Original Paper Gynecol Obstet Invest 1992:34:164-170

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Department of Obstetrics and Gynecology. Section of Reproductive Endocrinology and Infertility. University of Chicago. III., USA; Universitatsfrauenklinik. Frankfurt am Main. FRG: Universitatsfrauenklinik. Wur/.burg. FRG; Department of Obstetrics and Gynecology. Section of Maternal-Fetal Medicine, University of Chicago. 111.. USA

Suppression of Human Chorionic Gonadotropin in the Human Placenta at Term by Human Thyroid-Stimulating Hormone in vitro

Keyw ords

Abstract

Human thyroid-stimulating hormone Human chorionic gonadotropin Placenta

Human chorionic gonadotropin (hCG) exerts a clinically apparent negative feedback on the secretion of human thyroid-stimulating hormone (hTSH) in pregnancy, and the two have cross-reactivity for the TSH receptor in mem­ brane preparations of the thyroid. We examined whether hTSH. in turn, has an influence on the secretion and synthesis of hCG in short-term cultures of human placenta at term. A dose- and time-dependent decrease in the extracel­ lular hCG concentration caused by hTSH was demonstrated. To examine whether hTSH inhibits de novo synthesis of hCG or decreases hCG depletion, we determined the amount of hCG secreted and the size of the intracellular pool by using an enzyme immunoassay. By incorporating a radiolabeled amino acid in the hCG molecule, we measured the amount of hCG synthe­ sized de novo. We concluded that hTSH acts by decreasing the rate of de novo synthesis of placental hCG.

The possible mechanisms by which the placental pro­ duction of protein hormones, especially human chorionic gonadotropin (hCG), is regulated and influenced are intriguing because of their significance for the outcome of pregnancy. In placental expiants or cell cultures, hCG release is increased by luteinizing hormone releasing hor­ mone [1,2], cyclic AMP [3] and epidermal growth factor [4]. No increase has been reported with insulin, epineph­ rine or prostaglandins [5,6]. However, physiologic factors that regulate the synthesis and release of hCG in vivo are

still unknown [7], The site of hCG production in the pla­ centa has been identified by various methods: immuno­ histochemical staining [8. 9] and biochemical detection [26]. It is therefore believed that hCG is secreted by the syncytiotrophoblasts. In 1974. Nisula and Ketelslegers [10] demonstrated that the hCG molecule possesses thyroid-stimulating ac­ tivity, which seems to be species specific [II]. Molecular studies revealed that partially desialylated variants of hCG inhibited the response of human thyroid mem­ branes to human thyroid-stimulating hormone (hTSH) by interaction of hCG with a specific hTSH membrane

M.W.B. was supported by a fellowship (Be 1215/1-1) from ihe ‘Deutsche Forschungsgemeinschaft“and in part by the CLIH-Mothcrs’ Aid Research Fund, and W.W. by a fellow­ ship (Wu 166/1-1) from the ‘Deutsche Forschungsgemein­ schaft*.

Dr. Matthias Beckmann Frauenklinik der Johann-Wolfgang-Gocthc-Universität Abteilung Gynäkologie und Gynäkologische Onkologie Thcodor-Stern-Kai 7 D-W-6000 Frankfurt am Main 70(FRG)

Introduction

Received: October 18.1991 Accepted after revision: March 13. 1992

© 1992 S. Karger AG. Basel 0378-7346/92/0343-0164 $2.75/0

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Matthias W. Beckmann Wolfgang Wiirfel Robert J. Austin Vila Linkc Peter J. Albert

