all pcos patients met all three criteria of the revised 2003 rotterdam european society of human reproduction and embryology ( eshre)/american society of reproductive medicine ( asrm ) pcos consensus workshop group diagnostic criteria . the three criteria are 1 ) oligo- and/or anovulation , 2 ) clinical and/or biochemical signs of hyperandrogenism , and 3 ) polycystic ovaries ( 20 ) . furthermore , all subjects in the control arm had normal findings on pelvic ultrasound scan , regular periods , and no hirsutism / acne . exclusion criteria for the study included age > 40 years , known cardiovascular disease , thyroid disease , neoplasms , current smoking , diabetes , hypertension ( blood pressure > 140/90 mmhg ) , and renal impairment ( serum creatinine > 120 mol / l ) . none of these women were on any medications for at least 6 months prior to the study , including oral contraceptives , glucocorticoids , ovulation induction agents , antidiabetic and antiobesity drugs , and estrogenic , antiandrogenic , or antihypertensive medication . blood pressure was measured in a sitting position within a quiet and calm environment after a rest of at least 5 min . subjects were outpatients of the department of reproductive medicine and gynaecological endocrinology of magdeburg university and the department of obstetrics and gynaecology of martin - luther - university halle . the metabolic study was performed in the outpatient department of endocrinology and metabolism of magdeburg university . blood samples were collected between 0800 and 0900 h , after a 3-day normal carbohydrate diet and an overnight fast . blood samples were drawn into serum separator tubes that contain no additives or anticoagulants , allowed to clot , centrifuged ( 3,000 rpm for 10 min ) to separate sera , and stored at 80c . a treatment with metformin in an  off - label use was offered to all pcos women independently from the results of insulin sensitivity testing . in those pcos women that agreed , therapy was initiated after basal assessment , and the dose of metformin was increased to a maintenance dose of 850 mg twice daily . carotid intima media thickness ( imt ) was measured in pcos subjects , before and after metformin ( see below ) . a total of 83 women of caucasian origin with pcos were recruited , 49 of which did not participate in the metformin arm of the study . the baseline characteristics of the 49 pcos women were comparable to the 34 pcos women who participated in the metformin arm of the study . furthermore , the 21 women who completed the metformin arm of the study were comparable to the 13 pcos women who did not complete the metformin arm of the study . for the purposes of elucidating the effects of metformin in pcos women , the study design was approved by the local research ethics committee of the university of magdeburg , and written informed consent was obtained from all participants , in accordance with the guidelines in the declaration of helsinki 2000 . measurement of carotid imt is a widely accepted tool to provide information about preclinical vascular disease . b - mode sonography of the proximal part of the carotid bulb was conducted on both sides , and the segments of the common carotid arteries 1.0 cm proximal were scanned longitudinally with hitachi eub-5000 plus - g ( hitachi ltd . , all women were investigated in a supine position with the head slightly hyperextended and turned away from the side being scanned . the image was focused on the posterior wall , and the resolution function was used to magnify the arterial far wall . when an optimal image was obtained , it was frozen , and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end - diastolic phase , to minimize variability during the cardiac cycle . five measurements were conducted on each side , and the mean imt was calculated as the average of these measurements . all measurements were performed by an experienced ultrasound sonographer ( intra - observer coefficient of variation was 6.8% ) who was blinded to the diagnosis of subjects . assays for glucose , insulin , cholesterol , triglycerides , luteinizing hormone , follicular stimulating hormone , testosterone , and rostenedione , dehydroepiandrosterone - sulfate , and sex hormone  binding globulin were performed using an automated analyzer ( abbott architect , abbott laboratories , abbott park , usa ) . the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) score was calculated as previously described ( 21 ) . high - sensitivity c - reactive protein ( hs - crp ) was determined immunoturbidimetrically using a modular system random - access analyzer ( roche diagnostics ) . omentin-1 levels in sera were measured using a commercially available elisa kit ( axxora , nottingham , u.k . ) , according to manufacturer 's protocol , with an intra - assay coefficient of variation of < 6% . immunodepletion of serum omentin-1 and crp were performed using the immunoprecipitation