polycystic ovarian syndrome ( pcos ) is a complex disease characterized by hyperandrogenism and chronic anovulation .
the detection of traditional cardiovascular ( cv ) risk factors , that is , insulin resistance , diabetes , visceral obesity [ 26 ] , and a state of low - grade inflammation [ 710 ] , places these patients at a significantly higher risk for development of symptomatic atherosclerotic cv diseases .
the presence of a cluster of metabolic and inflammatory factors has often been described in obese pcos compared with age- and weight - matched controls .
higher serum levels of hs - crp , il-6 , and leptin have been reported , the latter being a proinflammatory molecule mainly produced by adipose tissue [ 1315 ] .
the latter alterations were all related to bmi , mainly increased visceral adipose tissue , frequently observed in these patients and which in turn correlates with insulin resistance [ 8 , 16 ] .
conversely , adiponectin , an anti - inflammatory lipokine was found to be decreased in all pcos patients [ 17 , 18 ] .
although the majority of related articles focus on cv risk in adult pcos , with this aspect currently being studied also in young pcos patients , the various inflammatory markers are frequently investigated in separate studies [ 2 , 9 , 19 , 20 ] . the aim of the present paper was to evaluate the most frequently investigated inflammatory cytokines during the early stages of reproductive life in a sardinian pcos population , both with or without insulin resistance .
indeed , the study evaluated cytokine production following microbial and mitogenic stimuli in lymphomonocytes from healthy and pcos subjects , as these molecules are primarily of immunological origin .
from january 2007 , 44 young girls aged 1523 affected by pcos were recruited at the adolescent centre for gynecological diseases , department of obstetrics and gynecology ,  san giovanni di dio  hospital , university of cagliari .
the diagnosis   of pcos was made according to rotterdam criteria   in the presence of at least two of the following : ( 1 ) oligomenorrhea ( 40 subjects ) and/or anovulation ( 4 subjects ) , ( 2 ) hyperandrogenism ( clinical and/or biochemical ) ; ( 3 ) polycystic ovaries with the exclusion of other etiologies .
all subjects had previously been screened to exclude other causes of hyperandrogenism , such as androgen - secreting tumors and congenital adrenal hyperplasia ( tested by evaluation of 17 - 4 hydroxyprogesterone ) , and for euthyroid status , hyperprolactinemia , diabetes , hypertension , and other cardiovascular diseases . over the three months preceding the study no subject had been on hormonal contraceptives , other medications or diet which could have affected lipid profile , carbohydrate metabolism , and insulin levels .
fifty - five healthy young female volunteers with regular menstrual cycles , matched for bmi and with no clinical or biochemical signs of hyperandrogenism and ultrasound exclusion of pco , took part as a control group .
hormonal study ( after 12 hours fasting ) included a baseline plasma determination of androstenedione ( a ) , testosterone ( t ) , and prolactin ( prl ) .
lh , fsh , estradiol ( e2 ) , dehydroepiandrosteronesulphate ( dheas ) ; 17-hydroxyprogesterone ( 17-ohp ) , and sex hormone binding globulin ( shbg ) levels were also assayed but are not reported in the present paper .
all hormones were measured by ria methods using commercial kits ( radim , pomezia , and ares serono , milan , italy ) .
samples were immediately processed in a refrigerated centrifuge , and the sera obtained stored at 20c until assay .
the intra - assay and interassay coefficients of variation obtained were < 9% for all hormones .
( dsl ) , webster , tx ) and glycemia ( glucosidase method , beckman glucose analyzer , fullertonn ca ) were measured during a 75  g oral glucose tolerance test ( ogtt ) , at 0 , 30 , 60 , 90 , 120 , and 180  min from glucose load . a normal glycemic response to ogtt
insulin and glucose responses were expressed as area under the curve ( auc-180 ) , calculated according to the trapezoidal rule .
the homeostatic index of ir ( homa ) was estimated considering fasting glucose ( mmol / l ) and insulin ( u / ml ) divided by a constant ( io  go/22.5 ) . to identify the prevalence of ir in young pcos patients , homa values obtained from control group
were pooled to establish upper control values using the mean + 2 standard deviation as previously described [ 19 , 24 , 25 ] . on the basis of 95% confidence limits ,
body mass index ( bmi ) was calculated according to the following formula : weight in kilograms/(height in meters ) 2 . to ascertain whether metabolic state could be associated with a peculiar inflammatory pattern , patients were divided into two groups according to homa status : nonresistant ( nir ) and insulin resistant ( ir ) .