Materials and Methods Materials McCoy’s 5A medium was obtained from Gibco Laboratories (Grand Island, N.Y., USA). Penicillin-streptomycin and L-glutamine were obtained from Gibco Laboratories. Ficoll type 400 (Histopaque®), bovine thyroid-stimulating hormone (bTSH), and hTRH were obtained from Sigma (St. Louis, Mo., USA). hTSH was pur­ chased from Calbiochem (La Jolla. Calif., USA). 35S-mcthionine was obtained from Amcrsham (Arlington Heights. 111., USA). The count­ ing cocktail used was a product of Research Products International (Mount Prospect, 111.. USA). Methods Collection and Preparation o f Human Placenta at Term. Placental tissue derived from 25 women with normal pregnancies at 37-40 weeks of gestation. After uncomplicated, spontaneous vaginal deliv­ ery' (without medication), the complete placenta was extensively washed and then precooled (+4 °C) in McCoy's 5A medium contain­ ing 4 mM L-glutamine, 100 U/rnl penicillin and 100 gg/ml strepto­ mycin (referred to below as the medium). Each placenta was stored at +4 °C prior to individual aseptic preparation within the next 3 h.

The human placental tissue was prepared mechanically as de­ scribed by Mathialagan and Rao [2] with the addition of a Percoll separation step [24]. We did not use the cellular isolation technique of enzymatic digestion as proposed by Zeitleret al. [25] and Kliman et al. [9] because we wanted to avoid degradation of cell surface pro­ teins. especially placental surface receptors, such as luteinizing hor­ mone (LH) CG or a possible TSH receptor [26]. The decidual membranes were removed. The superficial layers from the cotyledonary villi were scraped off. pooled, resuspended and passed twice through a brass metal sieve (500 pm pore size). After centrifugation (5 min. 1. 0 0 0 + 4 °C). the samples were resus­ pended in fresh, cold medium [2], Fifteen-milliliter aliquots of the cell suspension were layered onto 15-ml Ficoll (6.4% Ficoll with 10% sodium diatrizoate) and centrifuged for lOmin at 1,000g, +4°C [24]. With this procedure, red blood cells and polynuclear leukocytes were separated mainly from the two upper layers of cells and tissue: syncytiotrophoblasts were included in both layers, but were more abundant in the second layer [9], This layer was removed, washed again and resuspended in 100 ml of medium. Experimental Procedures In preliminary' experiments, we ensured that the second layer included the syncytiotrophoblasts by using methanol/acetic acid fix­ ation and hematoxylin/eosin staining [2, 9], For individual experiments, the 100 ml suspension of cells was divided into 0.5-ml aliquots, which were added to Eppendorf tubes together with 0.5 ml of prewarmed medium. The cultures were prein­ cubated for 0.5 h (37 °C. 95% humidified air. 5% COi) to ensure metabolic equilibrium of the cultured cells. The viability of the cells, as assessed by trypan blue staining [27] before the start of an experi­ ment, was 85-93%. Experiments on one placenta were each per­ formed in three replicates at several time points. At the end of the preincubation period, defined as Oh, various reagents diluted in incubation medium were added. The control group was kept without reagents. The amount of hCG secreted into the medium was defined as the amount of hCG in the extracellular compartment; the amount of hCG after cell lyses was defined as the amount of hCG in the intracellular compartment. Experiment I. The effect of the dose of hTSH on extracellular concentrations of hCG was assessed with three different doses (10, 100 and 200 ng hTSH/ml). The extracellular hCG concentration was examined at 0, 15 and 3 0 min, and I, 4. and 6h. At the specified times, the reactions were stopped by cooling of the Eppendorf tubes on ice for 10 min. The tubes were centrifuged (5 min. 14,000 g, +4 °C), and the hCG concentrations in the supernatants (extracellu­ lar compartment) were measured. Experiment 2. In a second experiment in which we evaluated the mode of action of hTSH. 200 ng hTSH/ml was added simultaneously with 100 pCi/ml -,5S-methionine at 0 h. At 15 and 30 min. and 1.2.4. 6. 12. and 24 h. the reactions were stopped as described above, and the extracellular compartment was removed. The pellet was redis­ solved in 1 ml cold sodium citrate solution (0.7%) and incubated on ice for 10 min. Trypan blue staining of an aliquot showed no detect­ able cell viability. After centrifugation (5 min. 14.000£. +4°C). the second supernatant, which represented the intracellular compart­ ment. was removed. We measured the extracellular and intracellular hCG concentrations as well as the amounts of radiolabeled hCG in both compartments. In addition, the total amount of protein in both compartments was determined in duplicate in aliquots by a modifi­ cation of the method of Lowry etal. [28], with bovine serum albumin

