in TC-199 media with or without TRH degrading enzyme inhibitors (1 mM of dithiothreitol, 200 µm O-phenanthroline, 0.8 mM EDTA) at 37 C for 24 h. TRH was quantitated by RIA. Release of ir-TRH was also studied in an in vitro model of dually-perfused isolated lobule of human term placenta. Cellular localization of TRH was performed by staining early-, mid- and late-gestation placentae with anti-TRH rabbit polyclonal antibody, using the indirect avidin-biotin complex immunoperoxidase method.

TRH was produced by placental fragments from 7 to 41 weeks’ gestation. Placental TRH secretion was maximal between 7–12 weeks gestation both in presence (655 ± 79 pg/10 mg protein) and absence (423 ± 75 pg/10 mg protein) of enzyme inhibitors. Secretion of TRH declined with increasing gestation both with (y = 779 - 15x; r = 0.90; P < 0.001; n = 15) and without (y = 525 - 12x; r = 0.87; P < 0.001; n = 15) enzyme inhibitors. In the perfusion experiments, endogenous TRH was released predominantly into the fetal circulation, and its concentration was markedly higher in the presence of enzyme inhibitors (146 ± 27 vs. 34 ± 7 pg/mL; P < 0.001). Immunostaining of chorionic villi localized TRH to the cytoplasm of the syncytial layer, and the intensity declined with advancing gestational age. These data suggest that immunoactive TRH is produced by human placenta begin at 7 weeks gestation, and production declines with gestational age.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RESPIRATORY distress syndrome (RDS) as a consequence of prematurity is a major cause of perinatal mortality and morbidity (1). Combination therapy with glucocorticoids and TRH has been shown effective in reducing the incidence and severity of RDS (2, 3, 4). However, the mechanism by which TRH enhances lung maturity remains unclear. It has generally been accepted that TRH crosses the placenta and stimulates the fetal pituitary-thyroid axis to produce thyroid end hormones, which then enhance surfactant production by the fetal lung (5). Given the recent finding that the human term placenta forms an enzymatic barrier to free passage of TRH (6, 7, 8), the mechanism by which maternal TRH administration in vivo leads to fetal TRH response remains unclear. In an attempt to elucidate the mechanism by which maternally administered TRH stimulates the fetal thyroid axis, we have shown that maternal TRH (9) and thyroxine (10) do not cross human term placenta in significant quantity. Animal experiments (11) and our data (8) suggest that fetal hypothalamic-pituitary-thyroid axis is independent of maternal thyroid hormones.

Another plausible route by which maternally administered TRH elicits fetal thyrotropic response could be the release of a TRH-like-substance from the placenta. High TRH concentrations are present in the fetal circulation at a time when a hypothalamic portal vascular system has not developed (12, 13). This evidence suggests that fetal pituitary TRH release is under extrahypothalamic TRH control. Elevated levels of immunoreactive (ir)-TRH have also been found in the fetal extrahypothalamic tissues such as brain, placenta, pancreas, and gastrointestinal tract (14, 15). Hypothalamic ablation, pancreatectomy, or administration of TRH antiserum to rat fetuses fails to alter serum TRH concentration (11, 16), indicating that the fetal pituitary TRH is likely to be regulated by the placental TRH (17). TRH is present in the human term placenta (18), and its concentration in rat placenta increases with gestational age (17). However, no information is available on the ontogeny of TRH regulatory dynamics in the human placenta. This information is pertinent before experiments are undertaken to determine whether exogenous TRH, by competing for degrading enzymes can regulate the placental secretion of ir-TRH. Two main pathways for the enzymatic degradation of TRH have been characterized. Pyroglutamyl peptidase 1, a cytosolic enzyme, actively degrades TRH to produce His-Pro-NH2, while type 2 membrane-bound enzyme causes breakdown of the molecule at p-Glu His bound. In addition, nonspecfic dipeptidase enzyme may also cause hydrolysis of TRH (19).

