{"paper_id":"7bedfab2-c306-4050-b3e6-66f74cd0dbc5","body_text":"R E S E A R C H Open Access\nEffect of phlebotomy versus oral\ncontraceptives containing cyproterone\nacetate on the clinical and biochemical\nparameters in women with polycystic ovary\nsyndrome: a randomized controlled trial\nSamira Behboudi-Gandevani 1, Hayedeh Abtahi 1, Navid Saadat 2, Maryam Tohidi 3 and Fahimeh Ramezani Tehrani 1*\nAbstract\nBackground: Reduction of the body iron stores can improve hyperandrogenemia and insulin resistance. This study\naimed to compare clinical and para-clinical responses to the treatment of phlebotomy using oral contraceptive pills\n(OCs) containing cyproterone acetate in women with PCOS.\nMethods: In this randomized clinical trial, 64 patients with PCOS were randomly assigned to the phlebotomy and\nOCs groups ( n = 32 in each group). The intervention group, using a single treatment procedure, underwent\nvenesection of 450 mL of whole blood at the early follicular phase of the spontaneous or progesterone-induced\nmenstrual cycle. The control group received OCs pills for 3 months from the 1th day of spontaneous or\nprogesterone-induced menstrual cycle onwards for 3 weeks, followed by a pill-free interval of 7 days. The women\nwere evaluated after the 3-month intervention. The primary outcome measure was a change in the HOMA-IR and\nfree androgen index (FAI). Secondary outcomes were changes in the Ferriman-Gallwey (FG) score and other clinical,\nbiochemical and hormonal changes from the baseline (pre-treatment) to week 12.\nResults: In the phlebotomy group, 27 (84.3%) and in the OCs group 30 (93.7%) of the women completed the\n3-month follow-up. The median HOMA-IR significantly decreased from 3.5 to 2.7 in the phlebotomy, and from\n3 . 1t o2 . 8i nt h eO C sg r o u p ,a n dt h ec h a n g e sw e r ec o m p a r a b l eb e t w e e nt h eg r o u p s .M e d i a nc h a n g e si nt h e\nFAI significantly decreased in both groups, but the diff erences were not statistically significant between the\ngroups ( P = 0.061). With regard to secondary outcomes, mean FG scores in both groups significantly\ndecreased [from 16.8 (6) to 13.3 (7.4), P < 0.028] in the phlebotomy group and [from 14.3 (7) to 9.8 (7.6) in\nthe OCs group, P = 0.001] after 3 months of treatment, but such c hanges had no statistically significant\ndifferences between the groups. During treatment, me nstrual cycles became regular in all women in the OCs\ngroup and in 12.27 (44.4%) of the women in the phlebo tomy group, and the difference was statistically\nsignificant ( P = 0.001). Despite no statistically significant diff erences in lipid profiles between the groups at the\nbaseline, triglycerides were significantly higher in the OCs group compared to the phlebotomy at end of\nfollow up ( p = 0.019).\n(Continued on next page)\n© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\n* Correspondence: fah.tehrani@gmail.com; ramezani@endocrine.ac.ir\n1Reproductive Endocrinology Research Center, Research Institute for\nEndocrine Sciences, Shahid Beheshti University of Medical Sciences, No 24,\nParvane Street, Yaman Street, Velenjak, P.O.Box: 19395-4763, Tehran, Iran\nFull list of author information is available at the end of the article\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 \nhttps://doi.org/10.1186/s13048-019-0554-9\n\n(Continued from previous page)\nConclusion: Both treatment modalities had similar beneficial effects on insulin resistance and on androgenic profiles.\nHowever, OCs was reported moreeffective in treating menstrual irregularities and phlebotomy had less adverse effects on\ntriglyceride concentrations.\nTrial registration:Code: IRCT2013080514277N1.\nKeywords:Cyproterone compound, Phlebotomy, Polycystic ovarian syndrome, Randomized control trial (RCT),\nBackground\nPolycystic ovarian syndrome (PCOS) is the most com-\nmon ovarian endocrinopathy affecting 6 –14% of repro-\nductive aged women [ 1]. It is characterized by ovulatory\ndysfunction, clinical or para-clinical hyperandrogenism,\nand polycystic ovary morphology [ 2]. Insulin resistance\n(IR) and obesity are closely associated with PCOS and\nfurther reinforce the severity of clinical manifestations\n[3–5]. Treatment of PCOS is symptom-based and aims\nto reduce the metabolic risk associated with HA, obesity,\nand insulin resistance [ 6–9].