protocol provided by dr . one ml of human serum was incubated with mouse - anti - human omentin-1 igg - protein g - sepharose conjugate ( 12 g/100500 g of total protein ) or control mouse igg - protein g - sepharose conjugate ; goat - anti - human crp iga - protein a - sepharose conjugate ( 10 g/100500 g of total protein ) or control goat iga - protein a - sepharose conjugate , mixed overnight at 4c and centrifuged to sediment the conjugated sepharose beads . supernatants and sera were subjected to immunoblotting for omentin-1 as previously described ( 13 ) . immunoblotting confirmed that these protocols decreased serum omentin-1 and crp by 80% ( data not shown ) , respectively . human microvascular endothelial cells ( hmec-1 ) were obtained from the center for disease control , atlanta , usa hmec-1 cells were cultured in mcdb medium ( sigma - aldrich , gillingham , u.k . ) supplemented with 10% fetal calf serum ( sigma - aldrich , gillingham , u.k . ) , 100 iu / ml penicillin ( sigma - aldrich , gillingham , u.k . ) , 100 g / ml streptomycin ( sigma - aldrich ) , 5 ml of 200 mmol / l l - glutamine/500 ml of media , hydrocortisone 2 mol / l , and epidermal growth factor 2 ng / ml ( invitrogen , paisley , u.k . ) at 37c in 5% co2/95% air . prior to each experiment , cells were fed with mcdb with addition of 1% fetal calf serum for 16 h. for endothelial cell signaling experiments , hmecs were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] at different time points ( 0 , 2 , 5 , 10 , 20 , 30 , and 60 min ) . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . angiogenesis was assessed by studying the formation of capillary tubelike structures by culturing hmec-1 cells on growth factor reduced matrigel ( bd biosciences , san jose , usa ) . hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera . trypsinized hmec-1 cells were then seeded onto the matrigel - coated plates at a density of 45  10/well in fresh media and incubated at 37c . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . capillary tube formation images were captured with a digital microscope camera system ( olympus , tokyo , japan ) . image pro plus software was used to quantify tube length formation ; the lengths of tubes in 34 randomly selected fields in each of the wells were measured ( 2326 ) . endothelial cell migration assay was performed in a modified boyden chamber using a protocol obtained from bd biocoat angiogenesis system ( bd biosciences , san jose , usa ) . endothelial cells were trypsinized ; a cell suspension of 4  10 cells / ml was prepared , 250 l of which was added to each of the trans - well inserts . starvation media ( 750 l ) was added to the lower chamber and incubated overnight . after this , the cells were labeled by incubating with hank 's balanced salt solution medium ( sigma - aldrich , gillingham , u.k . ) containing 4 g / ml calcein - am ( bd biosciences , san jose , usa ) for 90 min . the cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation / emission wavelengths of 494/517 nm . only those labeled cells that have migrated through the pores of the membrane will be detected ( 2326 ) . we studied nf-b activation by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites ( pnf-b - luc ; stratagene , la jolla , usa ) . hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera for 2 h. cells were lysed , and luciferase activities were measured . experiments were also performed with tk plasmid ( promega , southampton , u.k . ) as negative control and tumor necrosis factor  ( tnf- ) as positive control . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . endothelial cells were lysed with sds sample buffer ( 5 mol / l urea , 0.17 mol / l sds , 0.4 mol / l dithiothreitol , and 50 mmol / l tris - hcl , ph 8.0 ) , mixed , sonicated , boiled , centrifuged ( 5,000 rpm for 2 min ) , and then stored at 80c until use . eighty g of each sample , supernatants , and sera were subjected to sds - page ( 8% resolving gel ) and transferred to polyvinylidene fluoride ( pvdf ) membranes . pvdf membranes were blocked in tris - buffered saline containing 0.1% tween-20 and 5% bsa for 2 h. the pvdf membranes were then incubated with polyclonal primary rabbit - anti - human antibody for phospho - erk1/2 ( cell signaling technology inc . , beverly , usa ) ( 1:1,000 dilution ) or polyclonal primary rabbit - anti - human antibody for phospho - akt ( ser473 ) ( cell signaling technology inc . , beverly , usa ) ( 1:1,000 dilution ) or monoclonal primary goat - anti - human antibody for crp ( sigma - aldrich , gillingham , u.k . ) ( 1:1,000 dilution ) overnight at 4c . the membranes were washed thoroughly for 60 min with tris - buffered saline0.1% tween before incubation with the secondary anti - rabbit horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) or secondary anti - goat horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) for 1 h at room temperature . antibody