This study aims to evaluate ontogeny of placental production of TRH and its cellular distribution in the human placenta by using previously validated radioimmunoassay and immunocytochemistry methods (18, 20). Based on our recent data that TRH is actively metabolized by type 2 pyroglutamyl aminopeptidases (6, 7, 8), we also evaluated the effect of enzyme inhibitors such as EDTA, dithiothreitol (DTT) and o-phenanthroline on placental release of TRH (21, 22). We also made an attempt to determine whether TRH is released predominantly into the maternal or fetal circulation of the dually perfused isolated lobule of human term placenta.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TRH antibody raised in the rabbit for immunocytochemistry was obtained from Biogenesis. Creatinine and TC-199 perfusion media were purchased from Sigma Chemicals (Poole, Dorset, UK). All other reagents and chemicals were of analytical grade and obtained from BDH (Leicestershire, UK).

Production of endogenous TRH by placental fragments

First trimester human placentae between 7 and 12 weeks were obtained from women undergoing legal abortions (n = 5). Placentae were also obtained between 24 and 42 week’s gestation (five each for mid-trimester and term pregnancies) after normal vaginal deliveries. Placental tissues were collected for research purpose after obtaining oral consent from the mother’s, as required by hospital ethics committee. Tissues were collected from those subjects who were not on any medication and were euthyroid. Gestational age was determined in all cases by ultrasound scan. From each placenta fifteen to twenty 1 cm3 pieces of tissue were removed at random and placed in cold phosphate-buffered saline (PBS) containing 100 units/mL penicillin, 100 µg/mL streptomycin and 100 units/mL nystatin. The tissue samples were washed several times with PBS to remove as much blood as possible. The placental tissues were teased into small pieces, carefully removing any obvious blood vessels or clots. This tissue was rewashed several times with PBS. Approximately 15 mL of settled tissue was made up to 30 mL with TC-199 containing 15% fetal calf serum, 1% penicillin-streptomycin, 10 mM HEPES, with or without TRH degrading enzyme inhibitors (1 mM dithiothreitol, 200 µmol O-phenanthroline, 0.8 mM EDTA). This suspension was swirled to provide a homogenous preparation before incubation.) Then, 2 mL aliquots of this suspension was incubated in a 12-well culture plate at 37 C under 95% O2 and 5% CO2. At each sample point, an aliquot of tissue suspension heated to 60 C was included as a control. Tissue viability during incubation was assessed by measuring ß-HCG and lactate dehydrogenase (LDH) levels in the culture media.

Experimental protocol. Five experiments each were undertaken using placentae obtained from early (7–12 weeks), mid-trimester (24–32 weeks) and term (37–41 weeks) pregnancies. Placental tissues were incubated in duplicate for 24 h. TRH concentration was determined by collecting samples every two hours. At each sample point, the reaction was stopped by adding 2 mL ice cold methanol. To measure the amount of TRH secreted by the placenta, samples were centrifuged at 3,000 g for 15 min. The supernatant was decanted, and the pellet was discarded. Supernatant was then evaporated to dryness. The TRH extract was then resuspended in 1 mL TRH assay buffer and samples stored at -70 C until assayed. To elucidate whether placental metabolism of TRH might influence endogenous secretion, at each sample point an aliquot of placental tissue fragments were also incubated with aminopeptidase enzyme inhibitors (1 mM dithiothreitol, 200 µm O-phenanthroline, 0.8 mM EDTA) (21, 22). We only measured TRH secreted by the placenta, and no attempt was made to determine its tissue concentration.

Secretion of endogenous TRH by the perfused placenta

Placentae were obtained immediately following vaginal delivery from normal pregnancies of 37 to 42 weeks’ gestation. A closed circuit maternal and fetal perfusion of the isolated lobule of the placenta was established. Feto-placental perfusion was established within 5 min under optimal physiological conditions by cannulating the chorionic artery and vein as described previously (23). Closed-circuit materno-placental circulation was established by placing five cannulae in the intervillous space. Autologous maternal blood from the intervillous space and fetal blood from the cord vessels was collected and centrifuged, and the blood cells were washed and resuspended in the modified TC-199 media containing bovine albumin (0.5 g/L) to form the maternal and fetal perfusates (6, 7). The fetal and maternal perfusion pressure and venous outflow were monitored continuously throughout the experiments by an on-line pressure transducer and flow probe. The fetal perfusion pressure was 24–30 mm Hg with a venous outflow of 8–10 mL/min-1, while maternal arterial pressure and flow rate were 10–12 mm Hg and 20–24 mL/min-1 respectively. Maternal and fetal perfusates had mean hematocrit values of 7.6 (range 4–12) and 18 (range 15–25), respectively. Initially during establishment of perfusion, both maternal and fetal circuits were oxygenated. Thereafter, tissue oxygenation was maintained by oxygenating the maternal circulation with 95% oxygen and 5% carbon-dioxide. Perfusion efficiency and diffusional transfer rate was determined in each experiment by measuring the rate of transplacental transfer of a freely diffusable marker, creatinine (MW 113). Experiments were discarded if (a) fetal perfusion pressure increased by more than 10 mm Hg; (b) there was a shift in fetal circulating volume across the placental membrane in excess of 5 mL, (c) fetal circulating volume dropped by more than 2–3 mL; and (c) maternal-to-fetal transfer of creatinine was not linear or greater than predefined range as described previously (23).