\nCombined oral contraceptive pills (OCs) have been the\nkey component of relieving P COS symptoms specifically\nmenstrual irregularity, hirsutism, and acne through improv-\ning androgen excess and regulating menstruation [ 10, 11].\nHowever, there are doubts concerning their impacts on the\ncardio-metabolism outcomes, which may be aggravated or\neven triggered by the use of OCs [ 12, 13]. Nevertheless, the\nbenefits of OCs outweigh related risks in women with\nPCOS. Since patients with PCOS mostly use these drugs for\nseveral years [11], safer therapeutic options are considered\nsuitable for the treatment of PCOS symptoms.\nPCOS is associated with iron overload and, which can\nbe associated with IR and further glucose intolerance [ 14–\n17]. Factors contributing to potential iron overload in\nwomen with PCOS are (i) the iron saved secondary to\nchronic infrequent menstrual bleeding and (ii) compensa-\ntory hyperinsulinemia following insulin resistance. It is\nbelieved that insulin favors the intestinal and tissue depos-\nition of iron and can decrease hepcidin, that consequently\nleads to elevated iron absorption [ 14, 17, 18]. In addition,\nreduction of body iron stores can improve hyperandro-\ngenemia and adverse cardio-metabolic effects [ 19–21]. In\nearlier studies, iron-chelating agents and blood donation\ncan prevent the development of diabetes due to iron\noverload [ 22, 23]. Other studies also have shown that\ndepleting iron stores in diabetes through phlebotomy and\nblood donation can decrease insulin resistance [24, 25].\nThe effects of iron reductive therapy in the treat-\nment of PCOS symptoms have not been previously\naddressed. Therefore, this ra ndomized controlled trial\nwas conducted to investigate the effect of phlebotomy\non clinical and biochemical parameters in women\nwith PCOS compared to oral contraceptives contain-\ning cyproterone acetate.\nMaterials and methods\nThis parallel non-inferiority randomized controlled trial was\nconducted on women with PCOS to compare the usual\nPCOS therapy using OCs containing cyproterone acetate\nand phlebotomy as the assumed potential modality.\nThis clinical trial was approved by the ethics commit-\ntee of the Research Institute of Endocrine Sciences,\nShahid Beheshti University of Medical Sciences (decree\ncode: 17ECRIES91/12/08). The research protocol was\nregistered on the Iranian Registry of Clinical Trials at\nwww.irct.ir (decree code: IRCT2013080514277N1). The\nwritten informed consent form was signed by the\nwomen prior to conducting the study.\nParticipants\nThey were known cases of PCOS based on the Rotter-\ndam criteria [ 26] and aged 18 –45 years, who referred by\nthe gynecologist (FRT) and had at least two of the\nfollowing criteria: i: oligo/anovulation (defined as either\nregular or irregular menstruation ≥34 days or history of\neight or fewer menstrual cycles in a year); ii: clinical\nsymptoms of hyperandrogenism (including hirsutism\ndiagnosed based on a standardized scoring system of\nmodified Ferriman-Gallwey ≥8, acne and androgenic\nalopecia) or biochemical hyperandrogenism (defined as\nthe increase of one or more serum level of androgens in-\ncluding testosterone or androstenedione above the 95th\npercentile, presented in the selected healthy non-hirsute\neumenorrheic women of the study population) [ 27]; iii:\npolycystic ovaries (having polycystic ovaries with 12 or\nmore follicles in each ovary, 2 –9 mm in diameter and/or\nincreased ovarian volume (10 cm 3)) and exclusion of\nother etiologies. The women with PCOS with the follow-\ning criteria were excluded: i) clinically significant organic\ndiseases including malignancy; ii) history of chronic\ndisorders such as diabetes, cardiovascular diseases,\nhypertension, thyroid disorders, hepatic dysfunction or\nrenal disorder; iii) history of hemochromatosis or pres-\nence of the Cys282Tyr mutation; iv) history of smoking\nand drug or alcohol misuse; and v) history of distur-\nbances in iron balance e.g. iron overload or deficiency;\nvi) presence of anemia (hemoglobin < 12 g/dL) or serum\nferritin < 30 ng/mL.\nAt baseline hemoglobin, ferritin, 75 g oral glucose to-\nlerance test (OGTT), and the lipid panel were evaluated.\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 Page 2 of 9\n\nRandomization\nThe process of study has been shown in Fig. 1. Initially,\nof 82 women with PCOS that were invited to participate\nin the study, 11 women did not accept it. Of 71 partici-\npants, 7 women did not meet the inclusion criteria.