Evaluating the Roles of Follicle-Stimulating Hormone Receptor ...

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The Journal of Clinical Endocrinology & Metabolism 92(6):2312–2317 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2006-2086

Evaluating the Roles of Follicle-Stimulating Hormone Receptor Polymorphisms in Gonadal Hyperstimulation Associated with Severe Juvenile Primary Hypothyroidism Ginny L. Ryan, Xiuyan Feng, Catarina Brasil d’Alva, Meilin Zhang, Bradley J. Van Voorhis, Emilia Modolo Pinto, Ana Elisa F. Kubias, Sonir R. Antonini, Ana Claudia Latronico, and Deborah L. Segaloff Division of Reproductive Endocrinology and Infertility (G.L.R., B.J.V.V.), and Department of Molecular Physiology and Biophysics (X.F., M.Z., D.L.S.), University of Iowa Roy J and Lucille A Carver College of Medicine, Iowa City, Iowa 52242; Developmental Endocrinology Unit and Laboratory of Hormone and Genetic Molecular LIM/42 (C.B.d., E.M.P., A.C.L.), Sao Paulo University Medical School, Sao Paulo 05403-900, Brazil; and Division of Pediatric Endocrinology, Department of Pediatrics (A.E.F.K., S.R.A.), School of Medicine of Ribeira˜o Preto, University of Sao Paulo, Sao Paulo 1409-900, Brazil Context: Rare activating mutations of the human (h)FSHR have been reported in some women with spontaneous ovarian hyperstimulation in pregnancy, where follicular growth is inappropriately stimulated by elevated concentrations of human chorionic gonadotropin acting through the hFSHR. It is not known whether ovarian hyperstimulation in peripubertal girls with untreated primary hypothyroidism is caused by hFSHR mutations and/or influenced by hFSHR allelic variants, rendering the hFSHR more sensitive to circulating TSH. Objective: The aim of the study was to determine whether mutations of the hFSHR and/or hFSHR allelic variants are associated with greater sensitivity of the hFSHR to TSH. Design: The hFSHR gene was sequenced from eight pediatric patients displaying gonadal hyperstimulation due to primary hypothyroidism. HEK293 cells expressing different hFSHR allelic combinations were studied for their responsiveness to recombinant (r)hTSH. Setting: The study was conducted at university research centers.

T

HE CLINICAL SYNDROME of severe primary hypothyroidism associated with isosexual precocious puberty in females was first described by Kendle in 1905 (1). Since then, there have been many case reports of this phenomenon (2– 6). In all these cases, long-standing untreated primary hypothyroidism is associated with a distinct form of sexual precocity that may include breast development, vaginal bleeding, and/or multicystic ovaries, but does not include significant pubic hair development and, unlike other First Published Online March 13, 2007 Abbreviations: AS, Ala307-Ser680; CG, chorionic gonadotropin; FSHR, FSH receptor; h, human; HEK, human embryonic kidney; OHSS, ovarian hyperstimulation syndrome; r, recombinant; TN, Thr307-Asn680; TSHR, TSH receptor; wt, wild-type. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Patients: Eight unrelated patients (seven girls and one boy) who exhibited primary hypothyroidism and gonadal hyperstimulation were included in the study. Interventions: There were no interventions. Main Outcome Measure: DNA sequencing of the hFSHR gene was the main outcome measure. Basal, rhFSHR- and rhTSH receptor-stimulated cAMP levels were assayed in HEK293 cells transfected with the hTSH receptor or different hFSHR allelic combinations. Cell surface receptor numbers were also determined. Results: No hFSHR mutations were identified in the patient population, but we did identify two known polymorphisms. In vitro experiments demonstrated a dose-dependent and specific rhTSHdependent increase in cAMP production in HEK293 cells expressing the wild-type hFSHR, regardless of hFSHR isoform. Conclusions: Pediatric gonadal hyperstimulation associated with severe primary hypothyroidism is likely due to the actions of the elevated concentrations of TSH on the wild-type hFSHR, and this response is not dependent upon the hFSHR isoform. (J Clin Endocrinol Metab 92: 2312–2317, 2007)

