Tom Fiers

Divergences in Insulin Resistance Between the Different Phenotypes of the Polycystic Ovary Syndrome

CONTEXT/OBJECTIVE Current diagnostic criteria for polycystic ovary syndrome (PCOS) have generated distinct PCOS phenotypes, based on the different combinations of diagnostic features found in each patient. Our aim was to assess whether either each single diagnostic feature or their combinations into the PCOS phenotypes may predict insulin resistance in these women. PATIENTS/DESIGN A total of 137 consecutive Caucasian women with PCOS, diagnosed by the Rotterdam criteria, underwent accurate assessment of diagnostic and metabolic features. Insulin sensitivity was measured by the glucose clamp technique. RESULTS Among women with PCOS, 84.7% had hyperandrogenism, 84.7% had chronic oligoanovulation, and 89% had polycystic ovaries. According to the individual combinations of these features, 69.4% of women had the classic phenotype, 15.3% had the ovulatory phenotype, and 15.3% had the normoandrogenic phenotype. Most subjects (71.4%) were insulin resistant. However, insulin resistance frequency differed among phenotypes, being 80.4%, 65.0%, and 38.1%, respectively, in the 3 subgroups (P < .001). Although none of the PCOS diagnostic features per se was associated with the impairment in insulin action, after adjustment for covariates, the classic phenotype and, to a lesser extent, the ovulatory phenotype were independently associated with insulin resistance, whereas the normoandrogenic phenotype was not. Metabolic syndrome frequency was also different among phenotypes (P = .030). CONCLUSIONS There is a scale of metabolic risk among women with PCOS. Although no single diagnostic features of PCOS are independently associated with insulin resistance, their combinations, which define PCOS phenotypes, may allow physicians to establish which women should undergo metabolic screening. In metabolic terms, women belonging to the normoandrogenic phenotype behave as a separate group.

Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization

Measurement of estrone (E1) and estradiol (E2) values <1 pg/mL (3.7 pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC-MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5 pg/mL (1.9 pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CVs were 4.0% at 21 pg/mL (77 pmol/L) for E2, 7.6% at 25 pg/mL (93 pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.

Thyroid hormone status within the physiological range affects bone mass and density in healthy men at the age of peak bone mass

CONTEXT The hormonal factors involved in the regulation of peak bone mass (PBM) in men have not been fully investigated. Apart from gonadal steroids and somatotropic hormones, thyroid hormones are known to affect bone maturation and homeostasis and are additional candidate determinants of adult bone mass. OBJECTIVE We aimed to investigate between-subject physiological variation in free and total thyroid hormone concentrations, TSH, and thyroid binding globulin (TBG) in relation to parameters of bone mass, geometry, and mineral density in healthy men at the age of PBM. DESIGN AND SETTING We recruited 677 healthy male siblings aged 25-45 years in a cross-sectional, population-based study. Areal and volumetric bone parameters were determined using dual-energy x-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Total and free thyroid hormones, TBG, and TSH were determined using immunoassays. RESULTS Free and total thyroid hormone concentrations were inversely associated with bone mineral density (BMD) and bone mineral content (BMC) at the hip and total body (free triiodothyronine (FT(3)), total T(3) (TT(3)), and total T(4) (TT(4))) and at the spine (FT(3)). TBG was negatively associated with BMC and areal BMD at all sites. At the radius, cortical bone area was inversely associated with TT(3), TT(4), and TBG, and trabecular bone density was inversely associated with free thyroxine, TT(4), and TBG. We observed inverse associations between cortical bone area at the mid-tibia and FT(3), TT(3), TT(4), and TBG. No associations between TSH and DXA or pQCT measurements were found. CONCLUSION In healthy men at the age of PBM, between-subject variation in thyroid hormone concentrations affects bone density, with higher levels of FT(3), TT(3), TT(4), and TBG being associated with less favorable bone density and content.

