M. Ángeles Martínez-García

Prevalence of functional disorders of androgen excess in unselected premenopausal women: a study in blood donors

BACKGROUND The polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. On the contrary, the prevalences of other disorders of androgen excess such as idiopathic hyperandrogenism and idiopathic hirsutism remain unknown. We aimed to obtain an unbiased estimate of the prevalence in premenopausal women of (i) signs of androgen excess and (ii) PCOS, idiopathic hyperandrogenism and idiopathic hirsutism. METHODS A multicenter prevalence survey included 592 consecutive premenopausal women (393 from Madrid, Spain and 199 from Bologna, Italy) reporting spontaneously for blood donation. Immediately before donation, we conducted clinical and biochemical phenotyping for androgen excess disorders. We determined the prevalence of (i) hirsutism, acne and alopecia as clinical signs of androgen excess and (ii) functional disorders of androgen excess, including PCOS, defined by the National Institute of Child Health and Human Development/National Institute of Health criteria, idiopathic hyperandrogenism and idiopathic hirsutism. RESULTS Regarding clinical signs of hyperandrogenism, hirsutism and acne were equally frequent [12.2% prevalence; 95% confidence interval (CI): 9.5-14.8%], whereas alopecia was uncommon (1.7% prevalence, 95% CI: 0.7-2.7%). Regarding functional disorders of androgen excess, PCOS and idiopathic hirsutism were equally frequent (5.4% prevalence, 95% CI: 3.6-7.2) followed by idiopathic hyperandrogenism (3.9% prevalence, 95% CI: 2.3-5.4). CONCLUSIONS Clinical signs of hyperandrogenism and functional disorders of androgen excess show a high prevalence in premenopausal women. The prevalences of idiopathic hyperandrogenism and idiopathic hirsutism are similar to that of PCOS, highlighting the need for further research on the pathophysiology, consequences for health and clinical implications of these functional forms of androgen excess.

Proteomic analysis of plasma in the polycystic ovary syndrome identifies novel markers involved in iron metabolism, acute-phase response, and inflammation.

CONTEXT Hypothesis-free approaches such as proteomic analysis may identify novel biomarkers for disease. OBJECTIVE The objective of the study was to compare the plasma proteome of patients presenting with the polycystic ovary syndrome (PCOS) with that of women without hyperandrogenism. DESIGN This was a case-control study. SETTINGS The study was conducted at an academic hospital. PATIENTS Patients included 12 PCOS patients and 12 women without hyperandrogenism. INTERVENTIONS Interventions included basal blood sampling. MAIN OUTCOME MEASURES Two-dimensional difference in-gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, Western blot, and ELISA analyses were measured. RESULTS Two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analyses identified haptoglobin beta-chain and alpha2-macroglobulin as proteins underexpressed in PCOS samples, whereas transferrin and kappa-free light chain were overexpressed. We were able to confirm only the underexpression of haptoglobin beta-chain in subsequent Western blot and ELISA analyses. CONCLUSIONS Proteomic analysis of plasma from PCOS patients revealed changes in protein expression in several acute-phase response proteins including isoforms of plasma haptoglobin, alpha2-macroglobulin, and transferrin and in kappa-free light chain. In addition to their role as inflammatory markers, some of these molecules play major roles in iron metabolism, further suggesting that iron metabolism and low-grade chronic inflammation may be involved in the pathogenesis of insulin-resistant disorders such as PCOS.

Evidence for masculinization of adipokine gene expression in visceral and subcutaneous adipose tissue of obese women with polycystic ovary syndrome (PCOS).