complexes were visualized using chemiluminescence ( ecl+ ; ge healthcare , little chalfont , u.k . ) . for standardization , the same membranes were stripped and reprobed using polyclonal primary rabbit - anti - human antibodies for total erk1/2 ( cell signaling technology inc . the densities were measured using a scanning densitometer coupled to scanning software scion image ( scion corporation , frederick , usa ) . standard curves were generated to ensure linearity of signal intensity over the range of protein amounts loaded into gel lanes . comparisons of densitometric signal intensities for phospho - erk1/2 , phospho - akt , total erk1/2 , and total akt were made only within this linearity range . whitney u test or kruskal  wallis anova ( post hoc analysis : dunn 's test ) according to the number of groups compared . if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 . measurement of carotid imt is a widely accepted tool to provide information about preclinical vascular disease . b - mode sonography of the proximal part of the carotid bulb was conducted on both sides , and the segments of the common carotid arteries 1.0 cm proximal were scanned longitudinally with hitachi eub-5000 plus - g ( hitachi ltd . , all women were investigated in a supine position with the head slightly hyperextended and turned away from the side being scanned . the image was focused on the posterior wall , and the resolution function was used to magnify the arterial far wall . when an optimal image was obtained , it was frozen , and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end - diastolic phase , to minimize variability during the cardiac cycle . five measurements were conducted on each side , and the mean imt was calculated as the average of these measurements . all measurements were performed by an experienced ultrasound sonographer ( intra - observer coefficient of variation was 6.8% ) who was blinded to the diagnosis of subjects . assays for glucose , insulin , cholesterol , triglycerides , luteinizing hormone , follicular stimulating hormone , testosterone , and rostenedione , dehydroepiandrosterone - sulfate , and sex hormone  binding globulin were performed using an automated analyzer ( abbott architect , abbott laboratories , abbott park , usa ) . the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) score was calculated as previously described ( 21 ) . high - sensitivity c - reactive protein ( hs - crp ) was determined immunoturbidimetrically using a modular system random - access analyzer ( roche diagnostics ) . omentin-1 levels in sera were measured using a commercially available elisa kit ( axxora , nottingham , u.k . ) , according to manufacturer 's protocol , with an intra - assay coefficient of variation of < 6% . immunodepletion of serum omentin-1 and crp were performed using the immunoprecipitation protocol provided by dr . one ml of human serum was incubated with mouse - anti - human omentin-1 igg - protein g - sepharose conjugate ( 12 g/100500 g of total protein ) or control mouse igg - protein g - sepharose conjugate ; goat - anti - human crp iga - protein a - sepharose conjugate ( 10 g/100500 g of total protein ) or control goat iga - protein a - sepharose conjugate , mixed overnight at 4c and centrifuged to sediment the conjugated sepharose beads . supernatants and sera were subjected to immunoblotting for omentin-1 as previously described ( 13 ) . immunoblotting confirmed that these protocols decreased serum omentin-1 and crp by 80% ( data not shown ) , respectively . human microvascular endothelial cells ( hmec-1 ) were obtained from the center for disease control , atlanta , usa hmec-1 cells were cultured in mcdb medium ( sigma - aldrich , gillingham , u.k . ) supplemented with 10% fetal calf serum ( sigma - aldrich , gillingham , u.k . ) , 100 iu / ml penicillin ( sigma - aldrich , gillingham , u.k . ) , 100 g / ml streptomycin ( sigma - aldrich ) , 5 ml of 200 mmol / l l - glutamine/500 ml of media , hydrocortisone 2 mol / l , and epidermal growth factor 2 ng / ml ( invitrogen , paisley , u.k . ) at 37c in 5% co2/95% air . prior to each experiment , cells were fed with mcdb with addition of 1% fetal calf serum for 16 h. for endothelial cell signaling experiments , hmecs were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] at different time points ( 0 , 2 , 5 , 10 , 20 , 30 , and 60 min ) . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . angiogenesis was assessed by studying the formation of capillary tubelike structures by culturing hmec-1 cells on growth factor reduced matrigel ( bd biosciences , san jose , usa ) . hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera . vascular endothelial growth factor ( vegf ) served as positive control . trypsinized hmec-1 cells were then seeded onto the matrigel - coated plates at a density of 45  10/well in fresh media and incubated at 37c . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . capillary tube formation images were captured with a digital microscope camera system ( olympus , tokyo , japan ) . image pro plus software was used to quantify tube length formation ; the lengths of tubes in 34 randomly selected fields in each of the wells were measured ( 2326 ) . endothelial cell migration assay was performed in a modified boyden chamber using a protocol obtained from bd biocoat angiogenesis system ( bd biosciences , san jose , usa ) . endothelial cells were trypsinized ; a cell suspension of 4  10 cells / ml was prepared , 250 l of which was added to each of the trans - well inserts . starvation media ( 750 l ) after this , the cells were labeled by incubating with hank 's balanced salt solution medium ( sigma - aldrich , gillingham , u.k . ) containing 4 g / ml calcein - am ( bd biosciences , san jose , usa ) for 90 min . the cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) and/or with or without pi3k / akt inhibitor ( ly294002 ) ( calbiochem , san diego , usa ) for 30 min and 1 h , respectively , followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation / emission wavelengths of 494/517 nm . only those labeled cells that have migrated through the pores of the membrane will be detected ( 2326 ) . we studied nf-b activation by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites ( pnf-b - luc ; stratagene , la jolla , usa ) . hmec-1 cells were preincubated with or without omentin-1 ( kamiya biomedical company , seattle , usa ) for 30 min followed by treatment with or without 1% of human serum from normal ( n = 39 ; individually tested ) and pcos women [ before ( n = 21 ; individually tested ) and after ( n = 21 ; individually tested ) 6 months of metformin treatment ] or crp or omentin-1 [ pcos after metformin ( n = 21 ; individually tested ) ] and crp [ pcos before metformin ( n = 21 ; individually tested ) ] immunodepleted sera for 2 h. cells were lysed , and luciferase activities were measured . experiments were also performed with tk plasmid ( promega , southampton , u.k . ) as negative control and tumor necrosis factor  ( tnf- ) as positive control . dose- and time - dependent experiments were performed to determine the optimum concentration and time point . endothelial cells were lysed with sds sample buffer ( 5 mol / l urea , 0.17 mol / l sds , 0.4 mol / l dithiothreitol , and 50 mmol / l tris - hcl , ph 8.0 ) , mixed , sonicated , boiled , centrifuged ( 5,000 rpm for 2 min ) , and then stored at 80c until use . eighty g of each sample , supernatants , and sera were subjected to sds - page ( 8% resolving gel ) and transferred to polyvinylidene fluoride ( pvdf ) membranes . pvdf membranes were blocked in tris - buffered saline containing 0.1% tween-20 and 5% bsa for 2 h. the pvdf membranes were then incubated with polyclonal primary rabbit - anti - human antibody for phospho - erk1/2 ( cell signaling technology inc . , beverly , usa ) ( 1:1,000 dilution ) or polyclonal primary rabbit - anti - human antibody for phospho - akt ( ser473 ) ( cell signaling technology inc . , beverly , usa ) ( 1:1,000 dilution ) or monoclonal primary goat - anti - human antibody for crp ( sigma - aldrich , gillingham , u.k . ) ( 1:1,000 dilution ) overnight at 4c . the membranes were washed thoroughly for 60 min with tris - buffered saline0.1% tween before incubation with the secondary anti - rabbit horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) or secondary anti - goat horseradish peroxidase - conjugated immunoglobulin ( dako , ely , u.k . ) ( 1:2,000 ) for 1 h at room temperature . antibody complexes were visualized using chemiluminescence ( ecl+ ; ge healthcare , little chalfont , u.k . ) . for standardization , the same membranes were stripped and reprobed using polyclonal primary rabbit - anti - human antibodies for total erk1/2 ( cell signaling technology inc . the densities were measured using a scanning densitometer coupled to scanning software scion image ( scion corporation , frederick , usa ) . standard curves were generated to ensure linearity of signal intensity over the range of protein amounts loaded into gel lanes . comparisons of densitometric signal intensities for phospho - erk1/2 , phospho - akt , total erk1/2 , and total akt were made only within this linearity range . whitney u test or kruskal  wallis anova ( post hoc analysis : dunn 's test ) according to the number of groups compared . if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 . insulin , homa - ir , cholesterol , triglycerides , luteinizing hormone , testosterone , free androgen index , systolic blood pressure , diastolic blood pressure , imt , and hs - crp were significantly higher whereas dehydroepiandrosterone - sulfate and sex hormone  binding globulin were significantly lower in pcos women . clinical , hormonal , and metabolic features of women with pcos and control subjects data are median ( interquartile range ) . free androgen