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receptor [12. 13]. During normal human pregnancy, the thyroid-stimulating activity of hCG on the pituitary-thy­ roid axis is suggested by the observation that, at the time when hCG levels are highest, hTSH levels are lowest [14, 15]. During early pregnancy, the maternal thyroid gland is stimulated by hCG, and the level of free thyroxine is increased [16] and that of hTSH decreased. These hTSH levels were found to be significantly lower than those dur­ ing the remainder of the pregnancy or those in a nonpreg­ nant control group [15]. Winikoff and Malinek [17] pos­ tulated that this decreased hTSH value was a part of a thyroid ‘test profile’ at 8-9 weeks of pregnancy. In preg­ nant women with thyroid disorders (thyrotoxicosis or hypothyroidism), the hTSH level is elevated at that time. In this group of patients, repeated spontaneous abortions have been reported [18,19], The placenta is essentially impermeable to thyroxin and triiodothyronine, both are degraded in the placenta to inactive iodothyronines [20]. Human thyrotropin-releas­ ing hormone (hTRH) crosses the placental barrier [21], but hTSH does not. Evidence has been presented that hTSH binds to the syncytiotrophoblast [15. 22]. hCG and hTSH both have the a-subunit in common and perhaps have a similar functional control system [23]. but the possibility of a regulation of placental hCG pro­ duction by hTSH has never been examined critically. To persue this question, we, therefore, established an in vitro system of short-term cultures of human placenta at term to study an effect of hTSH on the production and/or secretion of hCG.

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used as standard. In the same samples, the total amount of radioac­ tivity (35S-methionine) incorporated into the proteins was deter­ mined in duplicate by trichloroacetic acid precipitation [25], Experiments 3 and 4. An experiment in which we tested the spe­ cies specificity of the hCG response with bTSH (experiment 3) as well as an experiment on the effect of the dose of hTRH on extracel­ lular concentrations of hCG (experiment 4) were performed in the same way as experiment l, except that the reagents added were bTSH (applied doses: 10. 100. 200. and 500 ng bTSH/ml) and hTRH (ap­ plied doses: 10, 100. and 200 ng hTRH/ml). respectively. Measurement o f hCG In all experiments, the amount of hCG in either the extracellular compartment or the extra- and intracellular compartments was determined by a solid phase enzyme immunoassay based on the sandwich principle (Abbott b-hCG 15715 bead assay) [29]. The coef­ ficient of variation of this assay at the low detection level is 6.7% for inter-assay and 4.4% for intra-assay variation. The cross-reactivity with hTSH at the concentration levels examined was less than 0.36%. The mean minimal sensitivity at the 95% confidence limit was 0.76 mlU/ml. Concentrations were determined by hCG binding to anti-P-hCG (goat)-coated beads, detected in a horseradish peroxi­ dase conjugate/o-phenylenediamine reaction and quantified by spectrophotometric measurement of the color absorbance at 492 nm. For the measurement of the de novo synthesized hCG (experi­ ment 2), the beads, to which the 35S-methionine-labeled hCG mole­ cules were bound, were removed and their radioactivity counted. The counting efficiency was 74-78%. Loss of radioactivity in the preceding colorimetric reaction was negligible; measurements on the solutions in which the beads had been bathed showed that less than 4% of the final counts remained in solution. Statistics Data are presented as means ± SEM. We used ANOVA to test for significant differences between groups. A p value < 0.05 was con­ sidered to indicate a significant difference.