CONTEXT Sex hormones, particularly androgens, may influence not only adipose tissue distribution but also its functions. OBJECTIVE We explored the possibility of sexual dimorphism in adipose tissue and skeletal muscle function. DESIGN This was a case-control study. SETTING The setting was an academic hospital. PARTICIPANTS Participants were severely obese men (n = 7), control women (n = 7), and hyperandrogenic women presenting with polycystic ovary syndrome (PCOS) (n = 7) submitting to bariatric surgery and an independent series of 40 patients with PCOS and 40 control women matched for age and body mass index. INTERVENTIONS Samples of subcutaneous (SAT) and visceral adipose tissue (VAT) and skeletal muscle were obtained during bariatric surgery in severely obese subjects. MAIN OUTCOME MEASURES Gene expression of chemerin, lipocalin-2, and omentin-1 in tissue samples was measured. We analyzed the effects of PCOS and obesity on serum concentrations of these adipokines in the larger series of women with PCOS and in control women. RESULTS Expression of chemerin and lipocalin-2 was higher in VAT than in SAT in men and women with PCOS; the opposite was observed in control women. Omentin-1 expression was higher in VAT than in SAT in the three groups. No differences were observed in the skeletal muscle expression of these adipokines. Obesity increased serum chemerin and lipocalin-2 levels and tended to decrease omentin-1, irrespective of PCOS. CONCLUSIONS The present results suggest that there is sexual dimorphism in some adipose tissue functions and that this dimorphism may be related to differences in androgen concentrations because women with PCOS show a masculinized pattern of expression of some adipokines.

Sexual dimorphism in adipose tissue function as evidenced by circulating adipokine concentrations in the fasting state and after an oral glucose challenge

STUDY QUESTION Do the circulating levels of a panel of adipokines involved in glucose metabolism exhibit sexual dimorphism in the fasting state and after an oral glucose load? SUMMARY ANSWER Our results indicate sexual dimorphism in the circulating concentrations of adipokines involved in intermediate metabolism in the fasting state and during an oral glucose load. This finding suggests an influence of sex steroids on adipose tissue function. WHAT IS KNOWN ALREADY Sexual dimorphism in adipose tissue distribution fully develops after puberty and modulates the risk for cardiometabolic disorders. However, the possibility that adipose tissue function exhibits sexual dimorphism as well as its distribution is unproved. STUDY DESIGN, SIZE, DURATION Cross-sectional case-control study including 32 subjects. PARTICIPANTS/MATERIALS, SETTING, METHODS Sixteen subjects with weight excess (8 men and 8 women, including 4 overweight and 4 obese subjects in each group) and 16 normal weight healthy volunteers (8 men and 8 women) presenting with similar age were submitted to a 75-g oral glucose tolerance test (oGTT). We measured circulating concentrations of insulin, glucose, chemerin, lipocalin-2, omentin-1, leptin and adiponectin and calculated their areas under the oGTT curve (AUC). MAIN RESULTS AND THE ROLE OF CHANCE Leptin and adiponectin concentrations were higher throughout the oGTT in women compared with men. Lipocalin-2 concentrations decreased during the oGTT in the whole group of study subjects. However, these levels remained higher in men with weight excess compared with normal weight men, whereas in women with weight excess lipocalin-2 levels at the end of the oGTT were lower compared with normal weight women. Sex was among the main determinants of the AUC of omentin-1 and leptin in linear regression models, and lower estradiol and testosterone concentrations were related to higher AUC of chemerin and omentin-1, respectively. Subjects with weight excess had higher AUC of chemerin and leptin and lower AUC of omentin-1 and adiponectin levels, independently of sex. LIMITATIONS, REASONS FOR CAUTION We included a relatively small sample size and, because this was a cross-sectional study, we cannot infer causality to the associations between the changes in circulating adipokine concentrations and the variables studied here. WIDER IMPLICATIONS OF THE FINDINGS Sexual dimorphism in adipose tissue function should be considered when studying adiposity and obesity, and also when designing strategies for their diagnosis and management.

A Nontargeted Proteomic Study of the Influence of Androgen Excess on Human Visceral and Subcutaneous Adipose Tissue Proteomes