index ( fai ) = t ( nmol / l)/shbg ( nmol / l )  100 . ns = not significant ; lh , luteinizing hormone ; fsh , follicular stimulating hormone ; dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone  binding globulin ; sbp , systolic blood pressure ; dbp , diastolic blood pressure . serum omentin-1 levels were significantly lower in pcos subjects compared with control subjects [ 23.7 ( 20.027.9 ) versus 27.6 ( 25.629.4 ) ng / ml ; p < 0.05 : table 1 ] . only 21 women completed the study and were investigated after 6 months of metformin treatment . reasons for subjects not completing study 2 were nausea and gastrointestinal side effects ( n = 4 ) , pregnancies ( n = 5 ) , incompliance ( n = 2 ) , and loss of contact ( n = 2 ) . clinical , hormonal , and metabolic features of women with pcos ( n = 21 ) before and after metformin treatment data are median ( interquartile range ) . free androgen index ( fai ) = t ( nmol / l)/shbg ( nmol / l )  100 . ns = not significant ; dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone  binding globulin ; sbp , systolic blood pressure ; dbp , diastolic blood pressure . after 6 months of metformin treatment , there was a significant increase in serum omentin-1 [ 23.7 ( 20.027.9 ) versus 59.2 ( 52.160.6 ) ng / ml ; p < 0.01 : table 2 ] . also , there were significant decreases in waist - to - hip ratio ( whr ) , testosterone , glucose , homa - ir , and imt . spearman rank analyses demonstrated that serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp ( p < 0.05 ) . however , when subjected to multiple regression analysis , none of these variables were significantly negatively correlated with serum omentin-1 levels ( p > 0.05 ) . furthermore , we analyzed the correlation between the change in serum omentin-1 levels before and after metformin therapy ( omentin-1 ) and the changes (  ) in other covariates ( table 3 ) . we found that omentin-1 was significantly negatively associated with whr , glucose , homa - ir , and hs - crp . when subjected to multiple regression analysis , only hs - crp was significantly negatively correlated with omentin-1 (  = 0.526 ; p = 0.036 ) . linear regression analysis of variables associated with changes in serum omentin-1 levels ( before and after metformin treatment ) , omentin-1 , in pcos subjects ( n = 21 ) spearman rank correlation was used for calculation of associations between variables . if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 . multiple regression analysis contained whr , glucose , homa - ir , and hs - crp . dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone  binding globulin ; fai , free androgen index ; sbp , systolic blood pressure ; dbp , diastolic blood pressure . given the above , we studied the effects of omentin-1 on in vitro migration and angiogenesis . capillary tube formation was optimal at 24 h , after which capillary tubes begin to disintegrate . in vitro migration and angiogenesis at 24 h was significantly increased when comparing sera of pcos women to normal control subjects ( figs . 1 and 2 ; p < 0.01 ) . also , in vitro migration and angiogenesis were significantly decreased in pcos women after 6 months of metformin treatment ( figs . 1 and 2 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) or the pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) were added to sera ( figs . 1 and 2 ; * p < 0.05 , * * p < 0.01 ) . furthermore , crp ( 1ug / ml ) and vegf ( 10 ng / ml ) induced in vitro migration , and angiogenesis was significantly decreased by omentin-1 ( figs . 1 and 2 ; * p < 0.05 ) . in vitro tube formation by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively . data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann  whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . endothelial cell migration by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively . data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann - whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , in vitro migration and angiogenesis were significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( figs . 1 and 2 ; nf-b was significantly activated by sera from normal and pcos women compared with control subjects ( fig . nf-b was significantly activated by sera from pcos women compared with normal women ( fig . 3 ; * * also , nf-b activation was significantly decreased in pcos women after 6 months of metformin treatment ( fig . 3 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) was added to sera ( fig . 3 ; furthermore , crp ( 1 ug / ml ) and tnf- ( 10 ng / ml ) induced nf-b activation was significantly decreased by omentin-1 ( fig . * p < 0.05 ) . effects of serum from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and tnf- ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively , on nf-b activation in hmec-1 cells stably transfected with pnf-b - luciferase at 2 h. cells were lysed , and luciferase activities ( rlu ) were measured . data are expressed as % difference of median of basal [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann  whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , nf-b activation was significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( fig . 