research article adrenocortical production is associated with...

Research ArticleAdrenocortical Production Is Associated withHigher Levels of Luteinizing Hormone in Nonobese Womenwith Polycystic Ovary Syndrome

Luciana Tock1 Glaacuteucia Carneiro1 Andrea Z Pereira1

Seacutergio Tufik2 and Maria Teresa Zanella1

1 Division of Endocrinology Department of Medicine Universidade Federal de Sao Paulo Sao Paulo SP Brazil2 Department of Psychobiology Sleep Disorders Center Universidade Federal de Sao Paulo Sao Paulo SP Brazil

Correspondence should be addressed to Luciana Tock lutockhotmailcom

Received 26 January 2014 Accepted 16 April 2014 Published 7 May 2014

Academic Editor Muhammad Shahab

Copyright copy 2014 Luciana Tock et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective Insulin resistance (IR) and ovarian and adrenal hyperandrogenism are a common finding in women with polycysticovary syndrome (PCOS) The aim of the present study was to access possible differences in insulin resistance gonadotropins andandrogens production in obese and nonobese PCOS women Study Design We studied 37 PCOS women (16 nonobese and 21obese) and 18 nonobese controls Fasting glucose insulin androgens and gonadotropins levels were determined Salivary cortisolwasmeasured basal and in themorning after dexamethasone (DEX) 025mg ResultsNonobese PCOSwomen showed higher basalsalivary cortisol and serum dehydroepiandrosterone sulfate and luteinizing hormone (LH) levels than controls and obese PCOSThese hormones levels did not differ between the obese and control groups After DEX administration no differences were foundbetween the three groups In PCOS women salivary cortisol levels showed negative correlation with BMI (119903 = minus052 119875 = 0001)and insulin (119903 = minus047 119875 = 0003) and positive correlation with LH (119903 = 040 119875 = 0016) Conclusion Our results show anincreased adrenocortical production in nonobese PCOS women not related to IR and associated with a normal hypothalamic-pituitary-adrenal suppression Higher LH levels might be involved in this event

1 Introduction

Ovarian hyperandrogenism is a hallmark of polycystic ovarysyndrome (PCOS) and insulin resistance (IR) is a commonfinding in these women [1ndash3] IR is present in 50 to 70 ofPCOSwomen and plays a significant role in the physiopathol-ogy of this syndromeThe resultant increases in insulin levelsstimulate ovarian androgen production acting directly onovarian theca cells and also reduces sex hormone-bindingglobulin (SHBG) which increases free androgens levels [1ndash3]Furthermore hyperinsulinism decreases hepatic productionof insulin-like growth factor-binding proteins (IGFBPs)leading to increase in insulin-like growth factor 1 (IGF-1) bioavailability which also stimulates ovarian androgenproduction [1ndash3] Insulin also can bind IGF-1 receptor andactivate the intracellular events contributing to the ovariansteroidogenic abnormalities found in PCOS women [1ndash3]

Functional adrenal hyperandrogenism is found inapproximately half of PCOS women [1] These womendemonstrate a generalized hypersecretion of adrenocorticalproducts both in a basal condition and in response toadrenocorticotropic hormone (ACTH) stimulation [4ndash8]although the mechanisms of these abnormalities are stillunclear Similarly unclear are the effects of insulin resistanceon adrenal function in PCOS women [1ndash9]

Although the prevalence of insulin resistance in womenwith PCOS is higher compared to healthy women with sim-ilar body mass index (BMI) there is a distinguished numberof PCOS women mainly the less obese that do not showinsulin resistance [3] Presumably a different physiopathologyis present in these women

The aim of the present study was to access possibledifferences in insulin resistance gonadotropins and andro-gens production in obese and nonobese PCOS women and

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2014 Article ID 620605 7 pageshttpdxdoiorg1011552014620605

2 International Journal of Endocrinology

the influence of insulin resistance on the hormonal profileof these two groups of women A low-dose (DEX) (025mg)suppression test with measurements of salivary cortisolwhich closely correlates with the concentration of serumfree cortisol [10] was used to evaluate the hypothalamic-pituitary-adrenal (HPA) axisThis test induces amoremodestACTH suppression than the standard DEX (10mg) testenabling the detection of subtle differences in HPA axisfeedback sensitivity to glucocorticoids [11]

2 Materials and Methods

21 Population Our study included 37 PCOS women whowere divided in 2 groups 16 nonobese (BMI lt30Kgm2) and21 obese (BMI ge30Kgm2) and 18 nonobese healthy womenwith age between 16 and 45 years Subjects were recruitedfrom the Endocrinology Division of Federal University ofSao Paulo (UNIFESP) Brazil The diagnosis of PCOS wasbased on the latest 2003 Rotterdam consensus [12] requiringthe presence of at least two of the following features (1)oligomenorrhea or chronic anovulation (2) clinical andorbiochemical hyperandrogenism and (3) ultrasound (US)appearance of polycystic ovaries after the exclusion of otherknown causes of hyperandrogenemia such as congenitaladrenal hyperplasia androgen-secreting tumors and Cush-ingrsquos syndrome Exclusion criteria included the use of oralcontraceptives corticosteroids and antidiabetic drugs in thelast 3 months We also excluded patients with diabetes mel-litus untreated hypothyroidism and renal hepatic cardiacor pulmonary disease The study was approved by the EthicsCommittee of UNIFESP and an informed written consentwas obtained from all subjects