CONTEXT Sex hormones, particularly androgens, may influence not only adipose tissue distribution but also its functions. OBJECTIVE We aimed to evaluate if sexual dimorphism in body composition is accompanied by differences in the protein abundance of adipose tissue by applying a nontargeted proteomic approach. DESIGN This was a case-control study. SETTINGS The setting was an academic hospital. PATIENTS Twenty-one morbidly obese patients, including 7 men, 7 women showing no evidence of androgen excess, and 7 hyperandrogenic women with polycystic ovary syndrome. INTERVENTIONS We obtained subcutaneous (SAT) and visceral (VAT) adipose tissue samples during bariatric surgery. MAIN OUTCOME MEASURES Protein abundance in VAT and SAT was analyzed by 2-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight coupled to mass spectrometry. Results were validated by RT-PCR. RESULTS The abundance of 2 spots of peroxiredoxin 6, creatine kinase B-type, 2 spots of selenium-binding protein 1, ruvB-like 2, 4-trimethylaminobutyraldehyde dehydrogenase, and albumin were higher in VAT compared with SAT in women with polycystic ovary syndrome. Men showed a similar pattern, whereas no difference between adipose tissue depots was observed in control women. Other proteins showed differences between VAT and SAT, confirming previous studies, or between the groups of subjects, without interaction between both effects. Several findings were confirmed by RT-PCR. CONCLUSIONS Sexual dimorphism influences the abundance of several proteins in VAT and SAT. The patterns of abundance in adipose tissue depots of several proteins involved in metabolic processes were similar in women with androgen excess and in men, suggesting that androgens influence adipose tissue function.

Common variants in the sex hormone-binding globulin gene (SHBG) and polycystic ovary syndrome (PCOS) in Mediterranean women

STUDY QUESTION Is there an association between polycystic ovary syndrome (PCOS) and the sex hormone-binding globulin (SHBG) rs1799941, rs6257, rs6259 and rs727428 variants in a large series of Mediterranean women? SUMMARY ANSWER The rs727428 and rs6259 variants are associated with PCOS in Mediterranean women. WHAT IS KNOWN ALREADY The level of SHBG, the primary plasma transport protein for sex steroids, which regulates the bioavailability of these hormones to target tissues, is reduced in patients with PCOS. Single-nucleotide polymorphisms in the SHBG gene influence circulating SHBG levels in American patients with PCOS and may predict the development of type 2 diabetes. STUDY DESIGN, SIZE AND DURATION This was a genetic case-control association study including 1004 premenopausal Mediterranean women. PARTICIPANTS/MATERIALS, SETTING AND METHODS In an Academic setting, we genotyped a clinical cohort consisting of 281 patients with PCOS and 142 women without any evidence of androgen excess, and a population-based cohort comprised of 581 unselected female blood donors from Spain and Italy. The latter included 31 patients with PCOS and 550 controls, of whom 298 had no evidence of any androgen excess disorder and were considered hyper-normal controls. MAIN RESULTS AND THE ROLE OF CHANCE Mutant alleles of the rs727428 variant were more frequent in patients with PCOS compared with controls and with hyper-normal controls. This association was independent of obesity. Carrying mutant alleles of rs727428 was found to be associated with a 1.29 odds ratio (OR) for PCOS, whereas carrying mutant alleles of rs6259 associated with a 0.68 OR for PCOS. The rs1799941 and rs6257 variants were not associated with PCOS. None of the SHBG variants influenced serum SHBG concentrations. LIMITATIONS AND REASONS FOR CAUTION The associations found here were relatively weak and, arising from a case-control study, do not necessarily indicate a causative role of the SHBG variants in the development of PCOS. Also, we studied different patients and controls from different sources, making some of the interpretations difficult. Finally, the rs1799941 variant was not in Hardy-Weinberg equilibrium in the small group of patients with PCOS recruited from the general population, yet this variant was not associated with PCOS. WIDER IMPLICATIONS OF THE FINDINGS SHBG variants that influenced circulating SHBG levels in American patients with PCOS are also associated with this syndrome in Mediterranean women, pointing to SHBG as a candidate gene for PCOS. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants PI080944 and PI110357 from Instituto de Investigación Carlos III, Spanish Ministry of Economy and Competitiveness. CIBERDEM is also an initiative of Instituto de Investigación Carlos III. The Authors have no competing interests to declare.

Impact of the storage temperature on human plasma proteomic analysis: Implications for the use of human plasma collections in research

Purpose: To analyze the impact of storage temperature on the 2D‐DIGE profile of human plasma.

Identification of reduced circulating haptoglobin concentration as a biomarker of the severity of pulmonary embolism: a nontargeted proteomic study.

