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Context: Adipose steroid metabolism modifies body fat development in polycystic ovary syndrome (PCOS)., Objective: To determine whether subcutaneous (SC) abdominal adipose aldo-keto reductase 1C3 (AKR1C3; a marker of testosterone generation) is increased in normal-weight women with PCOS vs age- and body mass index (BMI)-matched normoandrogenic ovulatory women (controls) and is related to SC abdominal adipose activator protein-1 (AP-1; a marker of adipocyte differentiation) and/or androgen receptor (AR) protein expression in predicting fat accretion., Design: Prospective cohort study., Setting: Academic center., Patients: Eighteen normal-weight PCOS women; 17 age- and BMI-matched controls., Interventions: Circulating hormone/metabolic determinations, intravenous glucose tolerance testing, total body dual-energy x-ray absorptiometry, SC abdominal fat biopsy, immunohistochemistry., Main Outcome Measures: Clinical characteristics, hormonal concentrations, body fat distribution, SC adipose AKR1C3, AR, and AP-1 protein expression., Results: Women with PCOS had significantly higher serum androgen levels and greater android/gynoid fat mass ratios than controls. SC adipose AKR1C3, AR, and AP-1 protein expressions were comparable between the study groups, but groups differed in correlations. In PCOS women vs controls, SC adipose AKR1C3 protein expression correlated positively with android and gynoid fat masses and negatively with SC adipose AP-1 protein expression. SC adipose AR protein expression correlated negatively with fasting serum free fatty acid and high-density lipoprotein levels. In both study groups, SC adipose AKR1C3 protein expression negatively correlated with serum cortisol levels., Conclusion: In normal-weight PCOS women, SC abdominal adipose AKR1C3 protein expression, in combination with intra-adipose AP-1 and AR-dependent events, predicts fat accretion in the presence of physiological cortisol levels., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Polycystic ovary syndrome (PCOS) is a common endocrinopathy of reproductive-aged women, characterized by hyperandrogenism, oligo-anovulation and insulin resistance and closely linked with preferential abdominal fat accumulation. As an ancestral primate trait, PCOS was likely further selected in humans when scarcity of food in hunter-gatherers of the late Pleistocene additionally programmed for enhanced fat storage to meet the metabolic demands of reproduction in later life. As an evolutionary model for PCOS, healthy normal-weight women with hyperandrogenic PCOS have subcutaneous (SC) abdominal adipose stem cells that favor fat storage through exaggerated lipid accumulation during development to adipocytes in vitro. In turn, fat storage is counterbalanced by reduced insulin sensitivity and preferential accumulation of highly lipolytic intra-abdominal fat in vivo. This metabolic adaptation in PCOS balances energy storage with glucose availability and fatty acid oxidation for optimal energy use during reproduction; its accompanying oligo-anovulation allowed PCOS women from antiquity sufficient time and strength for childrearing of fewer offspring with a greater likelihood of childhood survival. Heritable PCOS characteristics are affected by today's contemporary environment through epigenetic events that predispose women to lipotoxicity, with excess weight gain and pregnancy complications, calling for an emphasis on preventive healthcare to optimize the long-term, endocrine-metabolic health of PCOS women in today's obesogenic environment.

Context: Ovarian and adrenal steroidogenesis underlie endocrine-metabolic dysfunction in polycystic ovary syndrome (PCOS). Adipocytes express aldo-keto reductase 1C3 and type 1 11β-hydroxysteroid dehydrogenase, which modulate peripheral androgen and cortisol production., Objectives: To compare serum adrenal steroids, including 11-oxygenated androgens (11-oxyandrogens), cortisol, and cortisone between normal-weight women with PCOS and body mass index- and age-matched ovulatory women with normal-androgenic profiles (controls), and assess whether adrenal steroids associate with abdominal adipose deposition., Design: Prospective, cross-sectional, cohort study., Setting: Academic medical center., Patients: Twenty normal-weight women with PCOS and 20 body mass index-/age-matched controls., Interventions: Blood sampling, IV glucose tolerance testing, and total-body dual-energy x-ray absorptiometry., Main Outcome Measures: Clinical characteristics, hormonal concentrations, and body fat distribution., Results: Women with PCOS had higher serum total/free testosterone (T) and androstenedione (A4) levels and a greater android/gynoid fat mass than controls (androgens P < .001; android/gynoid fat mass ratio, P = .026). Serum total/free T and A4 levels correlated positively with android/gynoid fat mass ratio in all women combined ( P < .025, all values). Serum 11ß-hydroxyA4, 11-ketoA4, 11ß-hydroxyT, 11-ketoT, cortisol, and cortisone levels were comparable between female types and unrelated to body fat distribution. Serum 11-oxyandrogens correlated negatively with % total body fat, but lost significance adjusting for cortisol. Serum cortisol levels, however, correlated inversely with android fat mass ( P = .021), with a trend toward reduced serum cortisol to cortisone ratio in women with PCOS vs controls ( P = .075), suggesting diminished 11β-hydroxysteroid dehydrogenase activity., Conclusion: Reduced cortisol may protect against preferential abdominal fat mass in normal-weight PCOS women with normal serum 11-oxyandrogens., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Objective: To examine whether low-dose flutamide administration to normal-weight women with polycystic ovary syndrome (PCOS) reduces abdominal fat deposition, attenuates accelerated lipid accumulation in newly formed adipocytes derived from subcutaneous (SC) abdominal adipose stem cells (ASCs), and/or alters glucose-lipid metabolism., Design: A double-blind, placebo-controlled randomized clinical trial., Setting: An academic medical center., Patient(s): Twelve normal-weight women with PCOS and 12 age- and body mass index-matched controls., Intervention(s): Women underwent circulating hormonal and metabolic determinations, intravenous glucose tolerance testing, total body dual-energy roentgenogram absorptiometry, and SC abdominal fat biopsy. Interventions were repeated in women with PCOS after 6-month administration of flutamide (125 mg orally daily) vs. placebo., Main Outcome Measure(s): Clinical parameters and lipid accumulation in newly formed adipocytes derived from SC abdominal ASCs in vitro were compared between controls and the women with PCOS receiving flutamide vs. placebo., Results: Serum luteinizing hormone and androgen levels as well as lipid accumulation in newly formed SC abdominal adipocytes were greater in the women with PCOS than controls. Flutamide vs. placebo reduced percent android fat, lowered serum log low-density lipoprotein and log non-high-density lipoprotein levels, and increased fasting circulating glucose levels. In all women with PCOS, changes in percent android fat positively correlated with serum log non-high-density lipoprotein and log low-density lipoprotein levels, with correlations influenced by serum free testosterone levels. Flutamide vs. placebo also attenuated lipid accumulation in newly-formed PCOS SC abdominal adipocytes in vitro relative to controls, which was unrelated to serum lipid levels., Conclusion: Low-dose flutamide administration to normal-weight PCOS women reduces preferential abdominal fat deposition, attenuates accelerated lipid accumulation in newly-formed adipocytes derived from SC abdominal ASCs in vitro, and alters glucose-lipid homeostasis., Clinical Trial Registration Number: NCT01889199 (URL, clinicaltrials.gov; date of registration, 6/28/2013; enrollment date of first subject, 6/28/2013)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

As a common endocrinopathy of reproductive-aged women, polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, oligo-anovulation and polycystic ovarian morphology. It is linked with insulin resistance through preferential abdominal fat accumulation that is worsened by obesity. Over the past two millennia, menstrual irregularity, male-type habitus and sub-infertility have been described in women and confirm that these clinical features of PCOS were common in antiquity. Recent findings in normal-weight hyperandrogenic PCOS women show that exaggerated lipid accumulation by subcutaneous (SC) abdominal stem cells during development to adipocytes in vitro occurs in combination with reduced insulin sensitivity and preferential accumulation of highly-lipolytic intra-abdominal fat in vivo. This PCOS phenotype may be an evolutionary metabolic adaptation to balance energy storage with glucose availability and fatty acid oxidation for optimal energy use during reproduction. This review integrates fundamental endocrine-metabolic changes in healthy, normal-weight PCOS women with similar PCOS-like traits present in animal models in which tissue differentiation is completed during fetal life as in humans to support the evolutionary concept that PCOS has common ancestral and developmental origins., (© 2022. The Author(s).)