Metabolic Inflexibility Is a Feature of Women With Polycystic Ovary Syndrome and Is Associated With Both Insulin Resistance and Hyperandrogenism

CONTEXT Metabolic inflexibility, ie, the impaired ability of the body to switch from fat to carbohydrate oxidation under insulin-stimulated conditions, is associated with insulin resistance. This alteration in metabolic plasticity can lead to organ dysfunction and is considered a key issue among the abnormalities of the metabolic syndrome. It is still unknown whether this phenomenon occurs in women with polycystic ovary syndrome (PCOS). OBJECTIVE Our objective was to examine whether metabolic inflexibility is a feature of PCOS women and whether hyperandrogenism may contribute to this phenomenon. DESIGN AND PATIENTS Eighty-nine Caucasian women with PCOS were submitted to hyperinsulinemic-euglycemic clamp. Respiratory exchange ratios were evaluated at baseline and during hyperinsulinemia by indirect calorimetry to quantify substrate oxidative metabolism. Total testosterone was measured by liquid chromatography mass spectrometry and free testosterone by equilibrium dialysis. SETTING Outpatients were seen in a tertiary care academic center. MAIN OUTCOME MEASURE Metabolic flexibility was assessed by the change in respiratory quotient upon insulin stimulation. RESULTS Sixty-five of the 89 PCOS women (73%) had increased serum free testosterone, 68 (76%) were insulin resistant, and 62 (70%) had an impaired metabolic flexibility. Comparison of hyperandrogenemic and normoandrogenemic women showed that the 2 subgroups were of similar age but differed in terms of several anthropometric and metabolic features. In particular, hyperandrogenemic women had greater body mass index (32.9 ± 1.0 vs 24.7 ± 0.9 kg/m(2), P < .001) and lower glucose utilization during the clamp (9.2 ± 0.4 vs 10.9 ± 0.7 mg/kg fat-free mass · min, P = .023) and metabolic flexibility (0.09 ± 0.06 vs 0.12 ± 0.01, P = .014). In univariate analysis, metabolic flexibility was associated with several anthropometric, endocrine, and metabolic features. In multivariate analysis, this feature was directly associated with baseline respiratory quotient and insulin sensitivity and inversely with free testosterone and free fatty acids concentrations under insulin suppression (R(2) = 0.634, P < .001). CONCLUSIONS Metabolic inflexibility is a feature of PCOS women. Both insulin resistance and androgen excess might contribute to this abnormality.

Harmonized Reference Ranges for Circulating Testosterone Levels in Men of Four Cohort Studies in the United States and Europe

Background Reference ranges for testosterone are essential for making a diagnosis of hypogonadism in men. Objective To establish harmonized reference ranges for total testosterone in men that can be applied across laboratories by cross-calibrating assays to a reference method and standard. Population The 9054 community-dwelling men in cohort studies in the United States and Europe: Framingham Heart Study; European Male Aging Study; Osteoporotic Fractures in Men Study; and Male Sibling Study of Osteoporosis. Methods Testosterone concentrations in 100 participants in each of the four cohorts were measured using a reference method at Centers for Disease Control and Prevention (CDC). Generalized additive models and Bland-Altman analyses supported the use of normalizing equations for transformation between cohort-specific and CDC values. Normalizing equations, generated using Passing-Bablok regression, were used to generate harmonized values, which were used to derive standardized, age-specific reference ranges. Results Harmonization procedure reduced intercohort variation between testosterone measurements in men of similar ages. In healthy nonobese men, 19 to 39 years, harmonized 2.5th, 5th, 50th, 95th, and 97.5th percentile values were 264, 303, 531, 852, and 916 ng/dL, respectively. Age-specific harmonized testosterone concentrations in nonobese men were similar across cohorts and greater than in all men. Conclusion Harmonized normal range in a healthy nonobese population of European and American men, 19 to 39 years, is 264 to 916 ng/dL. A substantial proportion of intercohort variation in testosterone levels is due to assay differences. These data demonstrate the feasibility of generating harmonized reference ranges for testosterone that can be applied to assays, which have been calibrated to a reference method and calibrator.

Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis

Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology.

Implications of Androgen Assay Accuracy in the Phenotyping of Women With Polycystic Ovary Syndrome

CONTEXT/OBJECTIVE Hyperandrogenism is a common feature of women with polycystic ovary syndrome (PCOS) and is considered a cardinal element for the diagnosis and phenotyping of this condition. Unfortunately, routinely available methods for measuring serum androgens suffer from major limitations. No data are available on the impact of androgen assay quality on the assignment of PCOS women to the different clinical phenotypes of PCOS, when defined according to the Rotterdam criteria for diagnosis. PATIENTS Two hundred four consecutive Caucasian women with PCOS, diagnosed by the Rotterdam criteria participated in the study. DESIGN Assessment of total T (TT), free T (FT), and androstenedione (A) by both a chemiluminescent assay, routinely available in our hospital, and gold standard methodology, ie, liquid chromatography-mass spectrometry and equilibrium dialysis, was performed. The results were compared and the associations of these data with clinical and metabolic features of PCOS women were analyzed. RESULTS By using gold standard assays, TT was high in 36.3% of women, whereas A only marginally contributed to identifying hyperandrogenemic patients. However, gold standard FT measurement was elevated in 70.6% of the PCOS patients, identifying them as hyperandrogenemic. Routine TT and A assays, and the derived calculated FT, were strikingly inaccurate, with substantial overestimation. These assays erroneously classified 60 patients (29.4%), 32 as false hyperandrogenemic, and 28 as false normoandrogenemic, with incorrect assignment of many patients to the clinical phenotypes of PCOS and inappropriate estimation of their metabolic risk. In particular, women misclassified as normoandrogenic had a more severe metabolic profile than true normoandrogenic subjects. CONCLUSIONS FT alone, as measured by equilibrium dialysis or calculated by using the Vermeulen formula, provided that TT is assayed by gold standard methodology, can be used to identify hyperandrogenemic PCOS subjects. The use of routine androgen assays may misclassify the phenotype of many PCOS women, with errors in the estimation of individual metabolic risk.

Sex Steroids in Relation to Sexual and Skeletal Maturation in Obese Male Adolescents

BACKGROUND Childhood obesity is associated with an accelerated skeletal maturation. However, data concerning pubertal development and sex steroid levels in obese adolescents are scarce and contrasting. OBJECTIVES To study sex steroids in relation to sexual and skeletal maturation and to serum prostate specific antigen (PSA), as a marker of androgen activity, in obese boys from early to late adolescence. METHODS Ninety obese boys (aged 10-19 y) at the start of a residential obesity treatment program and 90 age-matched controls were studied cross-sectionally. Pubertal status was assessed according to the Tanner method. Skeletal age was determined by an x-ray of the left hand. Morning concentrations of total testosterone (TT) and estradiol (E2) were measured by liquid chromatography-tandem mass spectrometry, free T (FT) was measured by equilibrium dialysis, and LH, FSH, SHBG, and PSA were measured by immunoassays. RESULTS Genital staging was comparable between the obese and nonobese groups, whereas skeletal bone advancement (mean, 1 y) was present in early and midadolescence in the obese males. Although both median SHBG and TT concentrations were significantly (P < .001) lower in obese subjects during mid and late puberty, median FT, LH, FSH, and PSA levels were comparable to those of controls. In contrast, serum E2 concentrations were significantly (P < .001) higher in the obese group at all pubertal stages. CONCLUSION Obese boys have lower circulating SHBG and TT, but similar FT concentrations during mid and late puberty in parallel with a normal pubertal progression and serum PSA levels. Our data indicate that in obese boys, serum FT concentration is a better marker of androgen activity than TT. On the other hand, skeletal maturation and E2 were increased from the beginning of puberty, suggesting a significant contribution of hyperestrogenemia in the advancement of skeletal maturation in obese boys.