forms of precocious puberty, characteristically involves impaired growth and delayed skeletal maturation. Consistent with this apparently FSH receptor (FSHR)-mediated process, primary hypothyroidism in boys is associated with gonadal enlargement without virilization (6 – 8). The etiology of this unique sexual precocity remains the matter of some debate. The one consistent clinical finding is markedly elevated TSH levels and rapid improvement in symptoms with normalization of these levels. Other commonalities variably include the autoimmune nature of the thyroid disease, peripubertal onset of symptoms, elevation in prolactin levels, FSH levels in the high-normal range for age, and somewhat elevated estradiol levels (2– 6). Van Wyk and Grumbach (9), whose names are often associated with this clinical syndrome, first suggested in 1960 that the explanatory mechanism was an “overlap” in negative feedback response in which not only TSH but also other pituitary hor-

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Ryan et al. • Gonadal Hyperstimulation with Primary Hypothyroidism

mones such as FSH and LH are stimulated by TRH. Alternatively, others have suggested that the proximate nature of the TRH center to the GnRH center in the hypothalamus leads to excessive production of both releasing factors (2– 4). Still others have proposed that prolactin plays a primary role in the disease process, perhaps by sensitizing the ovaries to gonadotropins (2– 4), or that TSH itself sensitizes the ovaries to gonadotropin stimulation (2– 4). Although very rare, recent reports describe five naturally occurring mutations in the hFSHR that result in the inappropriate stimulation of the receptor by high concentrations of the related glycoprotein human chorionic gonadotropin (hCG), leading to spontaneous ovarian hyperstimulation in normal pregnancy (10 –14). In vitro analyses of these hFSHR mutations suggest that they are constitutively active and also susceptible to promiscuous activation by elevated concentrations of recombinant (r) human (h)TSH (10 –14). Recently, De Leener et al. (14) examined the hFSHR gene in two adult patients with ovarian hyperstimulation syndrome (OHSS) and hypothyroidism. They did not observe any mutations. However, they showed that heterologous cells expressing recombinant wild-type (wt) hFSHR respond to very high concentrations of rhTSH, confirming and expanding similar results reported earlier by Anasti et al. (6). These results suggest that the OHSS associated with hypothyroidism in these patients may be due to the inappropriate stimulation of the hFSHR by elevated TSH. An additional aspect of the hFSHR gene to consider is that there are two single nucleotide polymorphism sites in exon 10 of the hFSHR that are almost always in linkage disequilibrium, resulting in alleles Thr307-Asn680 and Ala307-Ser680. Studies have suggested that the nature of the hFSHR allelic variant in women impacts ovarian response to FSH as well as menstrual cycle dynamics (15–19). In a population study of adult women with iatrogenic OHSS, a correlation between

J Clin Endocrinol Metab, June 2007, 92(6):2312–2317

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OHSS and hFSHR polymorphisms was not observed; however, an association between the frequency of the Thr307Asn680 allele and the severity of symptoms was suggested (20). Our goal in the present study was to determine whether pediatric patients with primary hypothyroidism associated with gonadal hyperstimulation carry a mutant hFSHR or specific allelic variant of the hFSHR that results in a lower threshold to stimulation by TSH. Patients and Methods Plasmids and hormones The cDNA for the wt hFSHR (Thr307-Asn680 form) and highly purified rhFSH were generous gifts from Ares Advanced Technology (AresSerono Group, Randolph, MA). Highly purified rhTSH was purchased from Genyzme (Cambridge, MA). The specific activity for the rhTSH was 4 –12 IU/mg. For the purposes of this study, a specific activity of 4 IU/mg was used for calculating the concentrations of rhTSH. Pregnant mare serum gonadotropin (used to determine the nonspecific binding of 125 I-hFSH) and highly purified rhCG were purchased from Dr. A. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases’ National Hormone and Pituitary Program. The Thr307-Asn680 wt hFSHR cDNA was subcloned into pcDNA3.1/ neo (Invitrogen, Carlsbad, CA). The polymorphic variants Ala307 and Ser680 were introduced using the QuikChange site directed mutagenesis kit from Stratagene (La Jolla, CA). Plasmid was prepared using the QIAGEN maxiprep kit (QIAGEN, Hilden, Germany). DNA sequencing of the entire coding region was performed in the DNA Core of the University of Iowa.