Context: Increased aldo-keto reductase 1C3 (AKR1C3)-mediated conversion of androstenedione (A4) to testosterone (T) promotes lipid storage in subcutaneous (SC) abdominal adipose in overweight/obese polycystic ovary syndrome (PCOS) women., Objective: This work examines whether an elevated serum T/A4 ratio, as a marker of enhanced AKR1C3 activity in SC abdominal adipose, predicts metabolic function in normal-weight PCOS women., Methods: This prospective cohort study took place in an academic center and comprised 19 normal-weight PCOS women and 21 age- and body mass index-matched controls. Interventions included circulating hormone/metabolic determinations, intravenous glucose tolerance testing, total body dual-energy x-ray absorptiometry, and SC abdominal fat biopsy. Serum T/A4 ratios, hormone/metabolic measures, and AKR1C3 expression of adipocytes matured in vitro were compared between female types; serum T/A4 ratios were correlated with serum lipids, adipose insulin resistance (adipose-IR), homeostatic model assessment of insulin resistance (HOMA-IR) and insulin sensitivity (Si)., Results: Increased serum T/A4 ratios ( P  = .040) and log adipose-IR values ( P  = .002) in PCOS women vs controls were accompanied by AKR1C3 messenger RNA overexpression of PCOS adipocytes matured in vitro ( P  = .016). Serum T/A4 ratios in PCOS women, but not controls, negatively correlated with log triglycerides (TGs: R = -0.65, P  = .002) and the TG index (R   = -0.57, P  = .011). Adjusting for serum free T, serum T/A4 ratios in PCOS women remained negatively correlated with log TG (R = -0.57, P  = .013) and TG index (R = -0.50, P  = .036), respectively, without significant relationships with other metabolic measures., Conclusion: An elevated serum T/A4 ratio, as a marker of enhanced AKR1C3 activity in SC abdominal adipose, predicts healthy metabolic function in normal-weight PCOS women., (; The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Objective: To examine whether subcutaneous (SC) abdominal adipose stem cell differentiation into adipocytes in vitro predicts insulin sensitivity (Si) in vivo in normal-weight women with polycystic ovary syndrome (PCOS) and controls., Design: Prospective cohort study., Setting: Academic medical center., Patient(s): Eight normal-weight women with PCOS and 8 age- and body mass index-matched controls., Intervention(s): Women underwent circulating hormone/metabolic determinations, intravenous glucose tolerance testing, total-body dual-energy x-ray absorptiometry, and SC abdominal fat biopsy., Main Outcome Measure(s): PPARγ and CEBPa gene expression and lipid content of adipocytes matured in vitro were compared between women with PCOS and control women, and correlated with patient characteristics, systemic Si, and adipose insulin resistance (adipose-IR)., Result(s): Serum androgen levels, adipose-IR, and percentage of android fat were greater in women with PCOS than control women. Stem cell PPARγ and CEBPa gene expression increased maximally by day 12 without a female-type effect. In control cells, gene expression positively correlated with fasting serum insulin levels (both genes) and adipose-IR (CEBPa) and negatively correlated with Si (CEBPa). Conversely, CEBPa gene expression in PCOS cells negatively correlated with adipose-IR and serum free testosterone, whereas total lipid accumulation in these cells positively corelated with Si., Conclusion: In normal-weight women with PCOS, accelerated SC abdominal adipose stem cell differentiation into adipocytes in vitro favors Si in vivo, suggesting a role for hyperandrogenism in the evolution of metabolic thrift to enhance fat storage through increased cellular glucose uptake., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Normal-weight polycystic ovary syndrome (PCOS) women exhibit adipose resistance in vivo accompanied by enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with accelerated lipid accumulation per cell in vitro. The present study examines chromatin accessibility, RNA expression and fatty acid (FA) synthesis during SC abdominal ASC differentiation into adipocytes in vitro of normal-weight PCOS versus age- and body mass index-matched normoandrogenic ovulatory (control) women to study epigenetic/genetic characteristics as well as functional alterations of PCOS and control ASCs during adipogenesis., Results: SC abdominal ASCs from PCOS women versus controls exhibited dynamic chromatin accessibility during adipogenesis, from significantly less chromatin accessibility at day 0 to greater chromatin accessibility by day 12, with enrichment of binding motifs for transcription factors (TFs) of the AP-1 subfamily at days 0, 3, and 12. In PCOS versus control cells, expression of genes governing adipocyte differentiation (PPARγ, CEBPα, AGPAT2) and function (ADIPOQ, FABP4, LPL, PLIN1, SLC2A4) was increased two-sixfold at days 3, 7, and 12, while that involving Wnt signaling (FZD1, SFRP1, and WNT10B) was decreased. Differential gene expression in PCOS cells at these time points involved triacylglycerol synthesis, lipid oxidation, free fatty acid beta-oxidation, and oxidative phosphorylation of the TCA cycle, with TGFB1 as a significant upstream regulator. There was a broad correspondence between increased chromatin accessibility and increased RNA expression of those 12 genes involved in adipocyte differentiation and function, Wnt signaling, as well as genes involved in the triacylglycerol synthesis functional group at day 12 of adipogenesis. Total content and de novo synthesis of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), and oleic (C18:1) acid increased from day 7 to day 12 in all cells, with total content and de novo synthesis of FAs significantly greater in PCOS than controls cells at day 12., Conclusions: In normal-weight PCOS women, dynamic chromatin remodeling of SC abdominal ASCs during adipogenesis may enhance adipogenic gene expression as a programmed mechanism to promote greater fat storage.

Anti-Müllerian hormone (AMH) is produced by granulosa cells of pre-antral and small antral ovarian follicles. In polycystic ovary syndrome (PCOS), higher levels of serum AMH are usually encountered due to the ample presence of small antral follicles and a high AMH production per follicular unit which have led to the proposal of AMH as a serum diagnostic marker for PCOS or as a surrogate for polycystic ovarian morphology (PCOM). However, heterozygous coding mutations of the AMH gene with decreased in vitro bioactivity have been described in some women with PCOS. Such mutation carriers have a trend toward reduced serum AMH levels compared to noncarriers, although both types of women with PCOS have similar circulating gonadotropin and testosterone (T) levels. This report describes a normal-weight woman with PCOS by NIH criteria with severely reduced AMH levels (index woman with PCOS). Our objective was to examine the molecular basis for her reduced serum AMH levels and to compare her endocrine characteristics to similar-weight women with PCOS and detectable AMH levels. Twenty normoandrogenic ovulatory (control) and 13 age- and BMI-matched women with PCOS (19-35 years; 19-25 kg/m2) underwent transvaginal sonography and serum hormone measures including gonadotropins, sex hormone-binding globulin, total and free T, androstenedione, dehydroepiandrosterone sulfate, estrone, estradiol and AMH. The latter was measured by ELISA (Pico-AMH: Ansh Labs, Webster, TX, USA). Women with PCOS and detectable AMH had higher serum AMH (10.82 (6.74-13.40) ng/ml, median (interquartile range)), total and free T (total T: 55.5 (49.5-62.5) ng/dl; free T: 5.65 (4.75-6.6) pg/ml) levels and greater total antral follicle count (AFC) (46 (39-59) follicles) than controls (AMH: 4.03 (2.47-6.11) ng/ml; total T: 30 (24.5-34.5) ng/dl; free T: 2.2 (1.8-2.45) pg/ml; AFC 16 (14.5-21.5) follicles, P < 0.05, all values), along with a trend toward LH hypersecretion (P = 0.06). The index woman with PCOS had severely reduced serum AMH levels (∼0.1 ng/ml), although she also had a typical NIH-defined PCOS phenotype resembling that of the other women with PCOS and elevated AMH levels. All women with PCOS, including the index woman with PCOS, exhibited LH hypersecretion, hyperandrogenism, reduced serum estrogen/androgen ratios and PCOM. A homozygous Ala515Val variant (rs10417628) in the mature region of AMH was identified in the index woman with PCOS. Recombinant hAMH-515Val displayed normal processing and bioactivity, yet had severely reduced immunoactivity when measured by the commercial pico-AMH ELISA assay by Ansh Labs. In conclusion, homozygous AMH variant rs10417628 may severely impair serum AMH immunoactivity without affecting its bioactivity or PCOS phenotypic expression. Variants in AMH can interfere with serum AMH immunoactivity without affecting the phenotype in PCOS. This observation can be accompanied by discordance between AMH immunoactivity and bioactivity., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, oligo-anovulation and polycystic ovarian morphology, with metabolic dysfunction from insulin resistance and abdominal fat accumulation worsened by obesity. As ancestral traits, these features could have favored abdominal fat deposition for energy use during starvation, but have evolved into different PCOS phenotypes with variable metabolic dysfunction. Adipose dysfunction in PCOS from hyperandrogenemia and hyperinsulinemia likely constrains subcutaneous (SC) fat storage, promoting lipotoxicity through ectopic lipid accumulation and oxidative stress, insulin resistance and inflammation in non-adipose tissue. Recent findings of inherently exaggerated SC abdominal stem cell development to adipocytes in women with PCOS, and PCOS-like traits in adult female monkeys with natural hyperandrogenemia, imply common ancestral origins of PCOS in both human and nonhuman primates.