In a further five perfusion experiments we also added 200 µmol dithiothreitol, 1 mM o-phenanthroline and 0.8 mM EDTA to the perfusate, which was used to resuspend washed maternal and fetal blood cells.

Maternal and fetal samples were collected at 30 min intervals over 4 h. At the end of the perfusion period, both circuits were drained and their volumes measured. The perfused lobule was then excised and pressure blotted to remove the perfusate from the intervillous space. Samples were centrifuged at 3,000 g for 15 min and plasma stored at -70 C for subsequent analysis. Maternal and fetal concentrations of TRH were expressed in pg/mL.

Immunolocalization of endogenous TRH

Samples of chorionic villi (1 mm3) obtained from human first trimester, midtrimester, and term placentae were fixed in 10% formalin for 24 h and processed for histological analysis. The wax-embedded tissue blocks were sectioned at 4 µm and plated on poly-L-lysine coated slides. Immunostaining was carried out with anti-TRH rabbit polyoclonal antibody using the indirect avidin-biotin complex (ABC) immunoperoxidase method. Placental tissue sections were deparaffinized and rehydrated through graded alcohol and water, after which endogenous peroxidase activity was blocked by placing the slides in 3% H2O2 in tap water for 30 min at room temperature. The slides were then rinsed in PBS, pH 7.2 for 5 min. Nonspecific binding was thereafter blocked with 2% normal swine serum for 15 min at room temperature. The tissue sections were then incubated with anti-TRH antibody at 1/100 dilution overnight at 4 C. Adjacent tissue sections incubated with normal rabbit serum (1/100) served as negative controls. After incubation, the slides were rinsed in 3 x 5 min changes of PBS and incubated with biotinylated swine anti-rabbit IgG at 1/500 dilution, at room temperature for 45 min. After further 3 x 5 min rinses in PBS, the tissue sections were incubated with peroxidase labelled streptavidin, at 1/500 dilution for 45 min at room temperature. Final rinse of 3 x 5 minutes changes of PBS was carried out. Peroxidase activity was then demonstrated by developing the sections in 0.05% 3,3'-diaminobenzidine solution (DAB), with 0.03% H2O2 as substrate for 10 min at room temperature and rinsed in water. After checking the DAB development microscopically, the tissue sections were counterstained in Mayer’s hematoxylin for 15 sec and rinsed in water. The slides were then dehydrated in ascending concentrations of alcohol, mounted on coverslips with histomount, and allowed to air dry before microscopic examination.

Specificity of placental TRH

Column chromatography. To compare the molecular size of the TRH produced by the placenta with that of synthetic TRH, 100 µL of the samples containing 100 pg was applied to a sephadex G-10 column calibrated with 0.05 M phosphate buffer at pH 7.4. Immunoreactive TRH was measured in 1 mL fractions of the column elute.

Degradation of placental TRH by maternal serum. 100 pg of the placenta TRH or synthetic TRH was incubated in 0.5 mL of fresh maternal serum for 30, 60, 90, and 120 min at 37 C in a water bath. At each sample point, 1 mL ice cold methanol was added, and samples were centrifuged. The supernatant was dried under N2 and resuspended in 0.5 mL phosphate buffer for RIA for TRH.