a serum sample from each participant was stored at 80c for determination of il-1 , il-6 , tnf- , il-10 , tgf- , hs - crp , leptin , and adiponectin .
cytokines were assessed using a sandwich elisa test ( biosource , nivelles , belgium ) as reported previously .
absorbance at 450  nm for all cytokines was measured with a model 680 microplate reader ( bio - rad , hercules , ca ) .
a standard curve was prepared by plotting absorbance value of the standard cytokine versus the corresponding concentration ( pg / ml or ng / ml ) .
the range of the assay was 7.8500  pg / ml for il-1 , 0.1610  pg / ml for il-6 , 15.61000  pg / ml for tnf- , 31.22000  pg / ml for il-10 , 15.61000  pg / ml for leptin , 0.532  ng / ml for adiponectin , and 785000  pg / ml for crp .
the range of assay for cytokine cultures was 7.81000  pg / ml for il-1 , 7.81000  pg / ml for il-6 , 7.81000  pg / ml for tnf- , 31.22000  pg / ml for il-10 , and 62.54000  pg / ml tgf-. in some experiments the secretion of monocyte cytokines into medium was evaluated using samples obtained from several patients and controls .
cells were removed by centrifugation of 400  g for 10  min and supernatants frozen at 20c until assay .
informed consent for in vitro studies was obtained from 8 ir , 5 nir , and 8 control subjects .
each blood sample was diluted twice with rpmi 1640 before centrifugation on ficoll - hipaque ( density 1.077 ; sigma chemical co. ) at 800  g for 20  min at room temperature . the interphase band , containing the mononuclear cells , was collected and cells purified as previously described .
lymphomononuclear cells , at a density of 3  106/ml , were cultured in rpmi 1640 medium supplemented with 2% heat - inactivated human ab   serum , penicillin 100  u / ml , streptomycin 100  mg / ml and 2  mm l - glutamine , and were allowed to adhere at 37c in a 5% co2 atmosphere for 2 hours .
after removal of nonadherent cells , 8090% of the observed cells was represented by monocytes , as assessed by anti - cd-14b staining and flow cytometric analysis .
cells were washed twice with prewarmed medium and cultured with rpmi-1640 supplemented with heat inactivated 10% ab plasma . to avoid the effect of unknown inflammatory factors present in plasma from patients and/or volunteers , commercially obtained
adherent monocytes were activated with lps ( serotype 026:b26 , 100  ng / ml per 24 ) .   on completion of experiments
medium supernatants were stored at 70c and cytokines measured concomitantly to avoid variations in assay conditions . in some experiments , the suspended lymphocytes were lectin - stimulated ( pha 10  ng ) and utilized to evaluate proliferative and inflammatory response .
adherent cells were incubated for 4 hours in medium containing [ 14c]-oleate bound to bovine serum albumin ( bsa ) . to prepare the oleate - bsa complex ,
3.7  mbq of [ 14c]-oleic acid in ethanol ( dupont , nen specific activity 2.035  gbq / mmol ) was mixed with 1.4  mg of koh and the ethanol evaporated .
pbs ( 1.5  ml ) without ca and mg containing 4.24  mg of bsa ( fatty acid free , sigma ) was added and the mixture shaken vigorously .
this solution was added to each well at a final concentration of 74  kbq / ml .
after incubation , cells were washed with ice - cold pbs and lipids extracted with acetone .
neutral lipids were separated by tlc as previously described [ 28 , 29 ] and incorporation of [ 14c]-oleate into cholesterol esters was measured .
data were expressed as mean  sem , and differences between groups were evaluated by the student 's t - test . for systemic markers of inflammation , owing to the prominent skewness of distributions , data were expressed as median , interquartile and min - max ranges and differences between groups were evaluated using the nonparametric mann - whitney u test .
the study was approved by the ethical committee of the university of cagliari . written informed consent was obtained from all subjects and/or their parents or legal guardian before participating in the study . in line with the presence of peripheral insulin resistance ( homa > 65,6 ) ,
pcos subjects were divided into 2 subgroups : 19 insulin - resistant ( ir ) and 25 noninsulin - resistant ( nir ) subjects . as shown in table 1 ,
bmi , auc - i 180 , and homa-7,16 were significantly higher in ir pcos .
as expected , androgen and testosterone plasma levels were increased in all pcos patients ( table 2 ) .
