Mortality in Cushing's disease.

The causes of premature death in untreated Cushing's syndrome are vascular disease (myocardial infarction/stroke), uncontrolled diabetes mellitus and complications and infections. Long-term mortality outcome studies on pituitary-dependent Cushing's disease (CD) are limited to six studies in the English language literature. This paper reviews these studies on CD, other causes of Cushing's syndrome being excluded, because CD represents 80% of patients with the syndrome. The period covered by these studies (1970-1990) is when transsphenoidal surgery was well established as primary treatment for CD. Two studies were exclusively from surgical centres and are likely biased in favour of surgically resectable adenomas, so this needs to be borne in mind when interpreting their results. The criteria for remission of hypercortisolism and persistent disease were variable. The overall number of patients in each report is small, and the number of deaths even smaller by epidemiological standards giving very wide confidence intervals to the standardised mortality ratios (SMR). Moreover, follow-up time was relatively short (median 10-12 years) for a disease diagnosed in the patients' late 30s. Notwithstanding the above limitations of retrospective studies, and potential for positive bias, the overall SMR of around 1.5 was not significantly different from the relevant normal population for those patients deemed in remission. However, SMR was significantly worse for those patients with persistent disease. Where it was possible to analyse contributing factors to mortality, the presence of hypertension and diabetes mellitus, in addition to persistence of hypercortisolism, was shown to be significant. It remains possible that an overall SMR in 'cured' patients would be significant given a larger cohort, followed for longer, and with more deaths. What is clearly required is a multicentre prospective cohort study with >30 years' follow-up to answer the question definitively and identify the contributing factors in detail in order to achieve optimum long-term outcome.

Bromocriptine and dopamine mediate independent and synergistic apoptotic pathways in pituitary cells.

Dopamine (DA) agonists are the primary treatment choice for prolactinoma, effectively suppressing prolactin expression and reducing tumour size. However, the intracellular pathway(s) through which either DA or its agonists impact on proliferation or lead to tumour shrinkage are incompletely understood. To identify the mediators in the apoptotic cascades after DA or DA agonist challenges we used a well-characterized model system, the rodent somatolactotroph cell line GH3. In these cells, we show that apoptosis induced by the DA agonist bromocriptine (BC), but not DA, is initiated through activation of the JNK pathway. However, both DA and BC activate the terminal effector caspase, caspase-3. Kinetic studies and chemical inhibitor co-incubation experiments support a role for JNK activation preceding caspase-9 activation in BC challenged cells, however, engagement of these mediators was not apparent in DA challenge cells. These studies suggest that apoptosis induced by BC or DA is mediated through distinct and independent pathways that converge with activation of the terminal caspase, caspase-3. These observations were further reinforced by our findings that DA and BC, in co-incubation experiments, synergistically induce apoptosis. These findings raise the possibility that drugs acting through the same pathway as DA may be clinically beneficial when combined with BC.

Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma.

The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where approximately 50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.

Monitoring disease activity using GH and IGF-I in the follow-up of 501 patients with acromegaly.

CONTEXT: The aims of treatment in patients with acromegaly are to achieve serum GH/IGF-I concentrations associated with cure or normalization of mortality and alleviation of symptoms. OBJECTIVE AND METHODS: Using the West Midlands Acromegaly database (n = 501) we investigated the reliability of basal fasting GH in predicting nadir or mean GH during oral glucose tolerance test (OGTT) or GH day curve (GHDC), respectively, the degree of discordance between disease activity measured by GH and IGF-I values and the effect of radiotherapy on the above relationships. In total 773 OGTT and 507 GHDC were performed. RESULTS: Basal fasting GH was strongly correlated with nadir/mean GH on OGTT/GHDC (r = +0.87, P < 0.0001, r = +0.93, P < 0.0001, respectively). A basal GH < 2.5 microg/l was associated with a nadir/mean GH during OGTT/GHDC < 2.5 microg/l in 98.6% and 88.2% of cases, respectively. Elevated IGF-I was seen in 32.4% and 46.4% of patients with GH nadir values during OGTT < 1 and < 2.5 microg/l, respectively, and in 21.2% and 45.9% of GHDC with mean GH < 1 and < 2.5 microg/l, respectively. Radiotherapy increased the discordance in GH and IGF-I as markers of disease activity at GH < 2.5 microg/l (elevated IGF-I-values when OGTT nadir GH < 2.5 microg/l: radiotherapy 55.5%vs. no radiotherapy 36.9%, P = 0.002). CONCLUSIONS: There is a close relationship between a basal fasting GH < 2.5 microg/l and nadir/mean GH < 2.5 microg/l during OGTT/GHDC. There is a large discordance between disease activity when assessed by GH and IGF-I which is further increased by radiotherapy. These observations illustrate the challenge of defining appropriate biochemical end-points to achieve control of disease and normalization of mortality in acromegaly.