Calcium and bone homeostasis in heterozygous carriers of CYP24A1 mutations: A cross-sectional study.

BACKGROUND Bi-allelic CYP24A1 mutations can cause idiopathic infantile hypercalcemia (IIH), adult-onset nephrocalcinosis, and possibly bone metabolism disturbances. It is currently unclear if heterozygous carriers experience clinical problems or biochemical abnormalities. Our objective is to gain insight in the biochemical profile and health problems in CYP24A1 heterozygotes. STUDY DESIGN Cross-sectional evaluation of participants. Data of previously reported carriers are reviewed. SETTING AND PARTICIPANTS Outpatient clinic of a tertiary care hospital. Participants were eight family members of an infant with a well-characterized homozygous CYP24A1 mutation c.1186C>T p.(Arg396Trp). OUTCOMES Serum vitamin D metabolites. Symptoms or biochemical signs of hypercalcemia, hypercalciuria or nephrocalcinosis. Bone health in heterozygous as compared to wild type (WT) subjects. MEASUREMENTS Genotyping by Sanger sequencing; vitamin D metabolites by liquid chromatography tandem mass spectrometry; renal, calcium and bone markers by biochemical analyses; presence of nephrocalcinosis by renal ultrasound; bone health by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. RESULTS Six participants were heterozygous carriers of the mutation. None of the heterozygous subjects had experienced IIH. One had a documented history of nephrolithiasis, two others had complaints compatible with this diagnosis. No major differences between WT and heterozygous subjects were found regarding bone health, serum or urinary calcium or 25OHD/24,25(OH)2D ratio. Literature reports on three out of 33 heterozygous cases suffering from IIH. In all three, the 25OHD/24,25(OH)2D ratio was highly elevated. Nephrocalcinosis was frequently reported in family members of IIH cases. LIMITATIONS Small sample size, lack of a large control group. CONCLUSIONS Our and literature data suggest that most heterozygous CYP24A1 mutation carriers have a normal 25OHD/24,25(OH)2D ratio, are usually asymptomatic and have a normal skeletal status but may possibly be at increased risk of nephrocalcinosis. A review of the available literature suggests that an elevated 25OHD/24,25(OH)2D ratio may be associated with symptoms of IHH, irrespective of carrier status.

Functional and structural integrity of porcine pancreatic grafts subjected to a period of warm ischemia and cold preservation with histidine-tryptophan-ketoglutarate (Custodiol) or University of Wisconsin solution

Background. University of Wisconsin (UW) solution (Viaspan) is currently used to preserve organs from nonheartbeating donors. Histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) is of proven efficacy in experimental pancreas preservation, but its efficacy in combined warm ischemia (WI) and cold ischemia (CI) is unknown. The viability of HTK-preserved porcine pancreatic grafts was assessed after various periods of WI and compared with grafts flushed and preserved with UW solution. Methods. A total of 14 pigs were used: G1 (n=4, UW) and G2 (n=4, HTK) with 15-min WI and 16-hr cold storage; G3 (n=3, UW) and G4 (n=3, HTK) with 30-min WI and 16-hr cold storage. Results. All animals in G1 and G2 were normoglycemic, whereas only 66% of pancreases were functioning in G3 and G4. HTK perfusion was associated with increased wet weight. Transient hyperinsulinemia was noted in all the groups on postoperative day 1 (mean range: 8.9–12.4 &mgr;U/L). Postoperative serum amylase and lipase were more pronounced in G3 and G4. However, HTK-stored grafts exhibited less evidence of biochemical pancreatitis as compared with UW-stored grafts on the first postoperative day in the group with 15-min WI. Mean K values of intravenous glucose tolerance tests on postoperative day 14 were similar in both groups. Vascular congestion was uniformly observed and was considered a typical feature of WI. Conclusions. Porcine pancreatic grafts are viable after 16-hr CI following 15-min WI in this experimental nonheartbeating donor model. HTK solution seems to provide reliable graft function in this setting and to be equivalent to UW.