no significant differences were found in plasma lipids ( data not shown ) . in accordance with the four criteria established by cook ( 30 ) , no subjects were affected by metabolic syndrome . as shown in figure 1(a ) leptin levels were higher in all pcos groups , the higher values being found in ir patients ( median : 16.30  pg / ml ; min - max range : 3.6120.00 ; p <  .05 ) on comparison of single groups to controls ( median : 10.30  pg / ml ; min - max range : 4.0059.81 ) , thus displaying a trend similar to that observed for bmi ( figure 1(b ) ) . on the contrary , adiponectin ( figure 1(c ) )
was significantly decreased in all pcos groups : ir ( median : 11.40  ng / ml ; min - max range : 6.5024.80 ; p <  .05 ) , nir ( median : 14.15  ng / ml ; min - max range : 5.7034.98 ; p <  .05 ) , compared to control values ( median : 15.80  ng / ml ; min - max range : 4.8037.10 ) .
crp values , although revealing a trend similar to leptin and bmi , were significantly increased in ir ( median 555  pg / ml , min - max range : 54.351668 ) compared to nir ( median 189.67  pg , min - max : 41.2943 ) and control ( median : 139,6  pg min - max range : 41,21772 ) groups ( figure 1(d ) ) . no variation in tnf- ( figure 2(a ) ) was observed in any group , and similar observations were made for il-1 , il-10 , and tgf- ( data not shown ) .  
however , il-6 ( figure 2(b ) ) was significantly increased in all groups , when compared to controls ( median : 0.28  pg / ml ; min - max range : 0.1312 ) , with a peak in ir ( median : 2.79  pg / ml ; min - max range : 0.5612.00 ; p <  .001 ) .
when considering bmi and leptin ( r = 0.6071 ; p <  .001 ) , as well as bmi and adiponectin ( r   =   0.3387 ; p > 
.002 ) , both a positive and a negative correlation , respectively , was found in all subjects , independent of pcos status .
a correlation was detected between bmi and il-6 in the control group ( r2 = 0,64 ) but the range of il-6 values was definitely lower than that observed in both nir and ir groups .
in addition , in both pcos groups the correlation was very low ( r2 = 0.004 and 0.16 nir and ir , resp . ) .  
inflammatory response was evaluated in lymphocytes and monocytes challenged with microbial stimulus . tnf- and il- , as well as
the anti - inflammatory cytokine il-10 and tgf- secretion in 24-hour lps - activated monocytes , were similar in pcos and control groups ( data not shown ) . on the contrary ,
il-6 production was remarkably and significantly higher in all lps - treated monocytes from ir when compared to nir and controls ( figure 3 ) .  
inflammatory cytokines and proliferative response evaluated in lymphocytes from the same subjects did not reveal significant differences between groups ( data not shown ) .   as shown in figure 4
, lps did not modify the rate of cholesterol esterification , the pathway leading to foam cell formation , hallmark of atherosclerosis , either in pcos patients or controls .
the present study demonstrates that the presence of a cluster of metabolic and inflammatory factors in insulin resistant pcos girls is accompanied by an altered   immune response to microbial stimulus , contributing to higher levels of serum il-6 .
pcos patients , frequently affected by visceral obesity and insulin - resistance [ 30 , 31 ] , feature an increased risk of developing atherosclerotic vascular disease later in life [ 27 , 3234 ] . a large body of reports published to date has focused on the evaluation in middle - aged females of metabolic , hormonal , and inflammatory factors accounting for low - grade inflammation leading to atherosclerosis [ 810 , 3540 ] . although the majority of related articles focus on cv risk in adult pcos , a condition of low - grade inflammation has also been described in adolescent girls [ 2 , 20 , 30 , 40 , 41 ] . in the pcos population described in this study , bmi at times
accordingly , bmi and the prevalence of metabolic syndrome in the italian female population has already been reported to be lower than figures obtained for their american counterpart [ 42 , 43 ] .   in the present study
a cluster of molecules implicated in the onset of low - grade inflammation of the arterial wall was detected largely in adolescent girls developing insulin resistance .
as reported [ 17 , 18 , 44 ] , anti - inflammatory adiponectin alone was significantly lower in all pcos patients examined , irrespective of bmi and insulin resistance , whereas in pcos and normal healthy adults [ 11 , 13 , 14 , 35 ] , hs - crp and leptin were positively correlated with bmi .
the above molecules are likely related to the increased visceral adipose tissue often reported in the literature [ 8 , 18 , 45 ] .