A striking nitrogen isotope anomaly in the bencubbin and weatherford meteorites.

The stony-iron meteorites Bencubbin and Weatherford contain nitrogen with a ratio of nitrogen-15 to nitrogen-14 larger than normal by as much as a factor of 2. The excess nitrogen-15 may be due either to a nucleosynthetic origin or to extreme isotopic fractionation. In the former case, it may reflect failure to homogenize nitrogen-15 produced in nova explosions. In the latter case, it may reflect chemical processing at temperatures below 40 K in a presolar molecular cloud.

A component of primitive nuclear composition in carbonaceous meteorites.

The oxygen of anhydrous, high-temperature minerals in carbonaceous meteorites is strongly depleted in the heavy stable isotopes (17)O and (18)O. The effect is the result of nuclear rather than chemical processes and probably results from the admixture of a component of almost pure (16)O. This component may predate the solar system and may represent interstellar dust with a separate history of nucleosynthesis.

Oxygen Isotope Fractionation between Minerals and an Estimate of the Temperature of Formation.

Oxygen isotopic compositions of separated minerals from three type A and four type B rocks are very uniform. The delta(18)O values are: plagioclase, 6.20; clinopyroxene, 5.75; ilmenite, 4.45 (parts per thousand relative to Standard Mean Ocean Water). The isotopic distribution corresponds to equilibrium at 1120 degrees C. The isotopic composition of lunar pyroxenes falls within the range for pyroxenes of terrestrial mafic and ultramafic rocks, ordinary chondrites, enstatite chondrites, and enstatite achondrites, but above the range for basaltic achondrites, hypersthene achondrites, and mesosiderites. Glass isolated from the lunar soil has a delta(18)O value of 6.2, significantly richer in (18)O than the crystalline rock fragments in the soil.

Oxygen isotopes: experimental vapor fractionation and variations in tektites.

High-temperature (about 2800 degrees C) vapor fractionation of a silicate glass that originally contained 82 percent by weight of SiO(2) resulted in a decrease of the silica content to 45 percent. Oxygen isotope analyses show that the O(18)/O(16) ratio increased from 13.80 per mil in the starting material to 14.47 and 15.03 per mil in the residuum. This suggests that bediasites, which also exhibit an increase in this ratio with decreasing content of silica, have been subjected to a process of vapor fractionation.

Eolian origin of quartz in soils of hawaiian islands and in pacific pelagic sediments.

The particle-size distribution and oxygen-isotopic composition of quartz isolated from Hawaiian soils, east-central Pacific sediments, and tropospheric dusts are remarkably uniform. A common origin and tropospheric transport from continental land masses is suggested.

Water in SNC meteorites: evidence for a martian hydrosphere.

The Shergotty-Nakhla-Chassigny (SNC) meteorites, purportedly of martian origin, contain 0.04 to 0.4 percent water by weight. Oxygen isotopic analysis can be used to determine whether this water is extraterrestrial or terrestrial. Such analysis reveals that a portion of the water is extraterrestrial and furthermore was not in oxygen isotopic equilibrium with the host rock. Lack of equilibrium between water and host rock implies that the lithosphere and hydrosphere of the SNC parent body formed two distinct oxygen isotopic reservoirs. If Mars was the parent body, the maintenance of two distinct reservoirs may result from the absence of plate tectonics on the planet.