Developmental origins of adult disease (DoHAD) refers to critical gestational ages during human fetal development and beyond when the endocrine metabolic status of the mother can permanently program the physiology and/or morphology of the fetus, modifying its susceptibility to disease after birth. The aim of this review is to address how DoHAD plays an important role in the phenotypic expression of polycystic ovary syndrome (PCOS), the most common endocrinopathy of women characterized by hyperandrogenism, oligo-anovulation and polycystic ovarian morphology. Clinical studies of PCOS women are integrated with findings from relevant animal models to show how intergenerational transmission of these central components of PCOS are programmed through an altered maternal endocrine-metabolic environment that adversely affects the female fetus and long-term offspring health. Prenatal testosterone treatment in monkeys and sheep have been particularly crucial in our understanding of developmental programming of PCOS because organ system differentiation in these species, as in humans, occurs during fetal life. These animal models, along with altricial rodents, produce permanent PCOS-like phenotypes variably characterized by LH hypersecretion from reduced steroid-negative feedback, hyperandrogenism, ovulatory dysfunction, increased adiposity, impaired glucose-insulin homeostasis and other metabolic abnormalities. The review concludes that DoHAD underlies the phenotypic expression of PCOS through an altered maternal endocrine-metabolic environment that can induce epigenetic modifications of fetal genetic susceptibility to PCOS after birth. It calls for improved maternal endocrine-metabolic health of PCOS women to lower their risks of pregnancy-related complications and to potentially reduce intergenerational susceptibility to PCOS and its metabolic derangements in offspring.

Context: Ovarian hyperandrogenism from polycystic ovary syndrome (PCOS) and hyperinsulinemia from insulin resistance are modulators of ovarian follicle development. We report on a woman with PCOS and hyperandrogenism and severe insulin resistance from metabolic syndrome who received long-term GnRH analogue therapy preceding bilateral salpingo-oophorectomy for massive ovarian enlargement. Ovarian histological examination showed proliferating granulosa cells within antral follicles coexistent with serous cystadenofibromas, demonstrating a unique link between hyperinsulinemia and granulosa cell mitogenesis., Case Description: A 30-year-old woman with PCOS with hyperandrogenism, severe insulin resistance from metabolic syndrome, and nonalcoholic steatohepatitis experienced abdominal pain from bilaterally enlarged ovaries. She had previously experienced a pulmonary embolism while taking oral contraceptives and hepatotoxicity from metformin and spironolactone therapies. Long-term GnRH analogue therapy to induce pituitary desensitization to GnRH successfully decreased gonadotropin-dependent steroidogenesis without improving insulin resistance. Despite GnRH analogue therapy, progressive ovarian enlargement in the presence of hyperinsulinemia from worsening metabolic function eventually required bilateral salpingo-oophorectomy for removal of massively enlarged ovaries. Histological examination showed both ovaries contained proliferating granulosa cells within antral follicles coexistent with serous cystadenofibromas., Conclusions: In women with PCOS and hyperinsulinemia from severe insulin resistance due to metabolic syndrome, granulosa cell proliferation within antral follicles can occur despite long-term GnRH analogue therapy, implicating hyperinsulinemia as a granulosa cell mitogen in the absence of gonadotropin-dependent ovarian function., (Copyright © 2019 Endocrine Society.)

Context: Normal-weight women with polycystic ovary syndrome (PCOS) may have adipose tissue insulin resistance (adipose-IR)., Objective: To examine whether adipose-IR and subcutaneous (SC) abdominal adipose stem cell (ASC) gene expression are altered in normal-weight women with PCOS and correlated with hyperandrogenemia and/or whole-body IR., Design: Prospective cohort study., Setting: Academic medical center., Patients: Ten normal-weight women with PCOS and 18 control subjects matched for age and body mass index., Intervention(s): Women underwent circulating hormone and metabolic measurements, IV glucose tolerance testing, total-body dual-energy x-ray absorptiometry, and SC abdominal fat biopsy., Main Outcome Measure(s): Adipose-IR (fasting insulin × total fatty acid levels) and SC abdominal ASC gene expression were compared between groups and correlated with clinical outcomes., Results: Adipose-IR was greater in women with PCOS than in control subjects (P < 0.01), with 29 pmol/L × mmol/L providing 94% specificity and 80% sensitivity in discriminating the two groups (P < 0.001). Adipose-IR positively correlated with serum androgen and log of fasting triglyceride (TG) levels, percentage of small adipocytes (P < 0.01, all correlations), and acute insulin response to glucose (P < 0.05); and negatively correlated with insulin sensitivity (Si; P < 0.025) and serum adiponectin levels (P < 0.05). Adjusting for serum androgens, adipose-IR correlations with Si and log TG levels remained significant. ASC genes were differentially expressed by the two groups. Expression of functionally critical genes was associated with serum testosterone and/or fasting insulin levels., Conclusion: Normal-weight women with PCOS have increased adipose-IR and altered ASC gene expression related to hyperandrogenism and IR., (Copyright © 2019 Endocrine Society.)

Importance: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy of reproductive-aged women. Women with PCOS are at increased risk of developing several metabolic and reproductive abnormalities, including metabolic syndrome. Underlying the combined metabolic and reproductive dysfunction is lipotoxicity, defined as the ectopic deposition of lipid in nonadipose tissue where it induces oxidative stress linked with insulin resistance and inflammation., Objective: To examine what metabolic components underlie insulin resistance in PCOS, how lipotoxicity through insulin resistance impairs metabolism and reproduction in these women, and why evidence-based, individualized management is essential for their care., Evidence Acquisition: PubMed search was performed using relevant terms to identify journal articles related to the subject. Relevant textbook chapters were also used., Results: Polycystic ovary syndrome by Rotterdam criteria represents a complex syndrome of heterogeneous expression with variable adverse metabolic and reproductive implications. Women with classic PCOS are often insulin resistant and at greatest risk of developing metabolic syndrome with preferential fat accumulation and weight gain. Moreover, PCOS women may also have an altered capacity to properly store fat, causing ectopic lipid accumulation in nonadipose tissue, including the ovaries, where it can perpetuate insulin resistance and inflammation and harm the oocyte., Conclusions and Relevance: A personalized approach to managing PCOS is essential to improve the health of all PCOS women through cost-effective prevention and/or treatment, to minimize the risk of pregnancy complications in those individuals wishing to conceive, and to optimize the long-term health of PCOS women and their offspring.