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Results

The effects of the various reagents and of the concen­ trations applied are described separately for each experi­ ment. Extracellular ItCG Concentration after Addition o f hTSH We examined the extracellular concentration of hCG at three doses of hTSH (10. 100 and 200ng/ml; experi­ ment 1) in 11 placentas (fig. 1). At both Oh and 15 min from the start of the incubation, the baseline concentra­ tions of hCG in all treatment groups and in the control group were identical, and 0 h is therefore not included in figure 1. The addition of 10 ng/ml hTSH did not produce any significant change from the control value at any time during the 6-hour experiment. Upon addition of 100 and 200 ng hTSH/ml, a decrease in the extracellular hCG con­ centration was first noted at 30 min and continued to occur at the later times. The effect was proportional to the dose; 200 ng hTSH/ml always had a significantly greater effect than 100 ng. Inhibition ofde novo Synthesis ofhCG by hTSH To examine whether hTSH inhibited de novo synthe­ sis of hCG or influenced hCG depletion, we cultured 3 placentas by incubation in a combination of 200 ng hTSH/ml and 100 pCi/ml 35S-methionine (experiment 2). The total amounts of hCG in the intracellular and extra­ cellular compartments we measured by an enzyme immu-

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hTSH Effect on hCG in Short-Term Cultures of Human Placenta

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Fig. 1. Decrease in extracellular hCG concentration on incubation with liTSH. The graph depicts the results for controls (first column: 0 ng hTSH) and for increasing doses of hTSH: 10 (second column). 100 (third column) and 200 ng (fourth column). The same order of columns is used for all time points. Results for 11 placentas are combined. All values are expressed as means ± SEM. * p < 0.05 vs. the control values.

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Fig. 2. Inhibition of de novo synthesis of hCG by hTSH. Effect of 200 ng hTSH/ml on hCG production by human placentas in short-term culture, a Amount of hCG in the extracellular (control: left column; hTSH: second column) and intracellular (control: third column; hTSH: fourth column) com­ partments over time, as measured by en­ zyme immunoassay. The same order of col­ umns is used for all time points, b Amount of radioactivity/mlU hCG in extracellular and intracellular compartments, represent­ ing the amount of hCG synthesized dc novo. The order of columns is the same as in a. Results for 3 placentas are combined. All values are expressed as means ± SEM. * p < 0.05 vs. the control values.

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noassay (fig. 2a). In the extracellular compartment, the results of the incubation with 200 ng hTSH/ml incubation were similar to those in experiment 1. The intracellular compartment exhibited a significant decrease in the hCG content in the hTSH-stimulated group at 4 h and 6 h (fig. 2a). The amount of hCG synthesized de novo was determined by the measurement of the rate of incorpora­ tion of 35S-methionine (fig. 2b). In the intracellular com­ partment. there was no significant difference in the rates of 35S-methionine incorporation between the control and the hTSH treatment group at any of the times examined. In the extracellular compartment, however, the amount of radio­ labeled hCG in the hTSH treatment group decreased signif­ icantly from 30 min until 6 h. with the exception of 1 h.

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Effect o f hTSH on Total Protein Synthesis To determine whether the inhibiting effect of hTSH was specific for hCG synthesis or whether the hTSH also influenced the total cellular protein synthesis, we per­ formed trichloroacetic acid precipitation of the total 35Smethionine-labelcd proteins and a total protein determi­ nation in aliquots from experiment 2. The results (fig. 3) showed that, at 4 and 6 h, the incorporation of radiola­ beled 35S-methionine/mg total cellular protein was dimin­ ished significantly in the intracellular and extracellular compartments.

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Extracellular hCG Concentration after Addition o f Bovine TSH In experiment 3, we tested whether the inhibition of hCG secretion is species specific. We incubated cultures from 3 of the placentas with bTSH at the concentrations used for hTSH. 10. 100. and 200 ng/ml, and. in addition, 500 ng bTSH/ml. No significant difference in extracellu­ lar hCG concentrations between control and experimen­ tal groups was detectable at any time point (data not shown). Extracellular hCG Concentration after Addition o f hTRH To test the influence ofhTRH (experiment 4) on extra­ cellular hCG concentrations, we performed an experi­ ment identical to that described for the dose relationship with hTSH (experiment l ). We applied hTRH at concen­ trations of 10, 100. and 200 ng/ml to 3 placenta cultures. At 100 and 200 ng/ml. hTRH caused a significant in­ crease in hCG secretion at l and 4 h (data not shown).