Analytical methods

Radioimmunoassay for TRH. The concentration of TRH in maternal, fetal, and placental tissue was quantified by using an antisera raised in male New Zealand white rabbits against synthetic TRH conjugate prepared by the bisdiazotised benzidine method (7) and was obtained as gift from MRC unit for Reproductive Medicine, Edinburgh UK. Briefly, the assay buffer was 0.04 mol/L disodium phosphate/monosodium phosphate, pH 7.4, containing 1% (wt/vol) human serum albumin. 125I TRH was obtained from DuPoint (Stevenage, Hertfordshire, UK) with specific activity 4104 µCi/µg-1 and more than 99% pure on reverse phase high performance liquid chromatography. The final dilution of the antisera used in RIA was 1:5,000, while 100 µL of the sample was incubated with 100 µL antisera and 100 µL radiolabelled TRH (12,000 cpm 0.1 mL-1) overnight at 4 C. The bound-from-free fraction was separated by adding 5 volumes of ice cold ethanol followed by immediate mixing and centrifugation at 1500 rpm x 15 min. Under these RIA conditions the lower limit of detection was 10 pg in a 300 µl ethanol extract. Cross reaction of the TRH analogue, His-pro-NH2, p-Glu-pro, p-Glu-His, p-Glu-His-Pro, pGlu-Glu-Pro-NH2, pGlu-His-Pro-Gly, and noncyclizied TRH with anti-TRH antibody was consistently less than 0.001% to less than 0.0001%. The mean recovery of nonradioactive TRH from serum and tissue extract over the concentration range of 10–5000 pg was more than 95%. The mean coefficients of variation for different TRH concentrations in sera and tissue extract were 2.5–12.7%. The mean interassay coefficient of variation of TRH was 10%.

Creatinine assay. Creatinine concentration was determined by colorimetric assays (6, 7), with a coefficient of variation of 7–12%.

Protein estimation. The concentration of protein was measured spectrophotometrically by the method of Lowry et al. (24) using bovine serum albumin as a standard.

Data analysis

All values were expressed as mean ± SEM. Data between two groups as a function of time were compared by two-way ANOVA. One-way ANOVA was used to compare blocked variables between groups. P values of less than 0.05 were considered significant. Area under the curve (AUC) concentration of TRH was calculated by the trapezoidal rule (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Secretion of TRH by the placental slices

TRH was produced by the placenta throughout gestation. The secretion of TRH by first trimester placenta (7–12 weeks; n = 5) increased exponentially from undetectable levels to 423 ± 75 pg/10 mg protein at 24 hr with AUC of 3.2 ± 0.6 ng/mL/hr (Fig. 1AGo). When placental fragments were coincubated with enzyme inhibitors, concentration of TRH (655 ± 79 pg/10 mg protein; P < 0.01), AUC (5.0 ± 0.9 ng/mL/hr; P < 0.05), increased significantly compared with when no inhibitors were used. The secretion of ß-HCG (1201 ± 87 vs. 1159 ± 151 IU/L) and LDH (7.9 ± 1.2 vs. 8.6 ± 1.1 IU/L) by the placental fragments was comparable in both groups.



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  		Figure 1. Secretion of TRH (A) by the first trimester; (B) mid-trimester; and (C) third trimester placental fragments during incubation at 37°C with ({circ}) or without enzyme inhibitors (•). The statistically significant values at each time point with or without enzyme inhibitors are indicated by asterix. Fig 1DGo Compares the TRH concentration between early ({blacksquare}) mid () and term placentae () with or without enzyme inhibitors at 24 hr. Data were obtained from five experiments in each group.

 
The secretion of TRH by second trimester placenta (24–32 weeks; n = 5) increased from 5.5 ± 1.2 pg/10 mg protein at 0 min to 172 ± 43.0 pg/10 mg protein at 24 h, with AUC of 1.1 ± 0.2 ng/mL/hr. When placental tissue was coincubated with enzyme inhibitors, then the TRH concentration (339 ± 61.4 pg/10 mg protein; P < 0.001) and the AUC (2.6 ± 0.4 ng/mL/hr; P < 0.001) at 24 hr were significantly higher than when incubation was carried out without inhibitors (Fig. 1BGo). The secretion of TRH by second trimester placentae with (P < 0.001) or without enzyme inhibitors (P < 0.001) was less than in the first trimester placenta. The ß-HCG (654 ± 52 vs. 714 ± 79 IU/L) and LDH (7.6 ± 0.4 vs. 10.1 ± 0.5 IU/L) levels were comparable with first trimester placenta.