Objective: To examine whether abnormal subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes in polycystic ovary syndrome (PCOS) correlates with hyperandrogenism., Design: Prospective cohort study., Setting: Academic medical center., Patient(s): Eight normal-weight women with PCOS and eight normoandrogenic ovulatory (control) women matched for age and body mass index., Intervention(s): Circulating hormone and metabolic measurements, intravenous glucose tolerance testing, total body dual-energy X-ray absorptiometry, and SC abdominal fat biopsy., Main Outcome Measure(s): In vitro ASC commitment to preadipocytes (ZFP423 protein expression, day 0.5), preadipocyte differentiation to adipocytes (PPARγ gene expression, day 3) and adipocyte lipid content (Oil-Red-O fluorescence, day 12) comparisons correlated with clinical outcomes., Result(s): In women with PCOS, SC abdominal ASCs compared with those of control women showed exaggerated commitment to preadipocytes and had greater lipid content in newly formed adipocytes after in vitro maturation. In all women combined, ZFP423 protein expression negatively correlated with fasting plasma glucose levels whereas the lipid content of newly formed adipocytes positively correlated with both PPARγ gene expression and serum free testosterone levels., Conclusion(s): In normal-weight women with PCOS compared with the control group, exaggerated SC abdominal ASC commitment to preadipocytes and enhanced adipocyte lipid content during maturation in vitro negatively and positively correlate with circulating fasting glucose and androgen levels, respectively, as a possible mechanism to maintain glucose-insulin homeostasis when fat accretion is accelerated., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the degree to which E 2 hyperresponsiveness to FSH and antimüllerian hormone (AMH) overproduction in normal-weight women with polycystic ovary syndrome (PCOS) correlate with increased antral follicle number (AFN), hyperandrogenism, and/or metabolic dysfunction., Design: Prospective cohort study., Setting: Academic medical center., Patient(s): Seven normal-weight women with PCOS (1990 National Institutes of Health criteria) ages 20-34 years and 13 age- and body mass index- (BMI-; 18.5-25 kg/m 2 ) matched normoandrogenic ovulatory women were studied., Intervention(s): All women underwent basal serum hormone and metabolic measurements, FSH stimulation testing with transvaginal ovarian sonography, frequently sampled IV glucose tolerance testing, and whole-body dual-energy x-ray absorptiometry., Main Outcome Measure(s): Serum hormone/metabolite levels, 24-hour serum E 2 response to 150 IU recombinant human (rh) FSH infusion, AFN, insulin sensitivity, and body mass measurements., Result(s): Serum E 2 responsiveness to rhFSH and AMH levels were greater in women with PCOS than in BMI- and age-matched control women, as were serum androgen levels, AFN, and abdominal fat mass. In all women combined, serum E 2 responsiveness to rhFSH was associated with AFN. Serum AMH levels, however, positively correlated with AFN but remained positively correlated with serum LH and free T levels and negatively correlated with total body fat and percent body fat, adjusting for AFN., Conclusion(s): In normal-weight women with PCOS, serum E 2 hyperresponsiveness to rhFSH represents increased AFN, while elevated serum AMH levels reflect opposing effects of stimulatory reproductive (hyperandrogenism and increased AFN) versus inhibitory metabolic (body fat) factors. Given the small number of subjects reported, additional follow-up studies are required to confirm these data., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Multilineage differentiating stress enduring (Muse) cells, discovered in the spring of 2010 at Tohoku University in Sendai, Japan, were quickly recognized by scientists as a possible source of pluripotent cells naturally present within mesenchymal tissues. Muse cells normally exist in a quiescent state, singularly activated by severe cellular stress in vitro and in vivo. Muse cells have the capacity for self-renewal while maintaining pluripotent cell characteristics indicated by the expression of pluripotent stem cell markers. Muse cells differentiate into cells representative of all three germ cell layers both spontaneously and under media-specific induction. In contrast to embryonic stem and induced pluripotent stem cells, Muse cells exhibit low telomerase activity, a normal karyotype, and do not undergo tumorigenesis once implanted in SCID mice. Muse cells efficiently home into damaged tissues and differentiate into specific cells leading to tissue regeneration and functional recovery as described in different animal disease models (i.e., fulminant hepatitis, muscle degeneration, skin ulcers, liver cirrhosis, cerebral stroke, vitiligo, and focal segmental glomerulosclerosis). Circulating Muse cells have been detected in peripheral blood, with higher levels present in stroke patients during the acute phase. Furthermore, Muse cells have inherent immunomodulatory properties, which could contribute to tissue generation and functional repair in vivo. Genetic studies in Muse cells indicate a highly conserved cellular mechanism as seen in more primitive organisms (yeast, Saccharomyces cerevisiae, Caenorhabditis elegans, chlamydomonas, Torpedo californica, drosophila, etc.) in response to cellular stress and acute injury. This review details the molecular and cellular properties of Muse cells as well as their capacity for tissue repair and functional recovery, highlighting their potential for clinical application in regenerative medicine.

Context: Normal weight polycystic ovary syndrome (PCOS) women may have altered adipose structure-function underlying metabolic dysfunction., Objective: This study examines whether adipose structure-functional changes exist in normal weight PCOS women and correlate with hyperandrogenism and/or hyperinsulinemia., Design: This is a prospective cohort study., Setting: The setting was an academic medical center., Patients: Six normal weight PCOS women and 14 age- and body mass index-matched normoandrogenic ovulatory (NL) women were included., Intervention(s): All women underwent circulating hormone and metabolic measurements; frequently sampled intravenous glucose tolerance testing; total body dual-energy x-ray absorptiometry; abdominal magnetic resonance imaging; and SC abdominal fat biopsy., Main Outcome Measure(s): Circulating hormones and metabolites, body fat and its distribution, and adipocyte size were compared between PCOS and NL women, and were correlated with each other in all women., Results: Circulating LH and androgen levels were significantly greater in PCOS than NL women, as were fasting insulin levels, pancreatic β-cell responsiveness to glucose, and total abdominal fat mass. Intra-abdominal fat mass also was significantly increased in PCOS women and was positively correlated with circulating androgen, fasting insulin, triglyceride, and non-high-density lipoprotein cholesterol levels in all women. SC abdominal fat mass was not significantly increased in PCOS women, but contained a greater proportion of small SC abdominal adipocytes that positively correlated with serum androgen levels in all women., Conclusion: Hyperandrogenism in normal weight PCOS women is associated with preferential intra-abdominal fat deposition and an increased population of small SC abdominal adipocytes that could constrain SC adipose storage and promote metabolic dysfunction.

Context: Complex cumulus cell-oocyte interactions govern energy utilization during oocyte development., Objective: This study investigates the relationship of cumulus cell mitochondria with oocyte development during ovarian stimulation for in vitro fertilization (IVF)., Design: This is a prospective cohort study., Setting: The setting was an academic center., Patients: Thirty women underwent ovarian stimulation for IVF., Intervention(s): Pooled cumulus cells were collected; numbers of total and mature oocytes and two-pronuclear (day 1), six- to eight-cell cleavage (day 3), and blastocyst (day 5) embryos were recorded., Main Outcome Measure(s): A mitochondrial bioassay was developed with Jurkat cells and used with cumulus cells from IVF patients to correlate mitochondrial membrane potential resistance to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) stress with oocyte development and embryogenesis., Results: Adjusting for FSH administered and maternal age, cumulus cell mitochondrial membrane potential resistance to CCCP positively correlated with numbers of total (P < .025) and mature (P < .025) oocytes retrieved. The highest oocyte numbers that correlated with cumulus cell mitochondrial membrane potential occurred in women with the greatest ovarian response to FSH (mitochondrial membrane potential resistance to CCCP-log FSH interactions: total oocytes P < .025; mature oocytes P < .05). Multiple regression modeling of mature oocyte numbers, age, and cumulus cell mitochondrial membrane potential resistance to CCCP showed that numbers of mature oocytes best correlated with numbers of embryos at all stages (P < .0001)., Conclusion: During ovarian stimulation for IVF, cumulus cell mitochondrial membrane potential resistance to stress correlates with numbers of total and mature oocytes retrieved, suggesting that cumulus cell-oocyte interactions involving energy facilitate oocyte development.

Muse cells are a novel population of nontumorigenic pluripotent stem cells, highly resistant to cellular stress. These cells are present in every connective tissue and intrinsically express pluripotent stem markers such as Nanog, Oct3/4, Sox2, and TRA1-60. Muse cells are able to differentiate into cells from all three embryonic germ layers both spontaneously and under media-specific induction. Unlike ESCs and iPSCs, Muse cells exhibit low telomerase activity and asymmetric division and do not undergo tumorigenesis or teratoma formation when transplanted into a host organism. Muse cells have a high capacity for homing into damaged tissue and spontaneous differentiation into cells of compatible tissue, leading to tissue repair and functional restoration. The ability of Muse cells to restore tissue function may demonstrate the role of Muse cells in a highly conserved cellular mechanism related to cell survival and regeneration, in response to cellular stress and acute injury. From an evolutionary standpoint, genes pertaining to the regenerative capacity of an organism have been lost in higher mammals from more primitive species. Therefore, Muse cells may offer insight into the molecular and evolutionary bases of autonomous tissue regeneration and elucidate the molecular and cellular mechanisms that prevent mammals from regenerating limbs and organs, as planarians, newts, zebrafish, and salamanders do., Competing Interests: The authors declare that they have no competing interests. Gregorio D. Chazenbalk is a consultant for ClusterXStem Inc.