Patients This study received approval from the Institutional Review Boards of the University of Iowa and Sao Paulo University Medical School. Eight unrelated patients (seven girls and one boy; chronological age, 8.3–16.9 yr) with long-standing juvenile primary hypothyroidism associated with gonadal hyperstimulation were selected for this study from the United States and Brazil. Their clinical, hormonal, and ultrasonographic findings are summarized in Table 1. Clinical data of patients 1– 4 (21) and

TABLE 1. Clinical, hormonal, and ultrasonographic data of eight unrelated patients with primary hypothyroidism and gonadal stimulation Patient no.

Origin Sex Age (yr) Bone age (yr) Height (cm) Height SD Breastb Pubic hairb Menarche (yr) Goiter TSH (mU/liter) Free T4 (pmol/liter) LH (mIU/ml) FSH (mIU/ml) Estradiol (nmol/liter) Testosterone (nmol/liter) Prolactin (␮g/liter) Gonadal volume (cm3) FSHR polymorphisms

1a

2a

3a

4a

5

6

7

8a

Caucasian American Female 13.7

Asian American Female 16.9


Caucasian American Male 8.9


145.0 ⫺2.1 V IV 11 ⫺ 1058 2.5 ⬍0.2 6.8 3.0

151.4 ⫺1.8 V V Amenorrhea ⫹ 732 5.1 6.2 4.4 0.15

130.1 ⫺2.5 I I 11.5 ⫹ 526 2.5

132.4 ⫺0.2

132.0 0.3 I I Premenarchal ⫺ 436 5.1

Caucasian Brazilian Female 8.3 3.5 112.0 ⫺3.1 III I 8.3 ⫹ 1180 2.5 ⬍0.2 5.5 0.57

Caucasian Brazilian Female 8.6 7.9 132.0 0 III II 8.2 ⫺ 254 3.8 ⬍0.2 5.0 0.07

African Brazilian Female 9.7 9.0 125.3 ⫺1.4 IV II 9.1 ⫹ 1376 1.3 1.0 2.1 0.56

381 R: 830 L: 988 AS/AS

38 14.3 10.2 TN/AS

20 11 10.3 TN/AS

75 48 139 TN/TN

28 39 50 TN/AS

84.5 41.7 61.4 TN/TN

7.1 0.05

I ⫺ 366 2.5 0.13 0.9 4 5.0 4.0 TN/AS

54 TN/AS

Normal values: TSH, 0.3–5.0 mU/liter; free T4, 10.2–17.0 pmol/liter; estradiol (follicular phase), 0.07- 0.53 nmol/liter; testosterone, 3–30 nmol/liter; prolactin, ⬍ 20 ␮g/liter. R, right; L, left; ⫹, presence; ⫺, absence. a Previously reported patients (Refs. 21 and 22). b Breast and pubic hair development according to Tanner and Marshall criteria.