The secretion of TRH by third trimester placenta at 24 h (65.4 ± 12.6 vs. 193 ± 32.6 pg/10 mg protein; P < 0.01), AUC (0.7 ± 0.8 vs. 1.7 ± 0.2 ng/mL/hr; P < 0.01), was markedly increased in the presence of enzyme inhibitor (Fig. 1CGo). Concentration of ß-HCG (852 ± 76 vs. 743 ± 101 IU/L) and LDH (7.4 ± 0.3 vs. 9.5 ± 0.7 IU/L) levels were comparable with first and second trimester with or without inhibitors. The secretion of TRH by third trimester placentae with or without enzyme inhibitors was significantly less than the first (P < 0.001) and second trimester group (P < 0.001) (Fig. 1DGo). When placental tissue was boiled, TRH remained undetectable (data not shown).

The placental release of TRH at 24 h (y = 524.7 - 11.8x; r = 0.87; P < 0.001) and AUC (3625 - 75x; r = 0.93; P < 0.001) declined rapidly with advancing gestational age (Fig. 2AGo, B). A similar relationship between concentration of TRH (y = 779 - 15x; r = 0.90; P < 0.001) and AUC (y = 6310 - 123x; r = 0.92; P < 0.001) was found in the presence of enzyme inhibitors (Fig. 2CGo, D).



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  		Figure 2. Relationship between placental production of TRH and gestational age. Top panel shows correlation between gestational age and (A) TRH levels at 24 hr (524.7 - 11.8x; r = 0.87; P < 0.001); (B) area under the curve (3625 - 75x; r = 0.93; P < 0.001; n = 15), when placental tissue were incubated without inhibitors. The bottom panel shows effect of enzyme inhibitor on correlation between gestational age and (C) TRH levels at 24 hr (779 - 15x; r = 0.90; P < 0.001; n = 15); or (D) area under the curve (6310 - 123x; r = 0.92; P < 0.001; n = 15).

 
Secretion of TRH by the perfused placenta

During placental perfusion, TRH concentration in the fetal circulation increased from undetectable levels at 0 time to 34.4 ± 7.0 pg/mL at 4 h, with AUC of 5.25 ± 1.0 ng/mL/hr. TRH was undetectable in the maternal circulation. However, when enzyme inhibitors were added to the perfusate, then fetal concentration increased linearly to 146.1 ± 26.9 pg/mL at 4 h (P < 0.001), with AUC of 21.8 ± 4.0 ng/mL/hr (P < 0.001). The maternal concentration 19.4 ± 4.0 pg/mL and the AUC of TRH 2.2 ± 0.3 ng/mL/hr were significantly less than the fetal levels (Fig. 3Go). Concentrations of ß-HCG were comparable in groups with or without enzyme inhibitors, in both maternal (8845 ± 1023 vs. 7688 ± 1051 IU/L) and fetal circulations (72 ± 12 vs. 98.4 ± 14.8 IU/L).



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  		Figure 3. Release of placental TRH during perfusion of term placentae in (A) maternal and (B) fetal circulation. with ({circ}) or without (•) enzyme inhibitors.

 
Immunolocalization of TRH. In the first trimester placenta, TRH immunostaining was positive, intense, and localized to the covering syncytial layer (+++), whilst the underlying layer of cytotrophoblast cells failed to show any immunoreactivity (Fig. 4AGo). Although the second trimester placentae also showed positive TRH immunostaining (Fig. 4BGo), this was notably less intense than in the first trimester (+); however, positive TRH immunostaining was also detected in the fetal and maternal blood vessels musculature (Fig. 4CGo and D) as well as in the extravillous trophoblast cells (interstitial trophoblast) that had invaded the maternal decidua (Fig. 4CGo). In the third trimester placenta, TRH immunolocalization was no longer detectable in the syncytial layer (Fig. 4DGo) whereas the fetal and maternal vascular musculature (Fig. 4CGo and D) and the invasive extravillous trophoblast cells in the maternal decidual tissue continued to show intense TRH immunostaining (Fig. 4EGo). TRH immunoreactivity was not detected in any of the tissue sections, which served as negative control (Fig. 4FGo).