the increase revealed in waist to hip ( w / h ) ratio in pcos subjects is a common observation [ 4648 ] and is suggestive of a correlation with visceral adipose tissue , irrespective of bmi .  
since visceral adipose tissue is associated with an increased production of a large number of functional molecules , it may be implicated , at least in part , in both insulin resistance and low - grade inflammation . a significant increase in serum proinflammatory cytokines
was observed only for il-6 , featuring higher levels in all ir pcos , independent of bmi .
although described by others   no increase of serum levels and production of tnf alfa was detected in our population .
it is well acknowledged that il-6 has been implicated in cardiovascular atherosclerotic risk , dyslipidemia , and hypertension .
moreover , it is a potent inducer of hepatic crp , a molecule displaying increased levels in patients with severe atherosclerosis and following acute clinical events ( myocardial infarction and cerebral ictus ) .
it is a well - established fact that , in addition to visceral adipose tissue , activated lymphomonocytes produce large amounts of cytokines ( for review see ) .  
investigations were thus undertaken to evaluate cytokine production in the latter cells , strongly involved in atherosclerotic plaque formation .
indeed , in the presence of an appropriate stimulus , these are the first cells recruited in the arterial wall .
of all the pro- and anti - inflammatory cytokines evaluated , il-6 production alone proved to be invariably higher in lps - activated monocytes from ir subjects , whereas only 1 out of 5 nir produced il-6 comparably to ir cells .  
the results obtained suggest that an altered milieu due to insulin resistance may influence production of il-6 in these cells .
it may of course be argued that culture conditions do not reproduce the  in vivo 
environment , however , in an attempt to better resemble ex vivo conditions , following separation , cells were immediately cultured in presence of lps for 24 hours . nevertheless , neither the susceptibility of monocytes to evolve into foam cells , nor mitogenic and inflammatory response to lectin in lymphocytes differed between ir and nir pcos and controls .
nevertheless , it remains to be seen whether potential differences may be manifested later in life or on the manifestation of a metabolic disease .
accordingly , an abnormal production of tnf alfa has been described in monocytes from diabetic pcos .   to conclude , the results obtained in this study strongly suggest that hormonal and metabolic factors in young pcos subjects are strongly associated with insulin resistance , and may concur to determine a condition of low - grade inflammation eliciting an overproduction of il-6 monocytes in ir pcos subjects following microbial inflammatory stimulus .
this aspect should be carefully taken into consideration in view of recent evidence implicating infections as new atherosclerotic risk factors [ 53 , 54 ]
. should proof of the latter be provided , further studies should be undertaken to investigate immune response in pcos girls , particularly in view of the number of different microbial agents to which an individual is exposed during his or her lifetime .
finally , the results obtained suggest that high il-6 in young pcos subjects may also represent a signal of altered immune response that may enhance the identification of subjects featuring an increased risk of cardiovascular disease later in life .
the study was funded by fondazione banco di sardegna , regione autonoma della sardegna and nutrisearch srl ( italy ) .
there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported .
 polycystic ovarian syndrome ( pcos ) is frequently characterized by obesity and metabolic diseases including hypertension , insulin resistance , and diabetes in adulthood , all leading to an increased risk of atherosclerosis . 
 the present study aimed to evaluate serum and production of inflammatory markers in adolescent sardinian pcos . on the basis of homa findings , patients were divided into noninsulin resistant ( nir ) and insulin resistant ( ir ) , and were weight- and age - matched with healthy girls . 
 inflammatory cytokines ( tnf- , il-6 , il-10 , tgf- ) and lipokines ( leptin , adiponectin ) , the reactant hs - crp , and in vitro inflammatory lympho - monocyte response to microbial stimulus were evaluated . in healthy and pcos subjects , leptin and hs - crp were correlated with bmi , whereas adiponectin was significantly reduced in all pcos groups . 
 although cytokines were similar in all groups , interleukin-6 ( il-6 ) was significantly higher in ir pcos . 
 moreover , in the latter group lipopolysaccharide - activated monocytes secreted significantly higher levels of il-6 compared to nir and control subjects . to conclude , ir pcos displayed increased il-6 serum levels and higher secretion in lps - activated monocytes , whilst revealing no differences for other inflammatory cytokines . 
 these results suggest that in pcos patients an altered immune response to inflammatory stimuli is present in ir , likely contributing towards determining onset of a low grade inflammation . 
1. Introduction
2. Subjects and Methods
3. Results
4. Discussion
 Funding
 Conflict of Interests