3 ; p > 0.05 , * p < 0.05 ) . in hmec-1 cells , erk1/2 and akt pathways were significantly activated by sera from normal and pcos women compared with control subjects ( fig . erk1/2 and akt phosphorylation were significantly increased by sera from pcos women compared with normal women ( fig . also , erk1/2 and akt activation were significantly decreased in pcos women after 6 months of metformin treatment ( fig . 4a and b ; * p < 0.05 , * * p < 0.01 ) . furthermore , akt activation was significantly decreased when omentin-1 ( 200 ng / ml ) was added to sera from pcos women before metformin treatment ( fig . 4a ) . the detected proteins for phosphorylated erk1/2 , total erk1/2 , phosphorylated akt , and total akthad apparent molecular weights of 44/42 , 44/42 , 60 , and 60 kda , effects of serum from normal control subjects ( n = 39 ) , pcos women ( n = 21 ) , and pcos women after 6 months of metformin treatment with or without addition of omentin-1 ( 200 ng / ml ) ( n = 21 ) on erk1/2 and akt phosphorylation in hmec-1 cells at 2 and 5 min , respectively . data are expressed as % difference of median of basal [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann  whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . spearman rank analyses demonstrated that serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp ( p < 0.05 ) . however , when subjected to multiple regression analysis , none of these variables were significantly negatively correlated with serum omentin-1 levels ( p > 0.05 ) . furthermore , we analyzed the correlation between the change in serum omentin-1 levels before and after metformin therapy ( omentin-1 ) and the changes (  ) in other covariates ( table 3 ) . we found that omentin-1 was significantly negatively associated with whr , glucose , homa - ir , and hs - crp . when subjected to multiple regression analysis , only hs - crp was significantly negatively correlated with omentin-1 (  = 0.526 ; p = 0.036 ) . linear regression analysis of variables associated with changes in serum omentin-1 levels ( before and after metformin treatment ) , omentin-1 , in pcos subjects ( n = 21 ) spearman rank correlation was used for calculation of associations between variables . if individual bivariate correlations achieved statistical significance , multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1 . multiple regression analysis contained whr , glucose , homa - ir , and hs - crp . dhea - s , dehydroepiandrosterone - sulfate ; shbg , sex hormone  binding globulin ; fai , free androgen index ; sbp , systolic blood pressure ; dbp , diastolic blood pressure . given the above , we studied the effects of omentin-1 on in vitro migration and angiogenesis . capillary tube formation was optimal at 24 h , after which capillary tubes begin to disintegrate . in vitro migration and angiogenesis at 24 h was significantly increased when comparing sera of pcos women to normal control subjects ( figs . 1 and 2 ; p < 0.01 ) . also , in vitro migration and angiogenesis were significantly decreased in pcos women after 6 months of metformin treatment ( figs . 1 and 2 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) or the pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) were added to sera ( figs . 1 and 2 ; * p < 0.05 , * * p < 0.01 ) . furthermore , crp ( 1ug / ml ) and vegf ( 10 ng / ml ) induced in vitro migration , and angiogenesis was significantly decreased by omentin-1 ( figs . 1 and 2 ; * p < 0.05 ) . in vitro tube formation by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively . data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann  whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . endothelial cell migration by human serum at 24 h from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or pi3k / akt inhibitor ( ly294002 ; 10 mol / l ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and vegf ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively . data are expressed as % difference of median of normal control subjects [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann - whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , in vitro migration and angiogenesis were significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( figs . 1 and 2 ; p > 0.05 , * p < 0.05 ) . in hmec-1 cells , nf-b was significantly activated by sera from normal and pcos women compared with control subjects ( fig . nf-b was significantly activated by sera from pcos women compared with normal women ( fig . 3 ; * * also , nf-b activation was significantly decreased in pcos women after 6 months of metformin treatment ( fig . 3 ; * * p < 0.01 ) and when omentin-1 ( 200 ng / ml ) was added to sera ( fig . 