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  		Figure 4. A, Photomicrograph of first trimester placental villi (PV) showing intense TRH immunostaining in the syncytiotrophoblast layer (arrowheads) with absence of immunoreactivity in the cytotrophoblast cells (arrows). The negative control is shown as an insert. Magnification x400; (B) shows decreased TRH immunostaining in the syncytial layer (arrowheads) in the second trimester of pregnancy, magnification x200; (C) shows positive TRH immunostaining in the vascular musculature (arrowheads) surrounding the maternal vessel (BV) and also in extravillous trophoblast cells (arrows) of the second trimester of pregnancy, magnification x100; (D) photomicrograph of the third trimester placental villi (PV) showing positive TRH immunostaining in the vascular musculature (aterixs) surrounding the fetal blood vessel (BV). Note lack of TRH immunostaining in the syncytial layer (arrowhead), magnification x200; E, shows positive TRH immunostaining in extravillous trophoblast cells (arrows) that had invaded the maternal decidua (D) in the third trimester of pregnancy, magnification x200; F, negative control, shows lack of immunoreactivity in the syncytial layer (arrowheads) and in the maternal decidua (D) when non-immune serum was substituted for anti-TRH during immunostaining, Magnification x100.

 
Characterization of human placental TRH activity. The elution profiles of placental and synthetic TRH on a Sephadex G-10 column were identical. The ir-TRH was detectable in both groups by RIA in fractions numbering 25 to 35. Furthermore, the inhibition of binding of antibody and tracer in TRH RIA was increasing concentration of synthetic (y = 75.8 - 0.11x; r = 0.81) and placental TRH (y = 63.31 - 0.100x; r = 0.87) were comparable (Fig. 5AGo and B). Degradation of placental TRH when incubated in maternal serum for 2 h was similar to the synthetic TRH (Fig. 5CGo).



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  		Figure 5. Characteristics of placental TRH (A) a dilution curves of synthetic (•) and placental TRH ({circ}); (B) chromatography of synthetic (•) and placental TRH ({circ}) on sephadex G-10 column; (C) degradation of synthetic (•) and placental TRH ({circ}) by maternal serum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that ir-TRH is secreted by the human placenta from seven weeks gestation, thereafter declining rapidly with increased gestational age. Our data also show that ir-TRH is released predominantly in the fetal circulation and is present within the cytoplasm of the syncytiotrophoblast cells. Placental TRH dilutes in parallel with synthetic TRH standards in the RIA and coelutes with synthetic TRH on column chromatography. This observation indicates that immunoreactivity and molecular size of the placental TRH are similar to those derived from the hypothalamic source. In addition, incubation studies suggest that immunological activity of synthetic and placental TRH are comparable because of their similar degradation rates in maternal sera. It is therefore possible that immunoreactivity of placental TRH is similar to those of fetal pancreas (20) and stomach (25) and may be responsible for high circulating fetal TRH levels (26).

Although studies have shown the presence of TRH in the extra hypothalamic region of the human fetal central nervous system (14, 15), limited information is available on placental origin of TRH. Although one study suggests presence of ir-TRH in the human term placenta (18), it fails to provide comprehensive information necessary to understand its clinical significance. Therefore the current study focuses on the ontogeny, localization, and release of placental TRH in an in vitro model of placental fragments in culture. The recent literature suggest that TRH is metabolised by the placental amino-peptidase (6, 7). Therefore we also studied the effect of TRH degrading enzyme inhibitors on the secretion of placental TRH. Some studies have proposed that thyroid status can influence TRH levels in the hypothalamus (27), placenta (28), and peripheral blood (29) either by modulating the activity of degrading enzyme or by altering levels of pro-TRH and messenger RNA (30, 31). Therefore, placental tissues were obtained from subjects who had no thyroid disorders and, to maintain thyroid homeostasis during the experimental period, physiological concentrations of thyroxine were added to the culture media. The time-dependent exponential rise of TRH in culture media together with lack of release from the controls (tissue heated to 60 C) strongly suggests active production by the placenta and rules out the possibility that it was due to contamination with maternal or cord blood. The observed linear increase in ß-hCG level, without concomitant change in LDH by the placental tissue further suggests that the functional viability was preserved throughout the experiment.