22 Clinical Anthropometrical and Biochemical ParametersA questionnaire was used to document personal medicaland drug history regularity and length of menstrual cyclesand ovulation status Signs of androgen excess (hirsutismalopecia and acne) were noted in the physical examinationHirsutism with a Ferriman-Gallwey score of 8 or above wasconsidered as clinical evidence of androgen excess Bodyweight (in kilograms) body height (in meters) and waistand hip circumference (in centimeters) weremeasuredWaistcircumference was taken as the narrowest measurementmidway between the top of the iliac crest and the lowerrib margin whereas the hip circumference was taken as thewidest measurement at the level of the greater trochantersBMI was calculated from the ratio between weight and heightsquared

Blood specimens were obtained after a 12-hour overnightfast from all subjects in the early follicular phase of themenstrual cycle or after a period of amenorrhea above 3months for measurement of plasma glucose luteinizing hor-mone (LH) follicle-stimulating hormone (FSH) androgenicprofile and serum insulin A standard 75-gram oral glucosetolerance test (OGTT) was performed in PCOS patients forinsulin sensitive index (ISI) calculation

The assessment of HPA axis included low-dose (025mg)DEX suppression test with measurement of salivary cortisol

The women received two Salivettes (Sarstedt RommelsdorfGermany) which consist of a small cotton swab inside acentrifugation tube used to collect saliva and a half tablet of05mg of dexamethasone (Decadron Ache Brazil) Salivarysample was obtained between 700 and 800 am in the day ofthe administration of DEX which was taken between 1100and 1200 pm Another salivary sample was obtained on thenext morning between 700 and 800 am To analyze theresults we used an index of percentage of salivary suppression( cortisol suppression) calculated as the difference betweenthe post-DEX cortisol level and baseline cortisol levelsdivided by baseline cortisol levels and multiplied by 100

23 LaboratoryAnalysis Plasma glucosewasmeasured usingADVIA 2400 Chemistry System (Siemens Tarrytown NY)Dehydroepiandrosterone (DHEA) and 17OH-progesteronelevels were measured by enzyme-linked immunosorbentassays (Diagnostic Biochem Canada Ontario Canada andLabor Diagnostika Nod GmbH Nortdhorn Germany resp)Total testosterone (TT) dehydroepiandrosterone sulfate(DHEA-S) LH FSH and insulin were measured using Uni-Cel Dxl 800 Immunoassay System (Beckman Coulter BreaCA) The within-assay coefficient of variation of testosteronewas 199 and the between-assay coefficient was 422There are some limitations to measuring testosterone usinga chemiluminescence immunoassay but this was the onlylaboratory technique available Androstenedione and sexhormone-binding globulin (SHBG) were measured usingIMMULITE 2000 Immunoassay System (Siemens) Serum-free testosterone (FT) and bioavailable testosterone were esti-mated by the formula as previously validated by Vermeulenet al [13] (available at httpwwwissamchfreetestohtm)Free androgen index (FAI) was calculated according to theformula 100 times [TT (nmolL)SHBG] IR was estimatedusing the homeostasis model assessment for insulin resis-tance (HOMA-IR) by dividing the product of fasting insulin(microinternational units (120583IU)mL) and glucose (mmolL)by 225 ISI was calculated according to the formula devel-oped by Belfiore et al [14] 2((INSp times GLYp) + 1) whereINSp and GLYp are obtained by dividing the sum of plasmainsulin (120583IUmL) and glycemia (mmolL) (measured at 0and 2 hours after oral glucose load) by the sum of therespective values of the normal population Normal referencevalues were obtained in 35 normotensive individuals withnormal BMI All biochemical assays were performed at theSleep Institute Laboratory Salivary cortisol was measuredby radioimmunoassay in a single assay run in the SteroidLaboratory of the Endocrinology Division of UNIFESP Thelowest level of detection of salivary cortisol was 5 ngdL andthe between-assay coefficient of variation was 131

24 Statistical Analysis Data are expressed as mean (SD)Comparison of mean values between controls nonobeseand obese PCOS women was performed using ANOVACorrelation between variables was determined using linearcorrelation test The Fisher test was used to analyze theassociation between the presence of amenorrhea in the twogroups of PCOS Linear regression analysis was used to

International Journal of Endocrinology 3

0 10 20 30 40 50

LH (mIUmL)

Obese PCOSNon obese PCOS

r = 040

P = 0016

3

25

2

15

1

05

0

Basa

l sal

ivar

y co

rtiso

l (ng

dL)

times103

Figure 1 Correlation between basal salivary cortisol and luteinizinghormone (LH) levels in the two groups of women with polycysticovary syndrome

identify the hormones associated with cortisol levels amongPCOS subjects A 119875 value lt 005 was considered significantStatistical analyses were performed with the Statistical Pack-age for Social Sciences for Windows version 190 (SPSS IncChicago IL)