Objective: To determine whether follicular fluid (FF) cortisol levels affect cumulus cell (CC) lipid content during oocyte meiotic resumption, and whether CCs express genes for glucocorticoid action., Design: Prospective cohort study., Setting: Academic medical center., Patient(s): Thirty-seven nonobese women underwent ovarian stimulation for in vitro fertilization (IVF)., Intervention(s): At oocyte retrieval, FF was aspirated from the first follicle (>16 mm in size) of each ovary and pooled CCs were collected., Main Outcome Measure(s): Follicular fluid cortisol and cortisone analysis was performed with the use of liquid chromatography-tandem mass spectrometry. CCs were stained with lipid fluorescent dye Bodipy FL C16 to determine lipid content with the use of confocal microscopy. Quantitative real-time polymerase chain reaction was used to detect CC gene expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2, glucocorticoid receptor (NR3C1), lipoprotein lipase (LPL), and hormone-sensitive lipase (HSL)., Result(s): Adjusting for maternal age, FF cortisol levels negatively correlated with CC lipid content and positively correlated with numbers of total and mature oocytes. CCs expressed genes for 11β-HSD type 1 as the predominant 11β-HSD isoform, NR3C1, LPL, and HSL., Conclusion(s): FF cortisol levels may regulate CC lipolysis during oocyte meiotic resumption and affect oocyte quality during IVF., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Introduction: Embryonic stem cells and induced pluripotent stem cells have emerged as the gold standard of pluripotent stem cells and the class of stem cell with the highest potential for contribution to regenerative and therapeutic application; however, their translational use is often impeded by teratoma formation, commonly associated with pluripotency. We discuss a population of nontumorigenic pluripotent stem cells, termed Multilineage Differentiating Stress Enduring (Muse) cells, which offer an innovative and exciting avenue of exploration for the potential treatment of various human diseases., Areas Covered: This review discusses the origin of Muse cells, describes in detail their various unique characteristics, and considers future avenues of their application and investigation with respect to what is currently known of adult pluripotent stem cells in scientific literature. We begin by defining cell potency, then discuss both mesenchymal and various reported populations of pluripotent stem cells, and finally delve into Muse cells and the characteristics that set them apart from their contemporaries., Expert Opinion: Muse cells derived from adipose tissue (Muse-AT) are efficiently, routinely and painlessly isolated from human lipoaspirate material, exhibit tripoblastic differentiation both spontaneously and under media-specific induction, and do not form teratomas. We describe qualities specific to Muse-AT cells and their potential impact on the field of regenerative medicine and cell therapy.

Metabolic characteristics of polycystic ovary syndrome women and polycystic ovary syndrome-like, prenatally androgenized (PA) female monkeys worsen with age, with altered adipogenesis of sc abdominal adipose potentially contributing to age-related adverse effects on metabolism. This study examines whether adipocyte morphology and gene expression in sc abdominal adipose differ between late reproductive-aged PA female rhesus monkeys compared with age-matched controls (C). Subcutaneous abdominal adipose of both groups was obtained for histological imaging and mRNA determination of zinc finger protein 423 (Zfp423) as a marker of adipose stem cell commitment to preadipocytes, and CCAAT/enhancer binding protein (C/EBP)α/peroxisome proliferator-activated receptor (PPAR)δ as well as C/EBPα/PPARγ as respective markers of early- and late-stage differentiation of preadipocytes to adipocytes. In all females combined, serum testosterone (T) levels positively correlated with fasting serum levels of total free fatty acid (r(2) = 0.73, P < .002). PA females had a greater population of small adipocytes vs C (P < .001) in the presence of increased Zfp423 (P < .025 vs C females) and decreased C/EBPα (P < .003, vs C females) mRNA expression. Moreover, Zfp423 mRNA expression positively correlated with circulating total free fatty acid levels during iv glucose tolerance testing (P < .004, r(2) = 0.66), whereas C/EBPα mRNA expression negatively correlated with serum T levels (P < .02, r(2) = 0.43). Gene expression of PPARδ and PPARγ were comparable between groups (P = .723 and P = .18, respectively). Early-to-mid gestational T excess in female rhesus monkeys impairs adult preadipocyte differentiation to adipocytes in sc abdominal adipose and may constrain the ability of this adipose depot to safely store fat with age.

In 2010, Multilineage Differentiating Stress Enduring (Muse) cells were introduced to the scientific community, offering potential resolution to the issue of teratoma formation that plagues both embryonic stem (ES) and induced pluripotent (iPS) stem cells. Isolated from human bone marrow, dermal fibroblasts, adipose tissue and commercially available adipose stem cells (ASCs) under severe cellular stress conditions, Muse cells self-renew in a controlled manner and do not form teratomas when injected into immune-deficient mice. Furthermore, Muse cells express classic pluripotency markers and differentiate into cells from the three embryonic germ layers both spontaneously and under media-specific induction. When transplanted in vivo, Muse cells contribute to tissue generation and repair. This review delves into the aspects of Muse cells that set them apart from ES, iPS, and various reported adult pluripotent stem cell lines, with specific emphasis on Muse cells derived from adipose tissue (Muse-AT), and their potential to revolutionize the field of regenerative medicine and stem cell therapy.

The maternal-fetal environment plays an important role in developmental programming of adult disease. Metabolic and hormonal dysfunction during human fetal development accompanies gestational diabetes as a common occurrence in mothers with polycystic ovary syndrome (PCOS), while human fetal androgen excess from congenital adrenal hyperplasia or virilizing tumors precedes PCOS-like symptoms after birth. To date, clinical studies of infant blood levels at term have yet to confirm that human fetal androgen excess promotes PCOS development after birth. Earlier in development, however, circulating androgen levels in the second trimester female human fetus can normally rise into the male range. Furthermore, midgestational amniotic testosterone levels are elevated in female fetuses of PCOS compared with normal mothers and might influence fetal development because experimentally induced fetal androgen excess in animals produces a PCOS-like phenotype with reproductive and metabolic dysfunction. Such alterations in the maternal-fetal environment likely program adult PCOS by epigenetic modifications of genetic susceptibility of the fetus to PCOS after birth. Understanding this phenomenon requires advanced fetal surveillance technologies and postnatal assessment of midgestational androgen exposure for new clinical strategies to improve reproduction in PCOS women, optimize long-term health of their offspring, and minimize susceptibility to acquiring PCOS in future generations., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Context: Mineralocorticoid synthesis by the nonhuman primate periovulatory follicle enhances luteinization. Whether a similar event occurs in women undergoing in vitro fertilization (IVF) is unknown., Objective: The objective of the study was to determine whether human luteinized granulosa cells (LGCs) produce mineralocorticoids derived from 21-hydroxylase activity and also express mRNA for 21-hydroxylase and the mineralocorticoid receptor., Design: This was a prospective cohort study., Setting: The study was conducted at an academic center., Patients: LGC lipid content and follicle fluid (FF) hormone analysis was performed on 27 nonobese IVF women. LGCs from six additional nonobese IVF women were used for gene expression studies., Intervention: At oocyte retrieval, FF was aspirated from the first follicle (≥16 mm in size) of each ovary and pooled LGCs were collected., Main Outcome Measures: FF steroid analysis was performed by liquid chromatography-tandem mass spectrometry. LGCs were stained with lipid fluorescent dye BODIPY FL C16 to estimate lipid content by confocal microscopy as a cholesterol source for steroidogenesis in vivo. Quantitative real-time PCR was performed using LGCs to detect 21-hydroxylase and mineralocorticoid receptor mRNA expression. Pearson correlation coefficients determined associations between FF steroid levels and LGC lipid content., Results: FF levels of the 21-hydroxylase-derived steroids, 11-deoxycorticosterone [DOC, 39.97, median (13.94-63.02) ng/mL] and 11-deoxycortisol [11DOC, 2.07 (0.69-5.01) ng/mL], along with the 21-hydroxylase precursor 17-hydroxyprogesterone [1268.21 (493.26-3558.39) ng/mL], positively correlated with LGC lipid content (84 ± 43 fluorescent units/sample) (P ≤ .05, all steroids). 21-Hydroxylase and mineralocorticoid receptor mRNA expression was detected in LGCs., Conclusions: Human LGCs likely synthesize 21-hydroxylase-derived mineralocorticoids from cholesterol-containing lipid in vivo to promote postovulatory luteinization via mineralocorticoid receptor-mediated events.