\nFinally 64 women were randomly allocated in a 1:1 ratio\nto either the iron-reduction phlebotomy group and the\nOCs containing cyproterone acetate group ( n =3 2 i n\neach group) using a computer-generated randomization\ntable after signing the written informed consent form.\nAllocation of treatment was not blinded. All participants\nwere asked not to use any medication for PCOS for 6\nweeks, but they were advised to maintain their usual diet\nand physical activity, and also abstain from any other\nnew treatments for PCOS. They referred for phlebotomy\nto the “Blood Transfusion Unit of Tajrish Hospital ”,\nwhich was performed by a trained technician under the\nsupervision of a general practitioner.\nThe study group, in a single treatment procedure,\nunderwent venesection of 450 mL of whole blood (equiva-\nlent to 200 –220 mL of red blood cells), using a collection\nbag (Fenwal Europe®, SPRL, Belgium) during the early\nfollicular phase of spontaneous or progesterone-induced\nmenstrual cycle. To prevent volume imbalances, the\nbloodletting was conducted slowly over 10 min and\nphysiological saline was administered in compensation.\nThe administered volume was equivalent to that of the\nblood removed. The control group received OCs (35 μg\nFig. 1 The Consort flow diagram\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 Page 3 of 9\n\nethinyl estradiol and 2 mg cyproterone acetate; Aburaihan\npharmaceutical company®) for 3 months from day the 1th\nday of a spontaneous or progesterone-induced menstrual\ncycle onwards for 3 weeks followed by a pill-free interval\nof 7 days.\nIt should be noted that the adverse effects of the treat-\nments were monitored via telephone calls in a weekly\nmanner and at the final stage through interviewing.\nOutcome of interest\nThe primary outcome measure was a change in the\nHOMA-IR and free androgen index (FAI). Secondary\noutcomes were changes in the freeman-Galway score\nand other clinical, and biochemical and hormonal\nchanges from the baseline (pre-treatment) to week 12.\nClinical and biochemical measurements\nClinical and laboratory assessment was performed before\nand after the treatment by a trained single staff. In the\nstanding position and with participants wearing minimal\nclothes, weight and height were measured to the nearest\n100 g, following standardized procedures and using cali-\nbrated equipment. The waist circumference (WC) was\nmeasured midway between the lower rib margin and the\niliac-crest at the level of umbilicus and at the end of a\ngentle expiration. The hip circumference (HC) was\nmeasured using an unstretched measuring tape to the\nnearest 0.1 cm, following which the waist to hip ratio\n(WHR) was calculated. The body mass index (BMI) was\ncalculated through dividing weight (kg) by the square of\nheight (m).\nSystolic and diastolic blood pressure were assessed in\nthe sitting position using a standardized mercury sphyg-\nmomanometer which was calibrated by the Iranian Insti-\ntute of Standards and Industrial Researches.\nFasting blood samples were collected after 10 –12 h of\nfasting. Insulin was measured by Electrochemilumines-\ncence (ECLIA) (Roche Diagnostics GmbH, Mannheim®,\nGermany). HOMA-IR was calculated by the following\nformula: [glucose (nmol/L) × insulin ( μU/mL)/22.5]. Total\ncholesterol (TC), high-density lipoprotein cholesterol\n(HDL-C), and triglycerides were assayed using the enzym-\natic colorimetric method. Analyses were performed using\nrelated kits (Pars Azmon® Inc., Tehran, Iran) and a Selecta\n2 autoanalyzer (Vital Scientific®, Dieren, Netherlands). To\ncalculate LDL-C, a modified Friedewald equation was used\n[28]. The intra- and inter assay coefficients of variation\n(CV) were: 2.2 and 2.9 for FBS, 0.7 and 2.8 for TG, 2.1 and\n2.2 for TC, 2.1 and 2.8 for HDL-C, 1.7 and 1.7 for LDL-C,\nrespectively. The anti-mullerian hormone (AMH) by the\ntwo-site enzyme immunoassay method using Gen II kit\n(Beckman Coulter®, Inc., Fullerton, California) and the\nSunrise ELISA reader (Tecan Co®, Salzburg, Austria).\nTotal prostate specific antigen (PSA) levels were assessed\nby the Electrochemiluminescence (ECLIA) (Roche Diag-\nnostics GmbH®, Mannheim, Germany). Total testosterone\nwere measured by the Electrochemiluminescence (ECLIA)\n(Roche Diagnostics GmbH®, Mannheim, Germany).