3 ; * p < 0.05 ) . furthermore , crp ( 1 ug / ml ) and tnf- ( 10 ng / ml ) induced nf-b activation was significantly decreased by omentin-1 ( fig effects of serum from normal control subjects ( n = 39 ) with or without omentin-1 ( 200 ng / ml ) , pcos women ( n = 21 ) with or without omentin-1 ( 200 ng / ml ) or crp immunodepletion ( crp_ip ) , pcos women after 6 months of metformin treatment ( n = 21 ) with or without omentin-1 immunodepletion ( omentin-1_ip ) , crp ( 1ug / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , and tnf- ( 10 ng / ml ) with or without addition of omentin-1 ( 200 ng / ml ) ( n = 6 ) , respectively , on nf-b activation in hmec-1 cells stably transfected with pnf-b - luciferase at 2 h. cells were lysed , and luciferase activities ( rlu ) were measured . data are expressed as % difference of median of basal [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann  whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . moreover , nf-b activation was significantly decreased in crp ( pcos before metformin ) but not significantly altered by omentin-1 ( pcos after metformin ) immunodepleted sera ( fig . in hmec-1 cells , erk1/2 and akt pathways were significantly activated by sera from normal and pcos women compared with control subjects ( fig . erk1/2 and akt phosphorylation were significantly increased by sera from pcos women compared with normal women ( fig . also , erk1/2 and akt activation were significantly decreased in pcos women after 6 months of metformin treatment ( fig . 4a and b ; * p < 0.05 , * * p < 0.01 ) . furthermore , akt activation was significantly decreased when omentin-1 ( 200 ng / ml ) was added to sera from pcos women before metformin treatment ( fig . 4a ) . the detected proteins for phosphorylated erk1/2 , total erk1/2 , phosphorylated akt , and total akthad apparent molecular weights of 44/42 , 44/42 , 60 , and 60 kda , effects of serum from normal control subjects ( n = 39 ) , pcos women ( n = 21 ) , and pcos women after 6 months of metformin treatment with or without addition of omentin-1 ( 200 ng / ml ) ( n = 21 ) on erk1/2 and akt phosphorylation in hmec-1 cells at 2 and 5 min , respectively . data are expressed as % difference of median of basal [ medians ( interquartile range ) ] . wallis anova ( post hoc analysis : dunn 's test ) and mann  whitney u test , respectively . p < 0.05 , p < 0.01 ; * p < 0.05 , * * p < 0.01 . also , serum omentin-1 levels were significantly negatively correlated with bmi , whr , glucose , homa - ir , and hs - crp . we report that metformin ( 6 months treatment ; 850 mg twice daily ) significantly increases serum omentin-1 levels with concomitant decreases in insulin resistance and carotid imt in pcos subjects . although the change in serum omentin-1 levels were significantly negatively associated with changes in whr , glucose , homa - ir , and hs - crp , only the change of hs - crp was significantly negatively correlated with the change of serum omentin-1 levels when subjected to multiple regression analysis . the possible implication is that changes in omentin-1 levels associated with metformin treatment observed in our pcos subjects are more robustly linked to changes in inflammatory rather than metabolic parameters . of relevance , there is evidence that inflammatory markers other than crp are elevated in pcos subjects ( 35 ) ; also , studies have indicated metformin 's potential anti - inflammatory effects via modulation of tnf- , il-6 , il-18 , and il-1- levels ( 2729 ) . in pcos women , metformin corrects the associated endocrine and metabolic abnormalities ( 30 ) ; metformin counters ir by inhibiting hepatic glucose production ( 3133 ) . therefore , glucose may regulate circulating omentin-1 levels ; however , it is possible that the effect of metformin on omentin-1 levels could be via modulation of other circulating factors . we , like others ( 34 ) , observed a significant decrease in testosterone levels after metformin treatment . however , treatments of omental adipose tissue explants with gonadal and adrenal steroids revealed no meaningful effects on omentin-1 levels ( 13 ) . it is therefore unlikely that the effects of metformin on serum omentin-1 levels observed in this study are attributable to altered gonadal and adrenal steroids . furthermore , functional assays revealed that in vitro migration and angiogenesis were significantly increased by sera from both normal and pcos women ; in pcos women , in vitro migration and angiogenesis was attenuated after 6 months of metformin treatment . addition of omentin-1 to sera from pcos women before metformin treatment significantly decreased in vitro migration and angiogenesis . omentin-1 also decreased the effects of crp and vegf induced in vitro migration and angiogenesis . however , in vitro migration and angiogenesis were significantly decreased in crp but not significantly altered