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8 (22) were previously reported. Patients were referred to medical attention for different conditions, including symptoms and signs of hypothyroidism (patients 1, 3, and 4), menstrual abnormalities and galactorrhea (patient 2), intermittent ovarian torsion and pain (patient 5), occurrence of secondary sexual characteristics (patients 4 and 7), abdominal mass (patient 6), and decrease of growth velocity (patient 8). Short stature (sd ⱕ ⫺2) was observed in patients 1, 3, and 6. Bone age was performed in three patients, and it was much delayed (5 yr) in patient 6, suggesting a very long-standing hypothyroidism. Precocious puberty was diagnosed in four patients (no. 4 and 6 – 8), who showed low basal and GnRH-stimulated LH levels, indicating a gonadotropin-independent form of precocious puberty in these patients. Patient 5 had previously undergone surgical removal of her right ovary and fallopian tube for torsion of this ovary at age 5. All patients exhibited markedly elevated levels of TSH (254 –1376 mU/liter; normal values, 0.3–5.0 mU/ liter) and low levels of free T4 (1.3–5.1 pmol/liter; normal values, 10.2–17 pmol/liter). Goiter was detected in four patients on physical examination. Five patients had positive antiperoxidase and antithyroglobulin antibodies. Estradiol levels varied greatly from 0.05–3.0 nmol/liter in the female patients, whereas prolactin levels were elevated in five of six patients in whom prolactin was measured. Pelvic ultrasound revealed enlarged ovaries (10.2 to 988 cm3) in all female patients. Similarly, enlarged testes were palpable and confirmed by scrotal ultrasound in the male patient. Notably, thyroid hormone replacement corrected the clinical and hormonal manifestations of the gonadal hyperstimulation in all eight patients.

DNA analysis Genomic DNA was extracted from peripheral blood leukocytes using standard procedures from the eight patients with primary hypothyroidism and gonadal stimulation. The entire coding region of the hFSHR gene, including the 10 exons and exon-intron boundaries, was amplified using specific intronic primers. PCR was performed in a 100-␮l reaction mixture containing 200 ng genomic DNA, 10 mm/liter Tris-HCl (pH 8.3), 1.5 mm MgCl2, 200 ␮m each deoxynucleotide triphosphate, 0.5 U of Taq polymerase (Amersham Bioscience, Piscataway, NJ) and 30 pmol of each primer. After initial denaturation of 2 min at 94 C, samples were subjected to 35 cycles of 1 min at 94 C, 1 min at variable annealing temperature, and 2 min at 72 C, followed by a final extension step of 10 min at 72 C. All amplified fragments were examined after 1% agarose gel electrophoresis. The PCR products were pretreated with an enzymatic combination of exonuclease I and shrimp alkaline phosphatase (United States Biochemical Corp., Cleveland, OH) and directly sequenced using the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) in an ABI PRISM 3100 automatic sequencer (Perkin-Elmer Cetus, Norwalk, CT).

Cells and transfections Human embryonic kidney (HEK) 293 cells were maintained at 5% CO2 in growth media consisting of high-glucose DMEM containing 50 ␮g/ml gentamicin, 10 mm HEPES, and 10% newborn calf serum. Cells for experiments were plated onto 16-mm wells that had been precoated for 1 h with 0.1% gelatin in calcium- and magnesium-free PBS (pH 7.4). Cells were transiently transfected as previously described (23) and used for experiments 24 h after removing the cocktail. In the same experiment, cell surface expression of the hFSHR was determined by 125I-FSH binding to intact cells and basal and hormone-stimulated cAMP levels were determined (see below). 125

I-hFSH binding assays

HEK293 cells were plated and transfected as described above. On the day of the experiment, cells were washed two times with warm Waymouth’s MB752/1 containing 50 ␮g/ml gentamicin and 1 mg/ml BSA. To determine the maximal cell surface binding capacity, the cells were then incubated for 1 h at 37 C in the same media containing a saturating concentration of 125I-rhFSH (300 ng/ml final concentration) with or without an excess of unlabeled pregnant mare serum gonadotropin (250 IU/ml final concentration). The assay was terminated by washing the cells three times with cold Hanks’ Balanced Salt Solution modified to contain 50 ␮g/ml gentamicin and 1 mg/ml BSA. The cells were then


solubilized in 0.5 n NaOH, transferred to plastic test tubes with cotton swabs, and counted in a gamma counter.