3 Results

The anthropometrical and biochemical characteristics of allsubjects are summarized in Table 1 The proportions ofpatients with amenorrhea were not different between the twoPCOS groups of patients (25 in nonobese and 33 in obese119875 = 0580) Obese PCOS women had higher BMI and waist-to-hip ratio than nonobese PCOS subjects and controls butno differences were found between controls and nonobesePCOS women

Nonobese PCOS women had higher LH DHEA-S andbasal salivary cortisol (Figure 1) than control and obese PCOSwomenThese hormones levels were similar between controland obese PCOS women Although there was a trend for adifference inDHEA levels between the twoPCOS groups thisdifference did not reach statistical significance (119875 = 0072)

With regard to androgens profile total and free testos-terones and FAI were similar between PCOS groups andhigher than control group although bioavailable testosteroneshowed to be higher only when obese PCOS group wascompared to control with no difference between the twogroups of PCOS Androstenedione was higher in nonobesePCOS women when compared to controls with no differencebetween the two groups of PCOS

There were no differences in fasting glucose levels amongthe three groups although HOMA-IR and fasting insulin lev-els were higher in obese PCOS group compared to nonobese

3

25

2

15

1

05

0

Basa

l sal

ivar

y co

rtiso

l (ng

dL)


000 1000 2000 3000 4000 60005000

BMI (kgm2)

r = minus052

P = 0001

times103

Figure 2 Correlation between basal salivary cortisol levels andbody mass index (BMI) in women with polycystic ovary syndrome

PCOS and control There were no statistical differences insalivary cortisol levels after DEX suppression test among thethree groups

In the total group of PCOS salivary cortisol levels showedpositive and significant correlation with LH levels (119903 = 040119875 = 0016) (Figure 1) and negative correlation with HOMA-IR (119903 = minus048 119875 = 0003) fasting insulin levels (119903 = minus047119875 = 0003) and BMI (119903 = minus052 119875 = 0001) (Figure 2)Therewas a marginally significant positive correlation betweenbasal salivary cortisol and DHEA levels (119903 = 031 119875 = 0061)and DHEA-S (119903 = 031 119875 = 0063) levels LH levels alsoshowed negative and significant correlation with BMI (119903 =minus036 119875 = 0030)

In linear regression analysis among PCOS subjects withLH and fasting insulin as independent variables and salivarycortisol as dependent variable both hormones showed tohave significant and independent association with cortisollevels LH showed positive association and insulin negativeassociation (Table 2)

4 Discussion

In the present study we showed that there is no hyperactivityof HPA axis in PCOS women as the suppression of salivarycortisol after a low dose of DEX was similar in these womenand in controlsThe presence of obesity also did not interferein low-dose DEX suppression

Studies about cortisol production and metabolism inPCOS subjects demonstrate heterogeneous results Some ofthem show normal basal cortisol levels [15ndash19] while othersshow higher cortisol plasma levels [5ndash7 20] higher ACTHlevels [5 7] and hyperresponsiveness of adrenal to ACTH[4 7 8 17] A possible explanation for this heterogeneity isthe wide range of BMI included in these analyzes We found


Table 1 Clinical and biochemical characteristics of all subjects

Control PCOS non obese PCOS obese119899 18 16 21Presence of amenorrhea 0 25lowast 33

Age (years) 327 plusmn 62 261 plusmn 55lowast

304 plusmn 69

BMI (Kgm2) 239 plusmn 36 252 plusmn 33 379 plusmn 47sect

Waist (cm) 816 plusmn 80 861 plusmn 110 1144 plusmn 138sect

Waist-hip ratio 09 plusmn 01 09 plusmn 01 098 plusmn 01sect

LH (mIUmL) 56 plusmn 20 137 plusmn 113lowast

69 plusmn 36sect

FSH (mIUmL) 72 plusmn 14 61 plusmn 13 52 plusmn 12

Androstenedione (ngmL) 17 plusmn 08 33 plusmn 19lowast

28 plusmn 21

DHEA (ngmL) 55 plusmn 34 138 plusmn 108lowast

78 plusmn 83

DHEA-S (120583gdL) 1194 plusmn 617 1929 plusmn 679lowast

1366 plusmn 663sect

Total testosterone (ngdL) 338 plusmn 124 648 plusmn 296lowast

620 plusmn 406

SHBG (nmolL) 591 plusmn 248 371 plusmn 207lowast

267 plusmn 134

FAI 24 plusmn 12 85 plusmn 88lowast

100 plusmn 90

Free testosterone (ngdL) 05 plusmn 02 13 plusmn 09lowast

14 plusmn 11

Bioavailable testosterone (ngdL) 110 plusmn 43 230 plusmn 112 320 plusmn 248

Fasting glucose (mgdL) 906 plusmn 94 919 plusmn 113 927 plusmn 89

Fasting insulin (120583UmL) 66 plusmn 27 84 plusmn 57 135 plusmn 56sect

HOMA-IR (120583IUmL) 15 plusmn 06 20 plusmn 15 31 plusmn 14sect

Basal cortisol (ngdL) 10166 plusmn 3670 14730 plusmn 5391lowast

10114 plusmn 5128sect

Cortisol after DEX (ngdL) 1989 plusmn 2197 2827 plusmn 3291 1627 plusmn 1764

998779 cortisol (ngdL) 8177 plusmn 4067 11900 plusmn 5497 8488 plusmn 4896

Cortisol supression () minus787 plusmn 253 minus808 plusmn 207 minus825 plusmn 165

lowast119875 lt 05 PCOS non obese versus control

119875 lt 05 PCOS obese versus control

sect119875 lt 05 PCOS obese versus PCOS non obese

Table 2 Linear regression analysis of the hormomes that influencein cortisol levels among PCOS subjects