Ontogenesis of placental production of TRH was similar to that reported for human fetal pancreas, where TRH levels were maximal during first trimester and declined rapidly with gestational age (20). These findings contradict observations made in rat and ovine fetuses where placental production of TRH either increased progressively with gestational age or remained unaltered (18, 28). It is unlikely that the difference in ontogeny of TRH between human and other species could be attributed to the specificity of antibodies used to quantitate TRH, as we used a specific antibody with 100% cross-reactivity for TRH and minimal reactivity for other structurally related peptides. Furthermore using similar antibodies, we have shown that transmembraneous passage of nonlabeled TRH (estimated by RIA) is comparable to that of 125I TRH (32). Another possible explanation could be the differences in the ontogeny of TRH degrading enzymes in the various species. In rat no correlation was found between concentration of TRH degrading enzyme activity and tissue levels of TRH (33). Although no information is available on the ontogeny of TRH degrading enzyme activity in the human placenta, our data suggest that, regardless of gestational age, placental production of TRH in the presence of enzyme inhibitor was significantly higher than when experiments were done without inhibitors. Furthermore, as this effect was more marked in the third trimester, it is possible that decreased placental production of TRH at term also could be due to increased activity of enzymes responsible for TRH degradation.

We studied production of TRH by the perfused placenta. Although human fetal circulation is now accessible through invasive techniques, in vivo studies have failed to provide information on placental endocrine function. The placental perfusion model, which has been extensively validated by one of us (R.B.), provided a noninvasive method to evaluate whether TRH is released into maternal or fetal circulations under physiological conditions. As our recent findings suggest that TRH is rapidly degraded by maternal and cord blood, nonblood based media containing washed autologous maternal and fetal cells with physiological concentrations of T4 was used as a perfusate.

Our data suggest that ir-TRH is released predominantly into the fetal compartment, and this accords with our observation that the concentration of TRH in the fetal circulation is significantly higher than maternal levels (8). Given the evidence that TRH was mainly localized in the cytoplasm of the syncytiotrophoblast cells, one would have expected a higher level in the maternal circulation simply because of the close proximity of syncytiotrophoblast to the maternal blood. Accordingly, hormones such as ß-HCG (23) and CRF (34), which are primarily localized in the syncytiotrophoblast, are also released predominantly in the maternal circulation. It is possible that initially TRH is released in both maternal and fetal circulations because of its low molecular weight and the bipolar nature of the syncytial cells. But after its release, TRH is rapidly metabolized in the maternal circulation by the aminopeptidase enzymes that are predominantly present on the microvillous membrane. In keeping with this proposition, maternal concentrations of TRH were higher when enzyme inhibitors were added to the perfusate.

The placental production of TRH appears to be temporally regulated, as TRH expression in syncytiotrophoblast layer decreases with increasing gestational age. Immunostaining data also indicate for the first time that a) a switch in the phenotype of trophoblast cells expressing TRH occurs from the syncytiotrophoblast cells in the floating placental villi in the first trimester to differentiated cytotrophoblast cells (extravillous trophoblast cells) in maternal decidua in the second and third trimesters of pregnancy, and b) localization of TRH in the muscular layer of fetal and maternal blood vessel. The significance of this remains unclear. We contemplate that TRH may have a role in human placentation and angiogenesis. However, this remains to be determined by further studies. It is possible that TRH like other hypothalamic hormones (GnRH, CRF) (35), may regulate placental production of hormones like ß-HCG, and/or TSH-like substances. However, further studies are necessary to elucidate factors that control the secretion of TRH by the placenta. We believe that this may shed light on the mechanism of control of maternally administered TRH on neuroendocrine function of the fetal thyroid.

In conclusion, this study suggests that TRH is produced by the placenta in the early human pregnancy, and its concentration declines rapidly with increasing gestational age.


   Acknowledgments
 
We are grateful to Dr. H. Fraser, MRC unit for Reproductive Medicine, Edinburgh, UK, for providing rabbit anti-TRH antibody.

Received October 24, 1997.

Revised July 2, 1998.

Accepted July 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Office of Population Censuses and Surveys. 1992 Birth statistics. Review of the Registrar General on births and patterns of family building in England and Wales. London: HMSO.
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