A silicate inclusion in Puente del Zacate, a IIIA iron meteorite.

The IIIA and IIIB iron meteorites are considered to have formed in the cores of asteroids. A silicate inclusion within the IIIA meteorite Puente del Zacate consisting of olivine (Fa4), low-calcium pyroxene (Fs6Wo1), chromium diopside (Fs3Wo47), plagioclase (An14Or4), graphite, troilite, chromite, daubreelite, and iron metal resembles inclusions in IAB iron meteorites. The oxygen isotopic composition of the Puente del Zacate inclusion is like chromite and phosphate inclusions in other IIIA and IIIB irons. The Puente del Zacate inclusion may have been derived from the lower mantle of the IIIAB parent asteroid.

The fall, recovery, orbit, and composition of the Tagish Lake meteorite: a new type of carbonaceous chondrite.

The preatmospheric mass of the Tagish Lake meteoroid was about 200,000 kilograms. Its calculated orbit indicates affinity to the Apollo asteroids with a semimajor axis in the middle of the asteroid belt, consistent with a linkage to low-albedo C, D, and P type asteroids. The mineralogy, oxygen isotope, and bulk chemical composition of recovered samples of the Tagish Lake meteorite are intermediate between CM and CI meteorites. These data suggest that the Tagish Lake meteorite may be one of the most primitive solar system materials yet studied.

Cardiovascular function in acromegaly.

Even with modern treatment, acromegaly is associated with a 2- to 3-fold increase in mortality, mainly from vascular disease, which is probably a result of the long exposure of tissues to excess GH before diagnosis and treatment. There is accumulating evidence that effective treatment to lower serum GH levels to less than 1-2 ng/ml (glucose suppressed or random, respectively) and normalize IGF-I improves long-term outcome and survival. In addition to recognized cardiovascular risk factors of hypertension, type 2 diabetes mellitus, and dyslipidemia, there is accumulating evidence of specific structural and functional changes in the heart in acromegaly. Along with endothelial dysfunction, these changes may contribute to the increased mortality in this disease. There are specific structural changes in the myocardium with increased myocyte size and interstitial fibrosis of both ventricles. Left ventricular hypertrophy is common even in young patients with short duration of disease. Some of these structural changes can be reversed by effective treatment. Functionally, the main consequence of these changes is impaired left ventricular diastolic function, particularly when exercising, such that exercise tolerance is reduced. Diastolic function improves with treatment, but the effect on exercise tolerance is more variable, and more longitudinal data are required to assess the benefits. What scant data there are on rhythm changes suggest an increase in complex ventricular arrhythmias, possibly as a result of the disordered left ventricular architecture. The functional consequences of these changes are unclear, but they may provide a useful early marker for the ventricular remodeling that occurs in the acromegalic heart. Endothelial dysfunction, especially flow-mediated dilatation, is an early marker of atherosclerosis, and limited data imply that this is impaired in active acromegaly and can be improved with treatment. Similarly, early arterial structural changes, such as thickened intima media layer, appear more common in acromegalics, and there are hints that this may diminish with effective treatment, although more studies are required for a definite conclusion on this topic. In conclusion, impaired cardiac and endothelial structure and function in acromegaly are risk factors for vascular mortality and should be regarded as legitimate therapeutic targets in the overall management of this condition.

Association of somatotrophinomas with loss of alleles on chromosome 11 and with gsp mutations.