Beta 95 Confidence Interval 119875

LH 034 38ndash420 020Fasting insulin minus043 minus659ndashminus130 005

higher levels of basal salivary cortisol in nonobese PCOSwomen compared with obese PCOS and nonobese controlsOur results are partly in accordancewith a recent studywherelean PCOS had higher basal cortisol levels compared to obesePCOS and lean controls although LH levels did not showsimilar pattern [21]

Basal levels of DHEA-S also showed similar pattern forbasal levels of salivary cortisol with higher values in nonobesePCOS women compared to obese PCOS and nonobesecontrols The other androgens levels were similar in bothPCOS groups and as expected they were higher in PCOSwomen than in controls DHEA-S is primarily secreted bythe adrenals as there is maintenance of its levels in womenwith premature ovarian failure [22 23] and absence of thishormone in the ovarian vein after catheterization [20] Theseresults suggest that adrenals cortex has an important role inandrogen production only in nonobese PCOS women

Another substantial difference between PCOS groups inour study was related to LH levels Gonadotropin-secretorychanges with a characteristic increase in LH relative to FSH

release have long been appreciated in PCOS The increasein LH pulse frequency reflects an increase in GnRH releaseand suggests the presence of a hypothalamic defect in thisgroup of women [1] We found that nonobese PCOS womenhad higher levels of LH compared to the other two groupsand the level of this hormone was similar in controls andobese PCOS women We also found a negative correlationbetween LH levels and BMI in PCOS women Based on theseresults and in previous studies [22 24ndash26] we can assumethat there are two distinct groups of PCOS one composed ofnonobese noninsulin resistant women showing higher levelsof LH and adrenocortical hormones (cortisol and DHEA-S) and another composed of obese insulin resistant womenshowing lower levels of LH and adrenocortical products Ourcontrol group was composed only of nonobese women whoshowed levels of cortisol and LH comparable to those of obesePCOS women

We found a significant and positive correlation betweensalivary cortisol and LH levels in PCOS women This corre-lation was previously found in postmenopausal women whoalso present high levels of LH [27 28] Several studies havedemonstrated the presence of LH receptors in adrenocor-tical cells which also bind human chorionic gonadotropin(hCG) and their potential to induce local steroidogenesis[29ndash36] In situ hybridization and immunocytochemistryalso demonstrated that the adrenal zona reticularis and thedeeper layer of adrenal zona fasciculada contain LHhCG


receptors [31] In addition it has been demonstrated thatadministration of hCG stimulates cortisol production inguinea-pig adrenal cells [30] increases DHEA-S productionin human fetal adrenal gland [29] and stimulates DHEA-S production in human adrenocortical carcinoma cells [35]Study including 2 womenwith Cushingrsquos syndrome and bilat-eral adrenal hyperplasia demonstrated pronounced cortisolrise after GnRH administration and in vitro the adrenal cellsof these women also responded to hCG exposure increasingcortisol production Furthermore LH receptor mRNA wasdemonstrated in adrenal tissue of both patients [34] Lacroixet al [32] reported awomanwith bilateral adrenal hyperplasiawho presented high cortisol levels and Cushingrsquos syndromeduring her pregnancies andpermanent hypercortisolismonlyafter menopause This patient showed increased cortisolsecretion in response to LH and hCG administration Inaddition long-term suppression of LH by leuprolide acetateadministration resulted in complete reversal of Cushingrsquossyndrome It raises the possibility of an overexpression ofLH receptors in adrenal gland also in nonobese women withPCOS

Supporting the idea that high LH levels are responsible forthe adrenocortical hyperactivity in nonobese PCOS womenKero et al [33] showed the development of polycystic ovariesand bilateral adrenal hyperplasia with increased androgenand cortisol production in a transgenic mouse model witha high constitutive LH production Mazzuco et al [36]demonstrated in amousemodel transplantedwith geneticallymodified bovine adrenocortical cells with an overexpressionof LHhCG receptor gene that it was able to provoke subclin-ical or mild hypercortisolism

The finding in our study that nonobese PCOS patientshave increased adrenocortical production with normal sup-pressible cortisol production after low dose of DEX anda demonstration of a possible influence of LH in adrenalsteroidogenesis as described before suggests that LH mightinfluence adrenocortical production in nonobese PCOSwomen presumably increasing the adrenocortical responseto ACTH This hypothesis was proposed in 1999 by Lacroixet al [32] who showed increases in cortisol levels after LHadministration which were proportional to ACTH levels intwo healthy women in whom the secretion of endogenousLH had been previously suppressedThe same group demon-strated that the suppression of ACTH by pretreatment withDEX prevented the increase of cortisol after LH injection