Risk stratification of patients with pulmonary embolism (PE) may identify patients at high risk of early death who may benefit from more intensive surveillance or aggressive therapy. Nontargeted proteomics may identify biomarkers useful for the risk stratification of patients with acute symptomatic pulmonary embolism (PE). We studied 6 patients presenting with low-risk PE and 6 patients presenting with intermediate (n = 3) or high-risk (n = 3) PE. Two-dimensional difference gel electrophoresis was used to compare their plasma protein abundances. Candidate protein markers were identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry. A panel of four biomarkers (haptoglobin, hemopexin, α2-macroglobulin, and Ig α1-chain C region) showed differences in plasma abundance among patients with acute symptomatic PE of different severity. Haptoglobin and hemopexin were decreased, whereas α2-macroglobulin and Ig α1-chain C region were increased, in patients with high or intermediate-risk PE compared with low-risk PE patient. In a separate clinical population consisting of 104 adults with acute PE, serum haptoglobin concentrations had an 85% chance of correctly identifying patients with high-risk PE according to receiving operating characteristics curve analysis. Moreover, serum haptoglobin concentrations ≤1 g/l showed an 80% sensitivity and a 96% specificity for the diagnosis of high-risk PE. Nontargeted proteomics identified protein biomarkers for the severity of PE that are involved in iron metabolism pathways and acute-phase response. Among them, reduced serum haptoglobin concentrations show a high accuracy for the biochemical detection of high-risk PE.

A nontargeted study of muscle proteome in severely obese women with androgen excess compared with severely obese men and nonhyperandrogenic women

OBJECTIVE Androgen excess in women is frequently associated with muscle insulin resistance, especially in obese women with polycystic ovary syndrome. However, whether this is a primary event or the result of indirect mechanisms is currently debated. DESIGN This is an observational study. METHODS We obtained skeletal muscle biopsies during bariatric surgery from severely obese men (n=6) and women with (n=5) or without (n=5) androgen excess. We used two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry to identify muscle proteins showing differences in abundance between the groups of obese subjects. RESULTS Women with hyperandrogenism presented the lowest abundances of glycogen phosphorylase, pyruvate kinase, β-enolase, glycerol-3-phosphate dehydrogenase, creatine kinase M-type, and desmin, whereas the abundances of these molecules were similar in control women and men. CONCLUSION According to our nontargeted proteomic approach, women with hyperandrogenism show a specific alteration of the skeletal muscle proteome that could contribute to their insulin resistance. Because men do not show similar results, this alteration does not appear to be the direct effect on muscle of androgen excess, but rather the consequence of indirect mechanisms that merit further studies.

Association of TLR2 S450S and ICAM1 K469E polymorphisms with polycystic ovary syndrome (PCOS) and obesity

Toll-like receptors (TLRs) are activated by inflammatory stimuli and influence endothelial functions, contributing to the pathogenesis of atherosclerosis. We investigate the influence of polymorphisms in the genes encoding toll-like receptor 2 (TLR2) and 4 (TLR4) and endothelial adhesion molecules on polycystic ovary syndrome (PCOS) and its interaction with obesity. Ten single nucleotide polymorphisms were genotyped in 305 women with PCOS and 166 non-hyperandrogenic control women. In obese women, TLR2 S450S and ICAM1 K469E polymorphisms differently influenced metabolic variables and PCOS, respectively. Irrespective of PCOS, variant alleles of TLR2 S450S increased triglycerides, fasting insulin levels, and insulin resistance in obese women. TLR2 S450S interacted with obesity and PCOS on androstenedione levels, mutant alleles were associated with increased androstenedione concentrations in all women, with the exception of obese patients with PCOS (P=0.034). Regarding ICAM1 K469E, homozygosis for K469 alleles was more frequent in PCOS, but only in obese women (P=0.014). K469 alleles were also related to increased body mass index (P=0.017) and diastolic blood pressure (P=0.034). Moreover, ICAM1 K469E interacted with obesity and PCOS on serum triglyceride levels (P=0.019) and with PCOS on serum sex hormone-binding globulin concentrations (P=0.006). In conclusion, TLR2 S450S and ICAM1 K469E polymorphisms may be associated with PCOS and metabolic comorbidities in obese women.