The molecular pathology of somatotrophinomas has been investigated by a combined search for dominant mutations of the gene encoding the Gs alpha protein and for recessive mutations involving chromosome 11q13, which contains the gene causing multiple endocrine neoplasia type 1 (MEN1). Somatotrophinomas and peripheral leukocytes were obtained from thirteen patients with acromegaly; one patient also suffered from MEN1. Five DNA probes identifying restriction fragment length polymorphisms from 11q revealed allele loss in pituitary tumors from five (four non-MEN1 and one MEN1) patients. Deletion mapping revealed that the region of allele loss common to the somatotrophinomas involved 11q13. An analysis for similar allelic deletions at 12 other loci from chromosomes 1-5, 7-9, 12-14, and 17 did not reveal generalized allele loss in the somatotrophinomas. These results, which represent the first report of chromosome 11 allele loss occurring in non-MEN1 somatotrophinomas, indicate that a recessive oncogene on 11q13 is specifically involved in the monoclonal development of somatotrophinomas. In addition Gs alpha mutations were detected in two non-MEN1 somatotrophinomas, one of which also revealed allele loss of chromosome 11. Thus, our results reveal that the development of somatotrophinomas is associated with alterations in both dominant and recessive oncogenes and further characterization of these genetic abnormalities will help to elucidate the multistep etiology and progression of somatotrophinomas.

Loss of pRb expression in pituitary adenomas is associated with methylation of the RB1 CpG island.

We recently showed loss of pRb in a proportion of pituitary tumors that was not associated with loss of heterozygosity of an RB1 intragenic marker. To further define the mechanism responsible for loss of retinoblastoma protein (pRb) expression, we have investigated the methylation status of the CpG island contained within the promoter region of the RB1 gene, together with sequence analysis of the essential promoter region and exons coding for the protein-binding pocket domain. Methylation of the CpG island within the RB1 promoter region was detected in 6 of 10 tumors that failed to express pRb. In contrast, 18 of 20 tumors and all six histologically normal postmortem pituitaries that expressed pRb were unmethylated. No inactivating mutations were found within the RB1 promoter region in the four unmethylated tumors that failed to express pRB. However, one or more exons comprising the coding region for the protein-binding pocket domain were shown to be homozygously deleted in three of four tumors available for analysis. This study describes an additional tumor type, in addition to retinoblastoma, in which methylation of the RB1 promoter is associated with loss of pRb expression. Furthermore, we show that in addition to methylation of the RB1 promoter region, deletion within the protein-binding pocket domain is associated with a loss of detectable pRb expression. The reactivation of tumor suppressor genes, silenced through methylation, represents a promising therapeutic target in sporadic pituitary adenomas.

Chromosome 9p deletions in invasive and noninvasive nonfunctional pituitary adenomas: the deleted region involves markers outside of the MTS1 and MTS2 genes.

We have screened 57 cases of primary, nonfunctional, pituitary adenomas for loss of heterozygosity of markers on chromosome 9p. Using a panel of 11 microsatellite markers, we found hemizygous deletion with at least one of the markers in 18 tumors (31.5%). The frequency of loss was similar in both noninvasive (8 of 26; 31%) and invasive tumors (10 of 31; 32%), suggesting that loss on this chromosome might be an early event in pituitary tumorigenesis. Two discrete areas of loss were punctuated by a region of retention of heterozygosity between the markers D9S171 and IFNA, indicative of homozygous deletion. However, multiplex PCR analysis (MTS1 and MTS2) and the presence of a 3' untranslated region polymorphism in MTS1 suggested that neither of these tumor suppressor genes was homozygously deleted. In 6 of the 18 tumors showing LOH, sufficient DNA was also available for Southern blot analysis and, in all cases, showed retention of MTS1. Cell mixing experiments of tumor cell DNA homozygously deleted for MTS1 with DNA in which neither copy of the gene was deleted only gave rise to a signal at contamination levels greater than 30% and could discriminate homozygous and hemizygous loss. These studies support the recent findings that mechanisms other than hemi- and homozygous deletion are most likely responsible for the loss of MTS1 gene product in pituitary tumors (M. Woloschak et al., Cancer Res., 56: 2493-2486, 1996.). These data show that losses on either side of 9p21-22, both or either of which may be deleted, are involved in pituitary tumorigenesis and provide evidence for distinct suppressor gene loci, in addition to MTS1, on chromosome 9p.

Chromosome 13q deletion mapping in pituitary tumors: infrequent loss of the retinoblastoma susceptibility gene (RB1) locus despite loss of RB1 protein product in somatotrophinomas.