Our group of obese PCOSwasmore insulin resistant thanthe nonobese group and as we have already described theyhad lower levels of adrenocortical products (DHEA-S andcortisol)The effect of insulin on adrenal activity is controver-sial Some studies suggest that insulin is a negativemodulatorof adrenal androgen metabolism [37 38] although somestudies using insulin sensitizers in PCOS women showeda decrease of adrenal androgens after their administration[9 39] however this event could be consequent to decreasesin LH levels that also occurs after metformin administrationto these women [39] We found a significant and negativecorrelation between basal cortisol and HOMA-IR and fastinginsulin levels in PCOS women and a positive correlationbetween basal cortisol and ISI Our results are in accordance

with Gurusinghe et al [40] who found that morning cortisoland DHEA-S correlated with ISI in PCOS women After alinear regression analysis we showed that LH and insulinlevels may respectively positively and negatively predictcortisol levelsWe can thus raise the hypothesis that the lowerlevels of insulin in nonobese PCOS women might be alsocontributing to the higher adrenocortical production foundin this group compared to obese PCOS women On the otherhand the higher levels of insulin in the obese PCOS groupcould also be contributing to a lower androgen production ofthe adrenal gland

Also in accordancewith our results Saxena and Seely [28]demonstrated that hyperandrogenic women could be dividedinto two subgroups one with insulin resistance (IR) normalor minimally elevated LH and markedly elevated insulinlevels and one with elevated LH levels no insulin resistanceand normal insulin concentrations They found a negativecorrelation between LH and insulin but after eliminating theeffect of BMI this correlation was no longer significant Wedid not find any correlation between LH and HOMA-IR orISI suggesting a minor role of LH in PCOS pathophysiologyin obese women when compared to nonobese women

In conclusion our results indicate an increased adreno-cortical production not related to insulin resistance innonobese PCOS women and associated with a normal HPAsuppression after DEX administration A neuroendocrinedisturbance characterized by higher levels of LH in nonobesePCOS womenmight be involved in the increased adrenocor-tical production in this group Further studies are needed tobetter clarify the role of adrenal gland in the pathophysiologyof PCOS in nonobese women

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by Conselho Nacional de Pesquisa(CNPq) and AFIP (Associacao Fundo de Incentivo aPesquisa)

References

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[2] A K Schroder S Tauchert O Ortmann K Diedrich and JM Weiss ldquoInsulin resistance in patients with polycystic ovarysyndromerdquoAnnals ofMedicine vol 36 no 6 pp 426ndash439 2004

[3] E Carmina S Bucchieri A Esposito et al ldquoAbdominal fatquantity and distribution in women with polycystic ovarysyndrome and extent of its relation to insulin resistancerdquo Journalof Clinical Endocrinology and Metabolism vol 92 no 7 pp2500ndash2505 2007

[4] D A Ehrmann R L Rosenfield R B Barnes D F Brigell andZ Sheikh ldquoDetection of functional ovarian hyperandrogenism


in women with androgen excessrdquo The New England Journal ofMedicine vol 327 no 3 pp 157ndash162 1992

[5] C Invitti M de Martin G Delitala J D Veldhuis and F Cav-agnini ldquoAltered morning and nighttime pulsatile corticotropinand cortisol release in polycystic ovary syndromerdquoMetabolismClinical and Experimental vol 47 no 2 pp 143ndash148 1998

[6] R Luboshitzky A Ishai Z Shen-Or and P Herer ldquoEvaluationof the pituitary-adrenal axis in hyperandrogenic women withpolycystic ovary syndromerdquo Neuroendocrinology Letters vol24 no 3-4 pp 249ndash254 2003

[7] N Kamel V Tonyukuk R Emral D Corapcioglu M Bastemirand S Gullu ldquoRole of ovary and adrenal glands in hyper-androgenemia in patients with polycystic ovary syndromerdquoExperimental and Clinical Endocrinology and Diabetes vol 113no 2 pp 115ndash121 2005

[8] J Puurunen T Piltonen P Jaakkola A Ruokonen L Morin-Papunen and J S Tapanainen ldquoAdrenal androgen productioncapacity remains high up to menopause in women with poly-cystic ovary syndromerdquo Journal of Clinical Endocrinology andMetabolism vol 94 no 6 pp 1973ndash1978 2009

[9] B O Yildiz and R Azziz ldquoThe adrenal and polycystic ovarysyndromerdquo Reviews in Endocrine and Metabolic Disorders vol8 no 4 pp 331ndash342 2007

[10] J G Vieira K O Noguti J T Hidal E M Russo and R MMaciel ldquoMeasurement of saliva cortisol as a method for theevaluation of the free serum fractionrdquo Arquivos Brasileiros deEndocrinologia e Metabolismo vol 28 no 1 pp 8ndash10 1984

[11] NA TMHuizenga JWKoper P de Lange et al ldquoInterpersonvariability but intraperson stability of baseline plasma cortisolconcentrations and its relation to feedback sensitivity of thehypothalamo-pituitary-adrenal axis to a low dose of dexam-ethasone in elderly individualsrdquo Journal of Clinical Endocrinol-ogy and Metabolism vol 83 no 1 pp 47ndash54 1998

[12] The Rotterdam ESHREASRM-Sponsored PCOC ConsensusWorkshop Group ldquoRevised 2003 consensus on diagnosticcriteria and long-term health risks related to polycystic ovarysyndromerdquo Fertility and Sterility vol 81 no 1 pp 19ndash25 2004