Assay of intracellular cAMP production HEK293 cells were plated and transfected as described above. On the day of the experiment, cells were washed two times with warm Waymouth’s MB752/1 containing 50 ␮g/ml gentamicin and 1 mg/ml BSA and placed into the same medium containing 0.5 mm isobutylmethylxanthine. After a 15-min preincubation at 37 C, buffer alone (for basal cAMP levels), rhFSHR, or rhTSH was added to give the final concentrations noted, and the cells were incubated an additional 60 min at 37 C. The cells were then placed on ice, the media was aspirated, and intracellular cAMP was extracted by the addition of 0.5 n perchloric acid containing 180 ␮g/ml theophylline and measured by RIA. All assays were performed with triplicate wells, and all measurements were repeated in duplicate. Dose response curves were plotted, and EC50 and Rmax were determined using Prism (GraphPad Software, San Diego, CA).

Results

Automatic sequencing of the hFSHR gene of each of the patients was performed to determine whether they harbored a mutation of the hFSHR that rendered the hFSHR more sensitive to TSH. In all eight patients, no mutations of the hFSHR were identified. Two known hFSHR polymorphisms, Ala307Thr and Ser680Asn, were found in linkage disequilibrium (Table 1). Five of the patients were heterozygous for the Ala307-Ser680 and Thr307-Asn680 alleles (62.5%), two patients were homozygous for Thr307-Asn680 (25%), and one was homozygous for Ala307-Ser680 (12.5%), yielding an allelic frequency of 46% Thr307-Asn680 and 54% Ala307-Ser680. The genotype frequencies of the hFSHR polymorphisms in 50 Brazilian controls were 56% heterozygous for the Ala307Ser680 and Thr307-Asn680 alleles, 30% homozygous Thr307Asn680, and 14% homozygous Ala307-Ser680, and giving an allelic frequency of 58% Thr307-Asn680 and 42% Ala307-Ser680 (24). Although the patient group in our study is too small to make any conclusions regarding potential differences in the genotype frequency of the patients vs. the controls, we thought it important to determine whether the nature of the hFSHR polymorphisms might affect the signaling properties of the hFSHR with respect to its response to elevated concentrations of rhTSH. Therefore, we examined the rhFSH and rhTSH responses in HEK293 cells that were transfected with the hFSHR Thr307Asn680 only (TN/TN, to recapitulate the homozygous expression of this allele), with Ala307-Ser680 only (AS/AS, to recapitulate the homozygous expression of this allele), or with half Thr307-Asn680 and half Ala307-Ser680 (TN/AS, to recapitulate the heterozygous expression of the two hFSHR alleles). These experiments were performed under conditions where the densities of total hFSHR on the cell surface, as determined by 125I-hFSH binding to intact cells, were similar between the three groups of cells. As shown in Fig. 1, similar dose response curves to rhFSH were observed in the TN/TN, AS/AS, and TN/AS cells, demonstrating that the hFSHR allelic combinations do not affect responsiveness to FSH. When challenged with increasing concentrations of rhTSH, the TN/TN, AS/AS, and TN/AS cells each responded with increased cAMP, but these responses were observed at much higher concentrations of rhTSH than those needed to stimulate HEK293 cells expressing the hTSHR (Fig.


FIG. 1. Dose response analysis of HEK293 cells expressing rhFSHR isoforms to rhFSH. HEK293 cells were transiently transfected with empty vector, the Thr307-Asn680 isoform of the hFSHR only (TN/TN), the Ala307- Ser680 isoform of the hFSHR only (AS/AS), or half Thr307Asn680 and half Ala307- Ser680 (TN/AS). Cell surface 125I-hFSH binding levels in the experiment shown were 4.84, 4.76, and 5.0 ng/106 cells to the TN/TN, AS/AS, and TN/AS cells, respectively. The cells were incubated with the indicated concentrations of rhFSH for 1 h at 37 C, and intracellular cAMP was then assayed as described in Patients and Methods. The data shown are the mean ⫾ SEM of triplicate determinations within one experiment that is representative of two independent experiments.