by omentin-1 immunodepleted sera . it is important to consider that omentin-1 levels in tissues are unknown ; the levels are likely to be significantly higher than in sera as sequestration of omentin-1 in vascular tissue , in particular , within stromal - vascular cells , has been shown ( 10,35 ) . all of these effects appear to be associated with nf-b and akt signaling pathways , both well - established mediators of angiogenesis ( 3638 ) . taken together , increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women . it is important to bear in mind that metformin may also modulate angiogenesis via the vegf system . of relevance the link between angiogenesis and the pathogenesis of atherosclerosis has been well described ( 17 ) . given our data of an increase in circulating omentin-1 levels with concomitant decrease in imt , omentin-1 may have a potential role in the pathogenesis of atherosclerosis . omentin-1 , by promoting activation of the akt signaling pathway and in turn modulating the endothelial nitric oxide synthase in endothelial cells , may impact upon endothelial dysfunction , a step that is crucial in the pathogenesis of atherosclerosis ( 40,41 ) . our novel data of omentin-1 on angiogenesis in relation to the akt signaling pathway thus clarifies omentin-1 's potentially important role in the pathogenesis of cardiovascular disorders . a limitation of our study may relate to the lack of a lean pcos comparator group . only 3 of the 21 pcos subjects and 6 of the 39 control subjects had bmi < 25 kg / m ; thus , no meaningful result could be obtained . furthermore , given ethical constraints , that is , blood samples could and were only obtained at baseline and after 6 months of metformin treatment , it is therefore difficult to be certain as to whether changes in serum omentin-1 levels precede or follow changes in clinical and hormonal indexes as a consequence of metformin treatment . also , as diet and lifestyle modifications were only subjectively assessed , the changes in serum omentin-1 levels may be partially attributable to diet and lifestyle modifications . further studies are needed to elucidate this point . additionally , like others ( 42 ) , we used whr , a universally accepted , simple , and cost - effective measure of adiposity as a surrogate marker for central fat mass . however , cascella et al . has reported that whr does not distinguish between visceral and subcutaneous fat mass in pcos women ( 43 ) . therefore , more accurate and equally cost - effective methods of assessing central obesity are needed . finally , given that pcos is a proinflammatory state associated with clustering of cardiovascular risk factors , it would be of importance to study the effect of metformin therapy in the context of omentin-1 biology on reactive oxygen species in pcos women , as reactive oxygen species not only induces tissue damage and inflammation but also is increased in dysmetabolic states including pcos ( 44 ) . in conclusion , we provide evidence that increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women . our findings provide novel insights into the relationship between obesity , insulin resistance , and metformin therapy with dysregulated angiogenesis in pcos women in the context of omentin-1 biology .
objectivepolycystic ovary syndrome ( pcos ) is associated with the metabolic syndrome . decreased omentin-1 levels are associated with obesity and diabetes . to study the effects of metformin treatment on omentin-1 levels in pcos subjects and effects of omentin-1 on in vitro migration and angiogenesis.research design and methodsserum omentin-1 
 was measured by elisa . 
 angiogenesis was assessed by studying capillary tube formation in human microvascular endothelial cells ( hmec-1 ) on growth factor reduced matrigel . 
 endothelial cell migration assay was performed in a modified boyden chamber . 
 nuclear factor-b ( nf-b ) was studied by stably transfecting hmec-1 cells with a cis - reporter plasmid containing luciferase reporter gene linked to five repeats of nf-b binding sites . 
 akt phosphorylation was assessed by western blotting.resultsserum omentin-1 was significantly lower in pcos women ( p < 0.05 ) . 
 after 6 months of metformin treatment , there was a significant increase in serum omentin-1 ( p < 0.01 ) . 
 importantly , changes in hs - crp were significantly negatively correlated with changes in serum omentin-1 ( p = 0.036 ) . in vitro migration and angiogenesis 
 were significantly increased in serum from pcos women ( p < 0.01 ) compared with matched control subjects ; these effects were significantly attenuated by metformin treatment ( p < 0.01 ) plausibly through the regulation of omentin-1 levels via nf-b and akt pathways . 
 crp and vegf induced in vitro migration , and angiogenesis was significantly decreased by omentin-1.conclusionsincreases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin - treated pcos women .