Two recent studies have described allelic loss of an RB1 intragenic marker on chromosome 13q in aggressive and metastatic pituitary tumors that did not correlate with loss of pRB. The second report also showed that losses were more frequently associated with a more centromeric marker. Because both of these studies suggest the presence of another or other tumor suppressor genes (TSGs) on 13q, we carried out an allelotype analysis encompassing known and recently described TSG loci on 13q, together with immunohistochemical analysis of pRB. We analyzed 82 nonfunctional tumors and 53 somatotrophinomas subdivided into invasive and noninvasive cohorts. A significantly higher frequency of loss, at one or more of 13 markers, was evident in the invasive nonfunctional tumors (54%, 26 of 48) than in their noninvasive counterparts (29%, 10 of 34). An approximately equal frequency of loss was apparent in invasive (28%, 5 of 18) and noninvasive (31%, 11 of 35) somatotrophinomas at one or more markers. In those tumors harboring deletion, loss at two or more markers was more frequent in invasive nonfunctional tumors 65% (17 of 26) compared with 36% (4 of 11) of their noninvasive counterparts. In somatotrophinomas, 40% (2 of 5) of invasive tumors as compared with 64% (7 of 11) of noninvasive tumors had evidence of two or more deletions. In tumors showing loss at two or more loci, the majority showed large deletions; however, loss of the RB1 intragenic marker D13S153 was infrequent. In most cases, loss at individual markers was more frequent in invasive tumors than their noninvasive counterparts. A marker 3 cM telomeric to RB1 (D13S1319) showed the highest frequency of deletion in both invasive cohorts (29% of somatotrophinomas and 24% of nonfunctional tumors). Immunohistochemical analysis of pRB showed frequent loss in somatotrophinomas (27%, 9 of 33) in comparison with 4% (2 of 53) of non-functional tumors. Although loss of pRB did not correlate with loss of an intragenic marker or tumor grade, it was significantly associated with the somatotrophinoma subtype (P = 0.002). These data suggest that chromosome 13q is a frequent target for allelic deletion in pituitary tumors and point to another or other TSG loci in these regions.

Preferential loss of Death Associated Protein kinase expression in invasive pituitary tumours is associated with either CpG island methylation or homozygous deletion.

Death Associated Protein kinase (DAP kinase) a novel calmodulin-dependent serine/threonine kinase was first identified as a positive mediator of programmed cell death. Loss of DAP kinase expression was first demonstrated in highly metastatic cells, whilst re-expression of the protein resulted in delayed local tumour growth and a decreased incidence of metastasis. Although loss of DAP kinase expression has been reported in several cell lines derived from human malignancies the mechanisms responsible have not been defined. In this study we have examined 32 sporadic pituitary tumours for expression of the DAP kinase protein and transcript. In addition, we examined the methylation and deletion status of the DAP kinase CpG island as possible mechanisms for the inactivation of the DAP kinase gene. Eleven of 32 (34%) tumours had undetectable DAP kinase expression, by Western blot and/or RT-PCR analysis. Loss of DAP kinase expression was significantly (P=0.004) associated with invasive tumours (10 of 17; 59%) compared to their non-invasive (1 of 15; 7%) counterparts. Of 11 tumours that failed to express DAP kinase, five (45%) showed de novo methylation of the CpG island contained within the promoter region, while four (36%) had evidence of homozygous deletion of this region. Statistical analysis showed that loss of DAP kinase expression was significantly (P=<0.001) associated with methylation or deletion of the DAP kinase CpG island. With two exceptions, none of the remaining tumours or five histologically normal post-mortem pituitaries examined had evidence of methylation or deletion within this region. To our knowledge this is the first report that describes two mutually exclusive mechanisms associated with loss of DAP kinase gene expression. In addition, we also show that loss of the DAP kinase protein and associated genetic aberrations preferentially segregates with tumours that show an invasive phenotype.

Mutation and expression analysis of the p27/kip1 gene in corticotrophin-secreting tumours.