Comment

Up to now. a TSH binding site or receptor has not been identified in the placenta as it has been in the thyroid [ 13]. There is a remarkable structural homology in the a-subunit of glycoproteins, such as LH, follicle-stimulating hor­ mone. hCG and TSH, and also in their hormone-specific P-subunit. In spite of the different responses evoked by these hormones, their biological action is initiated by acti­

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vation of the same signaling system (i.e. adenylyl cyclase) in their respective targets cells. A conclusion arising from these structural and functional similarities is that the structures of the TSH and LH/CG receptors may be homologous [22], The finding that synthetic peptides derived from the a-subunits block the binding of LH/CG and TSH to their respective receptors supports this idea [ 12. 22 ], Even though we had not identified a specific placental TSH binding site or receptor, we were able to demonstrate that established short-term cultures of human placentas at term reacted in a time- and dose-dependent manner to incubation with hTSH. The 10 ng/ml dose of hTSH pro­ duced no significant effect, whereas 100 and 200 ng/ml caused a significant decrease in the extracellular hCG concentration from 30 min until 6h of incubation. To examine whether these changes in the extracellular hCG concentration were correlated with a decrease in the rate of de novo synthesis of hCG or were due to inhibition of hCG secretion, we performed a simultaneous incubation with 35S-methionine. In the intracellular compartment, the amount of radiolabel per IU hCG remained constant whereas the total concentration of hCG decreased at 4 and 6 h. These findings, together with the decreased amounts of radiolabeled hCG in the external compart­ ment in the hTSH treatment group, pointed to a reduced rate of de novo synthesis of hCG. At later times (12 and 24 h), significant differences from control values could not be demonstrated, suggesting that the inhibiting effect of a single application of hTSH is time limited.

Beckmann/Wiirfel/Austin/Link/Albert

hTSH Effect on hCG in Short-Term Cultures of Human Placenta

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Fig. 3. Effect of hTSH on total cellular protein production. Effect of 200 ng hTSH/ ml on 35S-methionine incorporation into to­ tal cellular protein in human placentas in short-term culture. The first columns repre­ sent total cpm/mg protein in controls, and the second columns that of the hTSH-treatcd group, both in the extracellular compart­ ment. The third columns show the amount of total cpm/mg protein in controls and the fourth columns the amount in the hTSHtreated group, both in the intracellular com­ partment. Results for 3 placentas are com­ bined. All values are expressed as means ± SEM. * p < 0.05 vs. control values.

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In vivo, the hTSH level in serum follows a diurnal rhythm, with values peaking after midnight and reaching a low mark in midafternoon [30]. This cyclical pituitary secretion could be important for the pathophysiology of the effect of hTSH in vivo. Perhaps the hTSH effect accu­ mulates over time, causing a chronic decrease in placental hCG synthesis. Besides the specific effect on hCG synthe­ sis. the generalized inhibiting effect of hTSH on the total secretion of cellular protein could serve as an additional explanation for the placental insufficiency and recurrent abortions that occur in women who have elevated hTSH levels. The species specificity of this hTSH effect or a placen­ tal hTSH binding site or receptor, as was demonstrated previously for binding of hCG to the TSH receptor in the thyroid [11], was demonstrated with bTSH at different doses, which produced no significant effect on the extra­ cellular hCG concentration. In the hypothalamic-pituitary-thyroid axis, there is an inverse correlation between the amount of peripheral cir­ culating TSH and the level of TRH released from the hypothalamus in vivo. When TRH was administered to pregnant women at term prior to delivery, it was found that hTRH crossed the human placenta, that the fetal pituitary' was responsive to TRH. and that endogenous fetal TSH, secreted as a result of maternal TRH stimula­ tion. increased the fetal thyroid activity [21]. High doses of hTRH stimulated hCG secretion in vitro, which is in accord with the effect in experiments with intravenous