White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The prevailing concept is that, in human thyroid tissue in vivo, all cell-surface TSH receptors (TSHR) cleave into disulfide linked A and B subunits. Because this viewpoint is based on studies using homogenized thyroid tissue and because of TSHR fragility, we studied TSHR subunit structure in intact thyroid cells, primary human thyrocyte cultures, FRTL-5 rat thyroid cells, and WRO (follicular) and NPA (papillary) thyroid cancer cell lines. To overcome the handicap of very low TSHR expression in thyroid cells, we generated a TSHR-expressing adenovirus (TSHR-Ad-RGD) with an integrin-binding RGD motif enabling efficient entry into cells lacking the coxsackie-adenovirus receptor. Two days after TSHR-Ad-RGD infection, [125I]TSH cross-linking to intact cells revealed uncleaved, single-chain TSHR as well as cleaved TSHR A subunits on the surface of all four thyroid cell types. The extent of TSHR cleavage, which is independent of the level of TSHR expression, was consistently lower in the human thyroid cancer cell lines than in the other cell lines. In flow cytometry studies after TSHR-Ad-RGD infection, strong signals were detected in all four thyroid cell types using a monoclonal antibody that primarily recognizes the uncleaved TSHR. Finally, using the same monoclonal antibody, confocal microscopy confirmed the presence of single-chain TSHR on TSHR-Ad-RGD-infected thyroid cells. In summary, TSH covalent cross-linking, flow cytometry, and confocal microscopy demonstrate the presence of uncleaved TSHR on the human thyrocyte surface. These data provide stronger evidence for this alternative than the contrary view based on the finding of only cleaved TSHR in homogenized thyroid cells.

The thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves' hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, "non-self" regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.

Thyroid autoantigens require internalization and processing by antigen-presenting cells to induce immune responses. Besides pinocytosis, antigen uptake can be receptor-mediated. The mannose receptor (ManR) has a cysteine rich domain (CR) and eight carbohydrate recognition domains (CRD) that bind glycosylated proteins. The TSH receptor (TSHR), thyroid peroxidase (TPO) and thyroglobulin (Tg) are glycoproteins. To investigate a role for the ManR in thyroid autoimmunity, we tested the interaction between these autoantigens and chimeric ManRs. Plasmids encoding the CR-domain linked to IgG-Fc (CR-Fc) and CDR domains 4-7 linked to IgG-Fc (CDR4-7-Fc) were expressed and purified with Protein A. Enzyme-linked immunosorbent assay (ELISA) plates were coated with human thyroid autoantigens and CR-Fc or CRD4-7-Fc binding detected with peroxidase-conjugated anti-IgG-Fc. CRD4-7-Fc binding was highest for the TSHR, followed by Tg and was minimal for TPO. CR-Fc bound to Tg but not to TSHR or TPO. The interaction between the TSHR and CRD-Fc was calcium-dependent; it was inhibited by mannose (not galactose), and required a glycosylated TSHR A-subunit. Moreover, precomplexing the TSHR A-subunit with CRD-Fc (but not CR-Fc), or adding mannose (but not galactose), decreased in vitro responses of splenocytes from TSHR-immunized mice. Our data indicate that the ManR may participate in autoimmune responses to Tg and the TSHR but not to TPO. Most important, ManR binding of heavily glycosylated TSHR A-subunits suggests a mechanism by which the minute amounts of A-subunit protein shed from the thyroid may be captured by antigen-presenting cells located in the gland or in draining lymph nodes.

Naked DNA vaccination with the TSH receptor (TSHR) does not, in most studies, induce TSHR antibodies and never induces hyperthyroidism in BALB/c mice. Proteins expressed endogenously by vaccination are preferentially presented by major histocompatibility complex class I, but optimal T cell help for antibody production requires lysosomal processing and major histocompatibility complex class II presentation. To divert protein expression to lysosomes, we constructed a plasmid with the TSHR ectodomain spliced between the signal peptide and transmembrane-intracellular region of lysosome-associated membrane protein (LAMP)-1, a lysosome-associated membrane protein. BALB/c mice pretreated with cardiotoxin were primed intramuscularly using this LAMP-TSHR chimera and boosted twice with DNA encoding wild-type TSHR, TSHR A-subunit, or LAMP-TSHR. With each protocol, spleen cells responded to TSHR antigen by secreting interferon-gamma, and 60% or more mice had TSHR antibodies detectable by ELISA. TSH binding inhibitory activity was present in seven, four, and two of 10 mice boosted with TSHR A-subunit, LAMP-TSHR, or wild-type TSHR, respectively. Importantly, six of 30 mice had elevated T4 levels and goiter (5 of 6 with detectable thyroid-stimulating antibodies). Injecting LAMP-TSHR intradermally without cardiotoxin pretreatment induced TSHR antibodies detectable by ELISA but not by TSH binding inhibitory activity, and none became hyperthyroid. These findings are consistent with a role for cardiotoxin-recruited macrophages in which (unlike in fibroblasts) LAMP-TSHR can be expressed intracellularly and on the cell surface. In conclusion, hijacking the TSHR to lysosomes enhances T cell responses and TSHR antibody generation and induces Graves'-like hyperthyroidism in BALB/c mice by intramuscular naked DNA vaccination.

The thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8alpha transmembrane region. For this purpose, we covalently cross-linked(125)I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8alpha-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8alpha) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8alpha (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts.

We used purified recombinant TSH receptor (TSHR) antigen prepared in mammalian cells to affinity-enrich TSHR autoantibodies from Graves' patients' IgG. Autoantibody enrichment, assayed by TSH binding inhibitory activity, was 20- to 1000-fold. Thyroid-stimulating antibody activity enrichment, although more difficult to quantitate, was comparable. TSHR-autoantibody approximate affinities for the holoreceptor assessed indirectly by TSH binding inhibition were 4-27 x 10(-9) m, an underestimate because 100% TSHR autoantibody purity was not attained. Consistent with previous data for serum, highly enriched TSHR autoantibodies in three of four patients showed lambda light chain bias. However, in contrast to expectations, antigen-enriched IgG was skewed primarily toward IgG2 and IgG3, subclasses associated with polysaccharides and microorganisms, respectively. Subclass depletion studies on antigen-enriched IgG indicated that TSHR autoantibodies were predominantly IgG1 and, surprisingly, IgG4. As controls, we affinity-enriched pooled IgG from normal individuals on TSHR antigen. This enriched IgG had detectable TSH binding inhibitory activity, although with lower specific activity than, and lacking the thyroid stimulatory activity of, Graves' IgG. Moreover, these natural IgG class autoantibodies largely recognized the same conformational variation in the TSHR N-terminal region as disease-associated TSHR autoantibodies. These studies suggest that TSHR autoantibodies may arise from natural autoantibodies, possibly by class switching from cross-reacting antibodies to microorganisms.

Whether monoclonal antibodies with thyroid-stimulating activity [thyroid-stimulating antibody/antibodies (TSAb)] from immunized animals are identical to human autoantibodies in Graves' disease is unknown. Here, we compared properties of a monoclonal hamster TSAb (MS-1) with human autoantibodies. The epitopes of neither MS-1 nor human autoantibodies can be determined by peptide scanning, indicating their conformational nature. A property of human TSAb is that their epitope is partially obscured on the TSH holoreceptor on the cell surface relative to the TSH receptor (TSHR) ectodomain tethered to the membrane by a glycosylphosphatidyl inositol anchor. On flow cytometry, as for human autoantibodies, MS-1 preferentially recognized the glycosylphosphatidyl inositol-anchored ectodomain vs. the TSH holoreceptor on Chinese hamster ovary cells. Also, as with human autoantibodies, only A-subunits with the active (but not the inactive) conformation adsorbed MS-1 binding activity. This difference localizes antibody binding to a cysteine-rich region at the TSHR N terminus. Remarkably, active TSHR A-subunit more effectively ( approximately 40-fold) neutralized human autoantibodies than it did MS-1. Therefore, MS-1 interacts less well than autoantibodies with the free A-subunit. In summary, we provide evidence that TSAb need not have identical epitopes. However, the TSAb epitope does appear to require involvement of the highly conformational N terminus of the A-subunit.