[13] A Vermeulen L Verdonck and J M Kaufman ldquoA critical eval-uation of simple methods for the estimation of free testosteronein serumrdquo Journal of Clinical Endocrinology and Metabolismvol 84 no 10 pp 3666ndash3672 1999

[14] F Belfiore S Iannello and G Volpicelli ldquoInsulin sensitivityindices calculated from basal and OGTT-induced insulinglucose and FFA levelsrdquo Molecular Genetics and Metabolismvol 63 no 2 pp 134ndash141 1998

[15] F Gonzalez D A Hatala and L Speroff ldquoAdrenal and ovariansteroid hormone responses to gonadotropin-releasing hormoneagonist treatment in polycystic ovary syndromerdquoTheAmericanJournal of Obstetrics andGynecology vol 165 no 3 pp 535ndash5451991

[16] P M Stewart R Penn R Holder A Parton J G Ratcliffe andD R London ldquoThe hypothalamo-pituitary-adrenal axis acrossthe normal menstrual cycle and in polycystic ovary syndromerdquoClinical Endocrinology vol 38 no 4 pp 387ndash391 1993

[17] A Lanzone F Petraglia A M Fulghesu M Ciampelli ACaruso and S Mancuso ldquoCorticotropin-releasing hormoneinduces an exaggerated response of adrenocorticotropic hor-mone and cortisol in polycystic ovary syndromerdquo Fertility andSterility vol 63 no 6 pp 1195ndash1199 1995

[18] L C Morin-Papunen I Vauhkonen R M Koivunen ARuokonen and J S Tapanainen ldquoInsulin sensitivity insulin

secretion and metabolic and hormonal parameters in healthywomen and womenwith polycystic ovarian syndromerdquoHumanReproduction vol 15 no 6 pp 1266ndash1274 2000

[19] D V Milutinovic D Macut I Bozic J Nestorov S Dam-janovic and G Matic ldquoHypothalamic-pituitary-adrenocorticalaxis hypersensibility and glucocorticoid receptor expressionand function in women with polycystic vary syndromerdquo Exper-imental and Clinical Endocrinology amp Diabetes vol 119 no 10pp 636ndash643 2011

[20] H Martikainen P Salmela S Nuojua-Huttunen et al ldquoAdrenalsteroidogenesis is related to insulin in hyperandrogenicwomenrdquo Fertility and Sterility vol 66 no 4 pp 564ndash570 1996

[21] I Shabir M A Ganie E P Praveen et al ldquoMorning plasmacortisol is low among obese women with polycystic ovarysyndromerdquo Gynecological Endocrinology vol 29 no 12 pp1045ndash1047 2013

[22] A N Elias M R Pandian and F Rojas ldquoSerum levelsof androstenedione testosterone and dehydroepiandrosteronesulfate in patients with premature ovarian failure to age-matched menstruating controlsrdquo Gynecologic and ObstetricInvestigation vol 43 no 1 pp 47ndash48 1997

[23] A Bachelot G Meduri N Massin M Misrahi F Kuttennand P Touraine ldquoOvarian steroidogenesis and serum androgenlevels in patients with premature ovarian failurerdquo Journal ofClinical Endocrinology and Metabolism vol 90 no 4 pp 2391ndash2396 2005

[24] L Anttila R Erkkola Y Ding K Irjala K Ruutiainen and IHuhtaniemi ldquoClinical features and circulating gonadotropininsulin and androgen interactions in women with polycysticovarian diseaserdquo Fertility and Sterility vol 55 no 6 pp 1057ndash1061 1991

[25] A Arroyo G A Laughlin A J Morales and S S C Yen ldquoInap-propriate gonadotropin secretion in polycystic ovary syndromeinfluence of adiposityrdquo Journal of Clinical Endocrinology andMetabolism vol 82 no 11 pp 3728ndash3733 1997

[26] M Zarkovic J Ciric Z Penezic B Trbojevic and M DrezgicldquoTemporal coupling of luteinizing hormone and follicle stim-ulating hormone secretion in polycystic ovary syndromerdquoGynecological Endocrinology vol 15 no 5 pp 381ndash388 2001

[27] M Alevizaki K Saltiki E Mantzou E Anastasiou and IHuhtaniemi ldquoThe adrenal gland may be a target of LH actionin postmenopausal womenrdquoEuropean Journal of Endocrinologyvol 154 no 6 pp 875ndash881 2006

[28] A R Saxena and E W Seely ldquoLuteinizing hormone correlateswith adrenal function in postmenopausal womenrdquoMenopausevol 19 no 11 pp 1280ndash1283 2012

[29] M Seron-Ferre C C Lawrence and R B Jaffe ldquoRole of hCGin regulation of the fetal zone of the human fetal adrenal glandrdquoJournal of Clinical Endocrinology andMetabolism vol 46 no 5pp 834ndash837 1978

[30] Y OrsquoConnell T J McKenna and S K Cunningham ldquoTheeffect of prolactin human chorionic gonadotropin insulin andinsulin-like growth factor 1 on adrenal steroidogenesis in iso-lated guinea-pig adrenal cellsrdquo Journal of Steroid Biochemistryand Molecular Biology vol 48 no 2-3 pp 235ndash240 1994