administration of hTRH to ovulatory, euthyroid and normoprolactinemic women, which resulted in stimulation of LH secretion [31]. The thyroid-stimulating activity of hCG has been stud­ ied extensively [12, 13, 32], One of the main effects of hCG in vivo on the pituitary-thyroid axis is thought to occur in early pregnancy (8-12 weeks), when the hTSH level is significantly lower than that during the remainder of the pregnancy. In patients with untreated thyroid dis­ ease. mainly those with hypothyroidism, the hTSH level remains high throughout pregnancy. In such patients, an increased incidence of miscarriages and spontaneous abortions has been reported [17. 19], Our study showed that, in cultures of human placenta at term, the secretion and de novo synthesis of hCG are decreased significantly by high levels of hTSH. Furthermore, the amount of total cellular protein produced decreases under the influence of hTSH. In vivo, these two findings could suggest a possible role of elevated hTSH levels in the downregulation of the production of hCG and of total cellular protein during pregnancy and could provide an explanation for placental insufficiency in women with thyroid disease.

Acknowledgments The authors are grateful for the technical advice of Prof. J.A. Holt and of the personnel of the CLI Endocrinology Laboratory. Depart­ ment of Obstetrics and Gynecology, University of Chicago.

References 5 Genbacev O. Patkovic M. Kraincanic M: Ef­ fect of prostaglandin PGF2aIpha on the synthe­ sis o f placental proteins and human placental lactogen (HPL). Prostaglandins 1977:13:723— 733. 6 Osathanondh R. Tulchinskv D: Placental poly­ peptide hormones; in Tulchinsky D. Ryan KJ (eds): Maternal-Fetal Endocrinology. Philadel­ phia. Saunders. 1980. pp 17-42. 7 Simpson ER. MacDonald PC: Endocrine phys­ iology o f the placenta. Annu Rev Physiol 1981; 43:163-188. 8 De Ikonicoff I.K. Cedard L: Localization of human chorionic gonadotropic and somatomammotropic hormones by the peroxidase immunohistoenzymologic method in villi and amniotic epithelium o f human placentas. Am J Obstct Gynecol 1973:116:1124-1132.

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Kliman 1IJ. Nestler JE. Sermasi E. Sanger JM. Strauss JF III: Purification, characterization, and in vitro differentiation o f cytotrophoblasts from human term placentae. Endocrinology 1986:118:1567-1582. Nisula BC. Ketelslegers JM: Thyroid-stimulat­ ing activity and chorionic gonadotropin. J Clin Invest 1974:54:494-499. AmirSM. Endo K. Osathanondh R. IngbarSH: Divergent responses by human and mouse thy­ roids to human chorionic gonadotropin in vi­ tro. Mol Cell Endocrinol 1985:39:31-37. Hoermann R. Amir SM. Ingbar SH: Evidence that partially desialylated variants o f human chorionic gonadotropin (hCG) are the factors in crude hCG that inhibit the response to thy­ rotropin in human thyroid membranes. Endo­ crinology 1988:123:1535-1543. Rees Smith B. McLachlan SM, Furmaniak J: Autoantibodies to the thyrotropin receptor. EndocrRev 1988:9:106-121.

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1 Khodr G. Siler-Khodr TM: The effect o f lutein­ izing hormone-releasing factor on human cho­ rionic gonadotropin secretion. Fcrtil Steril 1978:30:301-304. 2 Mathialagan N, Rao AJ: Gonadotropin releas­ ing hormone (GnRH) stimulates both secretion and synthesis o f human chorionic gonadotro­ pin (hCG) by first trimester human term pla­ cental minces in vitro. Biochem Int 1986:13: 757-765. 3 Handwerger S. Barrett J. Tyrev I . Schomberg D: Differential effect o f cyclic adenosine monophosphate on the secretion o f human pla­ cental lactogen and human chorionic gonado­ tropin. J Clin Endocrinol Mctab 1973:36: 1268-1270. 4 Benveniste R. Specg KV. Carpenter G. Cohen S. Lindner J. Rabinowitz D: Epidermal growth factor stimulates secretion o f human chorionic gonadotropin by human cultured choriocarci­ noma cells. J Clin Endocrinol Mctab 1978:46: 169-172.

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Suppression of human chorionic gonadotropin in the human placenta at term by human thyroid-stimulating hormone in vitro.

Human chorionic gonadotropin (hCG) exerts a clinically apparent negative feedback on the secretion of human thyroid-stimulating hormone (hTSH) in preg...
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