Immunization with adenovirus expressing the TSH receptor (TSHR) induces hyperthyroidism in 25-50% of mice. Even more effective is immunization with a TSHR A-subunit adenovirus (65-84% hyperthyroidism). Nevertheless, TSHR antibody characteristics in these mice do not mimic accurately those of autoantibodies in typical Graves' patients, with a marked TSH-blocking antibody response. We hypothesized that this suboptimal antibody response was consequent to the standard dose of TSHR-adenovirus providing too great an immune stimulus. To test this hypothesis, we compared BALB/c mice immunized with the usual number (10(11)) and with far fewer viral particles (10(9) and 10(7)). Regardless of viral dose, hyperthyroidism developed in a similar proportion (68-80%) of mice. We then examined the qualitative nature of TSHR antibodies in each group. Sera from all mice had TSH binding-inhibitory (TBI) activity after the second immunization, with TBI values in proportion to the viral dose. After the third injection, all groups had near-maximal TBI values. Remarkably, in confirmation of our hypothesis, immunization with progressively lower viral doses generated TSHR antibodies approaching the characteristics of autoantibodies in human Graves' disease as follows: 1) lower TSHR antibody titers on ELISA and 2) lower TSH-blocking antibody activity without decrease in thyroid-stimulating antibody activity. In summary, low-dose immunization with adenovirus expressing the free TSHR A-subunit provides an induced animal model with a high prevalence of hyperthyroidism as well as TSHR antibodies more closely resembling autoantibodies in Graves' disease.

A recent report of major pathophysiological significance, and opposed to present concepts, is that TSH (but not MS-1, a hamster monoclonal thyroid-stimulating antibody), cleaves the single-chain TSH receptor (TSHR) on the cell surface into its two-subunit form. We reassessed the issue using two approaches. First we wished to confirm the flow-cytometric assay previously used to quantitate TSHR cleavage. We used CHO cell lines expressing large (TSHR-10,000 cells) or conventional (TSHR-0 cells) numbers of TSHR. Cells were preincubated (16 h) in either control medium or medium supplemented with TSH (5 x 10(-8) m) or MS-1 (10 microg/ml). After stringent washing to maximize removal of residual ligand, we performed flow cytometry with two antibodies, one recognizing only the single-chain TSHR, the other recognizing all (cleaved and uncleaved) TSHRs. TSH pretreatment did not appear to increase TSHR cleavage. Instead we observed ligand occupancy of the TSHR (with MS-1) or fewer receptors on the cell surface (down-regulation), particularly with the TSHR-0 cells. Second, we covalently cross-linked [125I]TSH to monolayers of these cells, an unequivocal method to determine directly the proportion of single-chain and two-subunit TSHR forms. Pretreatment of TSHR-10,000 and TSHR-0 cells with TSH had no effect on the degree of TSHR cleavage. MS-1 slightly reduced spontaneous cleavage. In conclusion, in contrast to a recent report, we show that TSH does not alter the subunit structure of its cognate receptor, and we provide insight into the difficulties associated with the flow-cytometric assay for TSHR cleavage.

The TSH receptor (TSHR), unlike the LH receptor (LHR), has considerable ligand-independent adenylyl cyclase activity, a feature of pathophysiological importance. The TSHR ectodomain partially suppresses constitutive activity, an effect reversed by trypsin treatment of intact cells. Localizing the functional site of trypsin action would provide insight into how the TSHR ectodomain exerts its constraint. For this purpose, we examined the effect of trypsin on intact cells expressing a series of modified TSHR. Trypsin did not increase cAMP production by a chimeric TSH-LH receptor involving substitution of TSHR residues 261-418 (the ectodomain C terminus). In contrast, with the wild-type TSHR, trypsin enhanced constitutive activity despite mutation of the following potential tryptic cleavage sites [arginine (R) and lysine (K) residues]: 1) K565, K651, K660 in the extracellular loops of the serpentine region; 2) B subunit juxtamembrane residues K371, K401, K415; 3) A subunit residues R310, R312, K313. We previously excluded K337 and K339 from being implicated in TSHR tryptic activation. By exclusion, only one R/K cluster remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293. Mutation of this cluster is incompatible with TSHR cell surface expression. However, tryptic clipping at this locus would reproduce a previously demonstrated structural effect of trypsin on the TSHR, removal of about a 2-kDa polypeptide fragment extending downstream from the locus to the C terminus of the A subunit. Taken together, these data suggest that the C terminus of the A subunit functions as a suppressor of TSHR constitutive activity.

Two unusual features of the TSH receptor (TSHR) ectodomain are its intramolecular cleavage at the cell surface into disulfide-linked subunits and its constraint of ligand-independent (constitutive) activity inherent to the serpentine region. Whether ectodomain cleavage alters the level of TSHR constitutive activity is an important unanswered question. To address this issue, we used a TSHR engineered so as not to undergo spontaneous cleavage into subunits (deletion of amino acid residues 317-366 and GQE(367-369)NET substitution). Into this noncleaving TSHR (termed TSHR-D1-NET), we introduced thrombin recognition motifs (termed Thr 6 and Thr 18) at the site of spontaneous cleavage. Treatment of intact Chinese hamster ovary cells expressing TSHR-D1-NET-Thr 6 and -Thr 18 with thrombin induced cleavage into A and B subunits, as determined by (125)I-TSH covalent cross-linking. Nevertheless, constitutive activity of the thrombin-cleaved TSHR was unaltered. The level of TSHR constitutive activity was, therefore, fully dissociated from intramolecular cleavage into subunits. Trypsin treatment of the same cells expressing the noncleaving TSHR also generated disulfide-linked A and B subunits but, in contrast to thrombin, enhanced TSHR constitutive activity. Therefore, the activating effect of trypsin appears to involve clipping at an additional, as-yet unidentified, site. In summary, our data demonstrate that TSHR cleavage is, by itself, insufficient to reduce TSHR ectodomain constraint on ligand-independent constitutive activity. These data are consistent with other evidence that A subunit shedding consequent to TSHR cleavage is a critical factor in enhancing TSHR constitutive activity.

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb's) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb's]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb's, but not for TBAb's, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.

TSH receptor (TSHR) DNA vaccination induces high TSHR antibody levels in BALB/c mice housed in a conventional facility. However, under pathogen-free conditions, we observed a Th1 cellular response to TSHR antigen characterized by interferon-gamma (IFN gamma) production. In the present study we investigated the effect on TSHR DNA vaccination of diverting the cytokine milieu away from Th1 using 1) IFN gamma knockout BALB/c mice, and 2) wild-type mice covaccinated with DNA for the TSHR and for IFN gamma/receptor-Fc protein that prevents IFN gamma from binding to its receptor. Neither approach enhanced TSHR antibody levels, although splenocyte IFN gamma production in response to TSHR antigen was absent (IFN gamma knockouts) or reduced (IFN gamma receptor-Fc). Moreover, production of IL-2, another Th1 cytokine, but not Th2 cytokines, indicated that neither strategy overcame the Th1 bias of im DNA vaccination. Importantly, splenocyte production of IFN gamma and IL-2 provides a sensitive detection system for TSHR-specific T cells. Unexpectedly, higher TSHR antibody levels developed in rare mice. High titer animals had TSHR-specific responses of both Th2 and Th1 types, whereas low titer animals had Th1-restricted TSHR responses. The heterogeneity of responses induced by TSHR DNA vaccination in mice may provide insight into the titers and IgG subclasses of spontaneous autoantibodies in humans.

Recently, many exceptions have been reported that undermine the concept that the epitopes for thyroid-stimulating autoantibodies (TSAb) and thyrotropin-blocking autoantibodies (TBAb) lie within the N-terminal and C-terminal portions of the thyrotropin receptor (TSHR) ectodomain, respectively. Here we have used a new approach to examine the issue by attempting to neutralize autoantibodies with the purified, N-terminal 289 amino acids of the TSHR ectodomain (TSHR-289), essentially the A subunit. Immunoglobulin G (IgG) with TSAb activity from 10 patients with Graves' disease was preincubated with or without purified active or inactive TSHR-289. Active, but not inactive, TSHR-289 completely neutralized TSAb activity in all sera. We then tested the ability of active TSHR-289 to neutralize TBAb activity in two rare hypothyroid patients with pure TBAb activity lacking agonist activity. IgG from both patients was extremely potent in the TBAb assay. Unlike with TSAb, preincubation of the IgG with a large excess of active TSHR-289 (20 microg/mL) revealed a remarkable divergence. TBAb activity from one patient was totally neutralized whereas in the other patient TBAb activity was totally unaffected. In conclusion, using a new approach, we confirm that the major portion of the TSAb epitope in the 418 amino acid ectodomain lies upstream of residue 289 (within the A subunit). In contrast, TBAb that cause hyperthyroidism can be more heterogeneous, with epitopes that lie largely upstream (A subunit) or downstream (B subunit) of residue 289.