The molecular mechanisms leading to Cushing's disease are unclear. Inhibitors of cyclin-cyclin dependent kinase (CDK) complexes are regulators of the cell cycle and may function as tumour suppressor genes, many of which have been involved in the pathogenesis of several human malignancies. A member of this family, the p27/kip1 gene, maps to chromosome 12p13 and encodes an inhibitor of several cyclin-CDK complexes; these control the progression of the cell cycle from G1 to S-phase. Complete lack of p27/kip1 function, as occurs in the p27/kip1 'knockout' mouse, produces a complex phenotype associated with the development of pituitary tumours, specifically those of the intermediate lobe corticotrophs. We therefore investigated whether structural and functional abnormalities of the p27/kip1 gene and loss at the chromosome 12p13 region were present in human corticotrophin (ACTH)-secreting pituitary tumours. We studied 21 pituitary tumours, of which 20 were ACTH-secreting (two of these had biochemical and histological features of 'intermediate-lobe' tumours and one was malignant) while the remaining tumour was a prolactinoma; three ectopic secretors of ACTH (two bronchial and one thymic carcinoid); and a non-secretory thymic carcinoid. The whole coding region of the p27/ kip1 gene was screened for mutations by PCR-SSCP analysis and/or direct sequencing, while tumour mRNA expression was analysed by means of a semi-quantitative duplex PCR. Three polymorphic microsatellite markers of the 12p13 region were used to assess loss of heterozygosity (LOH) in 12 samples. Finally, tumour p27/kip1 protein expression was assessed by immunohistochemistry using a monoclonal antibody in 12 samples suitable for analysis. No sequence abnormalities were found in any of the samples other than a previously-described polymorphism. No LOH was observed in the tumours analysed. p27/kip1 mRNA expression was similar in tumour samples in comparison with normal pituitaries. Seven of the eight corticotroph tumours analysed by immunohistochemistry stained positive for p27/kip1, including the intermediate lobe. The only malignant pituitary tumour in the original series showed an absence of staining for p27/kip1. In addition, the three carcinoid tumours studied were negative on immunohistochemistry. Of a further three malignant pituitary tumours assessed, two (including a prolactinoma) were essentially negative, while the third was moderately positive. We conclude that mutations of the p27/kip1 gene, deletions of the 12p13 area or changes in expression, are not a general feature of corticotroph tumours, even those with intermediate lobe characteristics. However, other mechanisms of p27/kip1 inactivation, such as an abnormality at the post-translational level, may be related to more aggressive histological subtypes of ACTH-secreting and possibly other pituitary tumours.

Do polycystic ovaries on ultrasound scan indicate decreased insulin sensitivity in sisters of women with polycystic ovary syndrome?

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenemia, insulin resistance, altered lipid profile, and therefore with subsequently increased risk for metabolic complications such as type 2 diabetes and cardiovascular diseases. It has been reported that sisters of probands with PCOS, who themselves had PCOS or hyperandrogenemia with normal menses, were more insulin resistant than unaffected sisters. We have previously reported that 60% of first-degree relatives (premenopausal mothers and sisters) of PCOS probands had polycystic ovaries (PCO) on ultrascan. Sisters with PCO were more likely to have oligomenorrhea and increased androgen levels than sisters without PCO. The aims of this study were to assess insulin sensitivity status [homeostasis model of assessment, quantitative insulin sensitivity check index, glucose to insulin ratio (G/I)] and lipid profiles in probands with PCOS and their sisters with PCO and without PCO on ultrascan. Mixed model hierarchical regression analysis, to accommodate the nonindependent nature of the subjects (family relationships), with the three groups together did not show significant differences in insulin sensitivity, calculated as quantitative insulin sensitivity check index, homeostasis model of assessment, and G/I for PCOS probands, through sisters with PCO on ultrascan, to sisters without PCO on ultrascan. There was a significant association of measures of insulin sensitivity with body mass index. Lipid parameters did not differ between the groups. These data suggest that presence of PCO on ultrasound scan per se does not predispose to reduced insulin sensitivity in sisters of women with PCOS. Because about 20% of premenopausal women in the general population have PCO on ultrascan, and obesity/overweight is becoming more prevalent, it is important that future studies take full account of the contribution made by obesity to risk factors for metabolic/vascular complications.