[31] J E Pabon X Li Z M Lei J S Sanfilippo M A Yussman andC V Rao ldquoNovel presence of luteinizing hormonechorionicgonadotropin receptors in human adrenal glandsrdquo Journal ofClinical Endocrinology and Metabolism vol 81 no 6 pp 2397ndash2400 1996

[32] A Lacroix P Hamet and JM Boutin ldquoLeuprolide acetate ther-apy in luteinizing hormone-dependent Cushingrsquos syndromerdquo


The New England Journal of Medicine vol 341 no 21 pp 1577ndash1581 1999

[33] J Kero M Poutanen F P Zhang et al ldquoElevated luteinizinghormone induces expression of its receptor and promotessteroidogenesis in the adrenal cortexrdquo Journal of Clinical Inves-tigation vol 105 no 5 pp 633ndash641 2000

[34] R A Feelders SW J Lamberts L J Hofland et al ldquoLuteinizinghormone (LH)-responsive cushingrsquos syndrome the demonstra-tion of LH receptor messenger ribonucleic acid in hyperplasticadrenal cells which respond to chorionic gonadotropin andserotonin agonists in vitrordquo Journal of Clinical Endocrinologyand Metabolism vol 88 no 1 pp 230ndash237 2003

[35] C V Rao X L Zhou and Z M Lei ldquoFunctional luteinizinghormonechorionic gonadotropin receptors in human adrenalcortical H295R cellsrdquo Biology of Reproduction vol 71 no 2 pp579ndash587 2004

[36] T L Mazzuco O Chabre J J Feige and MThomas ldquoAberrantexpression of human luteinizing hormone receptor by adreno-cortical cells is sufficient to provoke both hyperplasia andcushingrsquos syndrome featuresrdquo Journal of Clinical Endocrinologyand Metabolism vol 91 no 1 pp 196ndash203 2006

[37] J E Nestler and J F Strauss III ldquoInsulin as an effector of humanovarian and adrenal steroid metabolismrdquo Endocrinology andMetabolism Clinics of North America vol 20 no 4 pp 807ndash8231991

[38] J Vrbıkova M Hill L Starka et al ldquoThe effects of long-termmetformin treatment on adrenal and ovarian steroidogenesis inwomen with polycystic ovary syndromerdquo European Journal ofEndocrinology vol 144 no 6 pp 619ndash628 2001

[39] A D Genazzani C Strucchi M Luisi et al ldquoMetforminadministrationmodulates neurosteroids secretion in non-obeseamenorrhoic patientswith polycystic ovary syndromerdquoGyneco-logical Endocrinology vol 22 no 1 pp 36ndash43 2006

[40] D Gurusinghe S Gill R U Almario et al ldquoIn polycysticovary syndrome adrenal steroids are regulated differently inthe morning versus in response to nutrient intakerdquo Fertility andSterility vol 93 no 4 pp 1192ndash1199 2010

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the influence of insulin resistance on the hormonal profileof these two groups of women A low-dose (DEX) (025mg)suppression test with measurements of salivary cortisolwhich closely correlates with the concentration of serumfree cortisol [10] was used to evaluate the hypothalamic-pituitary-adrenal (HPA) axisThis test induces amoremodestACTH suppression than the standard DEX (10mg) testenabling the detection of subtle differences in HPA axisfeedback sensitivity to glucocorticoids [11]

2 Materials and Methods

21 Population Our study included 37 PCOS women whowere divided in 2 groups 16 nonobese (BMI lt30Kgm2) and21 obese (BMI ge30Kgm2) and 18 nonobese healthy womenwith age between 16 and 45 years Subjects were recruitedfrom the Endocrinology Division of Federal University ofSao Paulo (UNIFESP) Brazil The diagnosis of PCOS wasbased on the latest 2003 Rotterdam consensus [12] requiringthe presence of at least two of the following features (1)oligomenorrhea or chronic anovulation (2) clinical andorbiochemical hyperandrogenism and (3) ultrasound (US)appearance of polycystic ovaries after the exclusion of otherknown causes of hyperandrogenemia such as congenitaladrenal hyperplasia androgen-secreting tumors and Cush-ingrsquos syndrome Exclusion criteria included the use of oralcontraceptives corticosteroids and antidiabetic drugs in thelast 3 months We also excluded patients with diabetes mel-litus untreated hypothyroidism and renal hepatic cardiacor pulmonary disease The study was approved by the EthicsCommittee of UNIFESP and an informed written consentwas obtained from all subjects

22 Clinical Anthropometrical and Biochemical ParametersA questionnaire was used to document personal medicaland drug history regularity and length of menstrual cyclesand ovulation status Signs of androgen excess (hirsutismalopecia and acne) were noted in the physical examinationHirsutism with a Ferriman-Gallwey score of 8 or above wasconsidered as clinical evidence of androgen excess Bodyweight (in kilograms) body height (in meters) and waistand hip circumference (in centimeters) weremeasuredWaistcircumference was taken as the narrowest measurementmidway between the top of the iliac crest and the lowerrib margin whereas the hip circumference was taken as thewidest measurement at the level of the greater trochantersBMI was calculated from the ratio between weight and heightsquared