Two approaches have been developed to induce TSH receptor antibodies in mice with properties resembling those in Graves' disease, the Shimojo model of injecting live fibroblasts coexpressing the TSH receptor and major histocompatibility complex antigen Class II, and TSH receptor-DNA vaccination. Thyroid-stimulating antibodies appear to occur less commonly after DNA vaccination, but there has been no direct comparison of these models. We performed a three-way comparison of 1) AKR/N and 2) BALB/c mice vaccinated with TSH receptor-DNA and 3) AKR/N mice injected with fibroblasts expressing the TSH receptor and the major histocompatibility complex antigen class II of AKR/N mice. TSH receptor-DNA vaccinated mice had low or undetectable levels of TSH receptor antibodies determined by ELISA or flow cytometry. Nonspecific binding precluded comparisons with sera from Shimojo mice by these assays. TSH binding inhibition and thyroid-stimulating antibody were undetectable in TSH receptor-DNA vaccinated mice. In Shimojo mice, TSH binding inhibition was positive in approximately 60%, and thyroid-stimulating antibodies were positive in hyperthyroid animals. Unlike the negative antibody data, splenocytes from TSH receptor-vaccinated (but not Shimojo) mice proliferated and produced the Th1 cytokine interferon-gamma in response to TSH receptor antigen. In conclusion, DNA vaccination is less effective at inducing TSH receptor antibodies than the Shimojo approach, but it permits the future characterization of TSH receptor-specific T cells generated without adjuvant.

We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin gamma1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. Removal in various permutations of the four cysteines at TSHR positions 24, 29, 31, and 41 (signal peptide residues are 1-21) revealed Cys(41) to be the key residue for receptor expression. Forced pairing of Cys(41) with any one of the three upstream Cys residues was necessary for trafficking to the cell surface of a TSHR with high affinity TSH binding similar to the wild-type receptor. However, for a full biological response to TSAb, forced pairing of Cys(41) with Cys(29) or with Cys(31), but not with Cys(24), retained functional activity comparable with the wild-type TSHR. These data suggest that an N-terminal disulfide-bonded loop between Cys(41) and Cys(29) or its close neighbor Cys(31) comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin gamma1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease.

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317-319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.

A secreted recombinant TSH receptor (TSHR) ectodomain variant (TSHR-289) neutralizes TSHR autoantibodies in Graves' disease, but is heterogeneous in containing both immunologically active and inactive molecules and is also unstable. We have now purified each form of TSHR-289 using sequential affinity chromatography with a mouse mAb (3BD10) specific for the inactive form, and a mAb to C-terminal His residues that recognizes both forms. The immunological difference between active and inactive TSHR-289 was unrelated to primary amino acid sequence or carbohydrate content and was, therefore, attributable to its folded state. The epitopes for Graves' autoantibodies and 3BD10 overlap, and both are destroyed by denaturation. Therefore, reciprocal binding by autoantibodies and 3BD10 to conformational determinants involving the same TSHR segment suggests a prion-like shift between two folded states of the molecule. Despite purification, immunologically active TSHR-289 remained labile, as determined by loss of autoantibody, and gain of 3BD10, recognition. However, using chemical chaperones we have, for the first time, been able to stabilize purified TSHR antigen in immunologically intact form. In summary, purification of immunologically active and stable antigen in milligram quantities provides a powerful tool for future diagnostic and therapeutic studies in Graves' disease.

Cleavage of thyrotropin receptors (TSHR) on the cell surface into disulfide-linked A and B subunits involves deletion of an intervening region that corresponds approximately to a 50 amino acid "insertion" in the TSHR relative to the noncleaving luteinizing hormone/choriogonadotropin receptor (LH/CGR). The location of this insertion is imprecise because of the relatively low homology between the two receptors in this region. We tested the hypothesis that the TSHR 50 amino acid insertion was further downstream than we previously concluded, a possibility that would relocate the crucial LH/CGR glycan at N291 relative to the position of the TSHR insertion, and that would mitigate against the 50 amino acid insertion playing a role in TSHR intramolecular cleavage. Thus, we transferred the LH/CGR glycan at amino acid 291 from downstream (N367) to upstream of the 50 amino acid insertion (N317) in the TSHR, leaving this insertion intact. TSHR cleavage persisted. Moreover, deletion of amino acid residues 320-366 in addition to the upstream N291 substitution (ALN317-319NET) also did not prevent cleavage. On the other hand, deletion of three contiguous downstream residues (GQE367-369) in the TSHR 50 amino acid insertion abolished receptor cleavage into subunits. In summary, the present data are consistent with our previous location of the TSHR 50 amino acid insertion and, therefore, do not undermine evidence for the involvement of this insertion in TSHR cleavage. In addition, the data regarding TSHR residues GQE367-369 (far downstream of cleavage site 1) support the controversial possibility of a secondary cleavage site downstream of the insertion.

Some TSH receptors (TSHR) on the cell surface cleave into A and B subunits. Cleavage at upstream Site 1 is followed by the proteolytic excision of an intervening C peptide region terminating at a downstream Site 2. Although present evidence suggests that Site 1 lies between amino acid residues 303 and 317, the mechanism and exact amino acid(s) involved in cleavage are unknown. Previous amino acid substitutions at Site 1 failed to abrogate cleavage. We, therefore, performed deletion mutations within this region. Cleavage of cell surface TSHR, detected by 125I-TSH cross-linking to intact cells, was not prevented by deletion of four individual segments within the Site 1 cleavage region (delta305-308, delta309-312, delta313-316, delta317-320). However, deletion of the entire region (delta305-320) reduced the extent of cleavage and shifted the cleavage site upstream of the glycan at amino acid residue N302. Elimination of this glycan (N302Q substitution) reversed the effect of deleting amino acid residues 305-320 on TSHR cleavage, suggesting that reduced cleavage at the new, upstream cleavage site was caused by steric hindrance by the glycan at N302. In summary, deletion, as opposed to mutagenesis, of the TSHR cleavage Site 1 region produces a spatial shift in TSHR cleavage Site 1 from downstream to upstream of the glycan at N302. These observations provide strong evidence that TSHR cleavage at this site does not occur at a particular amino acid motif and suggests that cleavage involves a "molecular ruler" mechanism involving cleavage at a fixed distance from a protease attachment site.

We studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305-316) would generate a B subunit of 51-52 kDa. However, only smaller B subunits (40-46 kDa) were detected, corresponding to N termini from residues approximately 370 (site 2) extending downstream to the region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45-52 kDa) than the majority of B subunits recovered with a C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding.

There is, at present, no assay in clinical use for the direct assay of autoantibody binding to the thyrotropin receptor (TSHR). We now describe a direct thyrotropin receptor autoantibody binding assay (DTAb) using a secreted form of the TSHR ectodomain (TSHR-289) without the need for antigen purification. The assay compensates for the low TSHR autoantibody concentration in serum by capturing a relatively large amount of patient immunoglobulin G (IgG) on high-capacity beads, a reversal of standard methods that typically first immobilize antigen. TSHR-289 captured by Graves' IgG was detected in a colorimetric reaction using a biotinylated murine monoclonal antibody to the poly-histidine tail engineered into the antigen. By this approach, sera from 11 normal individuals provided a mean optical density (OD) value of 0.20 +/- 0.08 SD (range 0.06-0.33). Of 38 sera from unselected patients with a history of Graves' disease (untreated and treated), 29 (76%) generated OD values > 0.37 (2 SD above the mean for the normal sera), the highest being OD 1.38. Surprisingly, 3 of 13 (23%) sera from TPO autoantibody-positive patients with Hashimoto's thyroiditis also provided values > 2 SD above the normal sera. The extent of direct autoantibody binding to the TSHR correlated closely with the thyrotropin binding inhibition (TBI) values (r = 0.881; p < 0.001). One serum was clearly positive in only the direct binding assay and another in only the TBI assay. The data obtained with the direct binding assay correlated less well with the thyroid-stimulating antibody (TSAb) assay (r = 0.582; p < 0.001). In summary, we describe a new direct DTAb assay that correlates more closely with the TBI than with the TSI assays. Future studies in a large series of clinically defined patients will be needed to evaluate the clinical utility of the DTAb assay.