2A). The responses of the hFSHR cells to rhTSH were specific because no increase in cAMP was observed in cells transfected with empty vector. Furthermore, the responses of the TN/TN-, AS/AS-, and TN/AS hFSHR-expressing cells to rhTSH were remarkably similar (Fig. 2B). In each case, the lowest concentration of rhTSH that elicited a response (⬃1.6fold) was 30 mIU/ml. At the highest rhTSH concentration used (700 mIU/ml), approximately an 18-fold increase in cAMP was observed. Discussion

Mutations of the hFSHR are very rare in the literature. However, recent studies have shown that certain mutations of the hFSHR stabilize the receptor in a conformation that permits its stimulation by high concentrations of hCG or TSH (10 –14). It has been reported that no detectable binding activity of radiolabeled hCG or TSH is observed in cells expressing these hFSHR mutants. Therefore, the constitutive activation of the hFSHR renders it more responsive to supramaximal concentrations of hCG or TSH, even when these hormones are binding at extremely low affinities. These promiscuous hFSHR mutants have been described in some women with OHSS, where the markedly elevated levels of hCG associated with pregnancy inappropriately cause ovarian hFSHR stimulation (10 –12, 14). Other examples of abnormalities associated with the lowaffinity binding of a very high concentration of a glycoprotein hormone to an inappropriate glycoprotein hormone receptor include molar pregnancy or choriocarcinoma, both conditions characterized by high hCG levels, causing hyperthyroidism (TSH-like activity) or theca lutein cysts (FSH-like activity) (14, 25, 26). In this study, we evaluated eight pediatric patients presenting with primary hypothyroidism and gonadal hyperstimulation, in whom the gonadal hyperstimulation resolved upon treatment of the hypothyroidism. Although these clinical features would be consistent with a mutation of the hFSHR that permitted promiscuous activation of the hFSHR


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FIG. 2. Dose response analysis of HEK293 cells expressing rhFSHR isoforms or recombinant hTSHR to rhTSH. HEK293 cells were transiently transfected with empty vector, the Thr307- Asn680 isoform of the hFSHR only (TN/TN), the Ala307- Ser680 isoform of the hFSHR only (AS/AS), or half Thr307- Asn680 and half Ala307- Ser680 (TN/AS). Cell surface 125I-hFSH binding levels in the experiment shown were 6.06, 6.24, and 5.45 ng/106 cells to the TN/TN, AS/AS, and TN/AS cells, respectively. In the same experiment, another group of cells was transfected with hTSHR (using a plasmid concentration that, based on flow cytometry, yielded similar cell surface expression as the three hFSHR cell groups). The cells were incubated with the indicated concentrations of rhTSH for 1 h at 37 C, and intracellular cAMP was then assayed as described in Patients and Methods. The data shown are the mean ⫾ SEM of triplicate determinations within one experiment that is representative of two independent experiments. Panel A depicts all the data from the experiment. In panel B, only those data from cells transfected with the hFSHR allelic combinations or empty vector are shown. Note the different scales on the x- and y-axes in the two panels.