Blood specimens were obtained after a 12-hour overnightfast from all subjects in the early follicular phase of themenstrual cycle or after a period of amenorrhea above 3months for measurement of plasma glucose luteinizing hor-mone (LH) follicle-stimulating hormone (FSH) androgenicprofile and serum insulin A standard 75-gram oral glucosetolerance test (OGTT) was performed in PCOS patients forinsulin sensitive index (ISI) calculation

The assessment of HPA axis included low-dose (025mg)DEX suppression test with measurement of salivary cortisol

The women received two Salivettes (Sarstedt RommelsdorfGermany) which consist of a small cotton swab inside acentrifugation tube used to collect saliva and a half tablet of05mg of dexamethasone (Decadron Ache Brazil) Salivarysample was obtained between 700 and 800 am in the day ofthe administration of DEX which was taken between 1100and 1200 pm Another salivary sample was obtained on thenext morning between 700 and 800 am To analyze theresults we used an index of percentage of salivary suppression( cortisol suppression) calculated as the difference betweenthe post-DEX cortisol level and baseline cortisol levelsdivided by baseline cortisol levels and multiplied by 100

23 LaboratoryAnalysis Plasma glucosewasmeasured usingADVIA 2400 Chemistry System (Siemens Tarrytown NY)Dehydroepiandrosterone (DHEA) and 17OH-progesteronelevels were measured by enzyme-linked immunosorbentassays (Diagnostic Biochem Canada Ontario Canada andLabor Diagnostika Nod GmbH Nortdhorn Germany resp)Total testosterone (TT) dehydroepiandrosterone sulfate(DHEA-S) LH FSH and insulin were measured using Uni-Cel Dxl 800 Immunoassay System (Beckman Coulter BreaCA) The within-assay coefficient of variation of testosteronewas 199 and the between-assay coefficient was 422There are some limitations to measuring testosterone usinga chemiluminescence immunoassay but this was the onlylaboratory technique available Androstenedione and sexhormone-binding globulin (SHBG) were measured usingIMMULITE 2000 Immunoassay System (Siemens) Serum-free testosterone (FT) and bioavailable testosterone were esti-mated by the formula as previously validated by Vermeulenet al [13] (available at httpwwwissamchfreetestohtm)Free androgen index (FAI) was calculated according to theformula 100 times [TT (nmolL)SHBG] IR was estimatedusing the homeostasis model assessment for insulin resis-tance (HOMA-IR) by dividing the product of fasting insulin(microinternational units (120583IU)mL) and glucose (mmolL)by 225 ISI was calculated according to the formula devel-oped by Belfiore et al [14] 2((INSp times GLYp) + 1) whereINSp and GLYp are obtained by dividing the sum of plasmainsulin (120583IUmL) and glycemia (mmolL) (measured at 0and 2 hours after oral glucose load) by the sum of therespective values of the normal population Normal referencevalues were obtained in 35 normotensive individuals withnormal BMI All biochemical assays were performed at theSleep Institute Laboratory Salivary cortisol was measuredby radioimmunoassay in a single assay run in the SteroidLaboratory of the Endocrinology Division of UNIFESP Thelowest level of detection of salivary cortisol was 5 ngdL andthe between-assay coefficient of variation was 131

24 Statistical Analysis Data are expressed as mean (SD)Comparison of mean values between controls nonobeseand obese PCOS women was performed using ANOVACorrelation between variables was determined using linearcorrelation test The Fisher test was used to analyze theassociation between the presence of amenorrhea in the twogroups of PCOS Linear regression analysis was used to


0 10 20 30 40 50

LH (mIUmL)


r = 040

P = 0016

3

25

2

15

1

05

0

Basa

l sal

ivar

y co

rtiso

l (ng

dL)

times103



3 Results





3

25

2

15

1

05

0

Basa

l sal

ivar

y co

rtiso

l (ng

dL)


000 1000 2000 3000 4000 60005000

BMI (kgm2)

r = minus052

P = 0001

times103





4 Discussion







304 plusmn 69





69 plusmn 36sect



28 plusmn 21


78 plusmn 83


1366 plusmn 663sect


620 plusmn 406


267 plusmn 134


100 plusmn 90


14 plusmn 11































Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 10 20 30 40 50

LH (mIUmL)


r = 040

P = 0016

3

25

2

15

1

05

0

Basa

l sal

ivar

y co

rtiso

l (ng

dL)

times103



3 Results





3

25

2

15

1

05

0

Basa

l sal

ivar

y co

rtiso

l (ng

dL)


000 1000 2000 3000 4000 60005000

BMI (kgm2)

r = minus052

P = 0001

times103





4 Discussion







304 plusmn 69





69 plusmn 36sect



28 plusmn 21


78 plusmn 83


1366 plusmn 663sect


620 plusmn 406


267 plusmn 134


100 plusmn 90


14 plusmn 11































Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















304 plusmn 69





69 plusmn 36sect



28 plusmn 21


78 plusmn 83


1366 plusmn 663sect


620 plusmn 406


267 plusmn 134


100 plusmn 90


14 plusmn 11































Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article adrenocortical production is associated with...

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