by elevated concentrations of TSH, we did not detect a mutation in the hFSHR gene in any of these patients. De Leener et al. (14) also failed to find a mutation in the hFSHR gene of two adult patients presenting with spontaneous ovarian hyperstimulation and severe primary hypothyroidism. Therefore, hFSHR mutations are unlikely to be the cause of the relatively common phenomena of juvenile primary hypothyroidism and ovarian hyperstimulation and the less common finding of gonadal hyperstimulation in hypothyroid adults. The hFSHR has been shown to have polymorphisms at codons 307 and 680, and the hFSHR alleles exist as either Thr307-Asn680 (TN) or Ala307-Ser680 (AS) (27). Therefore, individuals are homozygous TN/TN, homozygous AS/AS, or heterozygous TN/AS for the hFSHR. Although our patient population was too small to determine whether there is a relatively higher allelic frequency of a given hFSHR polymorphism associated with primary hypothyroidism and ovarian hyperstimulation than normal, in vitro experiments were performed to determine whether a given hFSHR allelic combination rendered the hFSHR more sensitive to TSH. Examining HEK293 cells expressing the wt hFSHR as TN/ TN, AS/AS, or TN/AS, we observed specific and dose-dependent increases in cAMP in response to supramaximal

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concentrations of rhTSH. Our results further showed that, at the concentrations of rhTSH used, there were no differences in the sensitivities of the hFSHR allelic combinations to rhTSH. The concentrations of rhTSH required to elicit a response of the wt hFSHR, although much higher than those required to stimulate the hTSHR (Fig. 2A), are similar to those reported by De Leener et al. (14) when testing the rhTSH responsiveness of one (not specified) polymorphic variant of the wt hFSHR. Our data suggest that the ovarian hyperstimulation mechanism in patients with severe primary hypothyroidism occurs via the activation of the wt hFSHR by the abnormally high concentrations of TSH. Our results further demonstrate that the relative sensitivity of the wt hFSHR to TSH is independent of hFSHR polymorphisms. It should be noted, though, that the concentrations of rhTSH required to stimulate the wt hFSHR in vitro are higher than the levels of TSH measured in our hypothyroid patients with OHSS. This apparent discrepancy may be due to different assays used to determine the specific activities of the TSH standard used in clinical assays compared with the commercial preparation of rhTSH. Also, the relative expression of receptor, Gs, and adenylyl cyclase may be different between the transfected cells and the gonadal cells, perhaps causing the gonadal hFSHR to be more sensitive to TSH. Finally, there may be other actions mediated by high levels of TSH that cause the gonadal hFSHR to be more sensitive to TSH in vivo than would be predicted by the in vitro experiments. It should be pointed out, however, that very low elevations in cAMP may have profound physiopathological consequences. For example, activating mutations of the hLH receptor cause gonadotropin-independent precocious puberty in young boys, although in vitro many of these mutations only cause 2- to 4-fold increases in intracellular cAMP levels (see Refs. 28 –31 for examples). Therefore, the manifestation of OHSS may arise when the hFSHR is stimulated only modestly by TSH. It is most likely, therefore, that the extremely high concentrations of TSH in patients with severe primary hypothyroidism are sufficient to cause promiscuous activation of wt hFSHR, regardless of the allelic variant, and to produce gonadal enlargement with multiple cysts, with or without downstream estrogenic effects mimicking puberty. As others have previously suggested, it may be that the peripubertal gonad is particularly susceptible to stimulation by TSH because it is primed to be activated by the very low concentrations of FSH that initially occur only nocturnally with the onset of puberty (5, 6). Thus, this clinical phenomenon is most commonly seen in the pediatric and adolescent population rather than in adults. Acknowledgments We thank Luciana Sarmento Drumond, Elizabeth A. Suarez, and Francesco De Luca for referring their patients for molecular analyses, and Nathan Johnson for technical assistance. Received September 25, 2006. Accepted March 7, 2007. Address all correspondence and requests for reprints to: Deborah L. Segaloff, Ph.D., Department of Molecular Physiology and Biophysics, The University of Iowa Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52246. E-mail: [email protected].


This work was supported by National Institutes of Health Grant HD22196 (to D.L.S.) and Conselho Nacional de Desenvolvimento em Cieˆncias Tecnolo´gicas-CNPq Grant 300469/2005-5 (to A.C.L.). Disclosure Summary: All authors have nothing to disclose.

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