\nAndrostenedione and SHBG were measured using the en-\nzyme-linked immunosorbent assay (ELISA) (Pars Azmun\nCo®. Tehran, Iran). The intra and inter assay coefficients\nof variation (CV) were: 4 and 4.1 for tT, 3.4 and 3.6 for\nandrostenedione, 3.4 and 5 for SHBG, respectively. The\nFAI was calculated by the following formula: TT/SHBG ×\n100 [29].\nSample size estimation and data analysis\nNo data from controlled trials were available for estimat-\ning of the effect of phlebotomy in women with PCOS;\nhowever in a previous trial conducted on the effect of\niron reduction on insulin sensitivity, as measured by an\nintravenous insulin tolerance test in patients with type 2\ndiabetes, a standardized effect of d = 0.78 was verified\n[30]. Since the intravenous insulin tolerance test is more\nsensitive than the HOMA-IR, a hypothetical estimated\neffect size (mean differences) of 0.2, standard deviation\nof 0.47 and non-inferiority margin of 0.5 for HOMA-IR\nas the primary outcome was considered in this study.\nUnder the non-inferiority assumption for this trial, 64\npatients ( n = 32 per group) could provide at least 80%\npower at a one-sided significance level of 0.05.\nAn intention to treat analysis was performed and base-\nline data of the women who had not completed the\nstudy process were used as final data. 20 Related statistics\nwere based on the total number of recruited patients.\nDescriptive values were presented as mean (SD) or\nnumber (percentage). Distribution of data was assessed\nusing the Kolmogorov –Smirnov test for normality. The\nt-test and nonparametric were used based on the normal\ndistribution of data. Demographic data and baseline\ncharacteristics between the groups were compared using\nthe χ 2 test, t-test, or Mann-Whitney U test. The paired\nt-test or the Wilcoxon Signed Ranks test were utilized to\ncompare measurements between the baseline and fol-\nlow-up examinations. Differences between the interven-\ntion and control groups in the changes from the baseline\nto the follow-up examinations were assessed using the t-\ntest or Mann-Whitney U test. All statistical analyses\nwere performed using the SPSS version 11.5 software for\nWindows (SPSS Inc., Chicago, IL, USA). A significance\nlevel of < 0.05 was used during the data analysis.\nResults\nIn the phlebotomy group, 27 (84.3%) and in the OCs\ngroup 30 (93.7%) of the women completed the 3-month\nfollow-up. Five women in the phlebotomy group and 2\nwomen in the OCs group withdrew from the study (Fig.\n1). However, the basic characteristics of those women\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 Page 4 of 9\n\nwho lost to follow were not significantly different from\nremaining ones.\nThe demographic characteristics of the women were\nshown in Table 1. The mean age and BMI in the OCs\nvs. the phlebotomy group were 24 (4.06) vs. 24.2 (5.2),\nyears and 27.1 (5.04) vs. 25.4 (5.3), kg/m 2, respectively at\nthe initiation of the study.\nAt baseline, all parameters were comparable between\nthe groups and no statistically significant differences be-\ntween the groups with regard to age, BMI, androgenic\nprofile, and other carbohydrate or metabolic profile were\nreported.\nPrior to the treatment, 77.7% (21 of 27) of the phlebot-\nomy group and 80% (24 of 30) of the OCs group had\noligo-menorrhea or amenorrhea. While all women in the\nOCs group had a normal menstruation after treatment,\nbut 12/27 (44.4%) in the phlebotomy group still suffered\nfrom oligo-menorrhea or amenorrhea, which was statis-\ntically significant ( P = 0.002).\nEffects of both treatments on HOMA-IR were pre-\nsented in Table 2. A statistically significant decline of\nHOMA-IR from the baseline compared to the end of the\n3th month of the treatment in both groups [3.5 (1.3 –4.5)\nvs. 2.7 (1.2 –4.3), P = 0.047 and 3.1 (1.4 –3.5) vs. 2.8 (1.8 –\n3.9), P = 0.032, in the phlebotomy and the OCs groups,\nrespectively] was reported, but it was not statistically\nsignificant ( P = 0.516).\nBoth treatments significantly decreased insulin levels\nthroughout the study (9.5 (6.8 –20) vs. 8.1 (6.5 –15.7) P =\n0.025 and 10.8 (7.6 –14.9) vs. 9.8 (7.8 –16.1), P = 0.015 in\nthe phlebotomy and the OCs group, respectively). More-\nover, mean differences were not statistically significant\nbetween the groups.\nAll androgenic parameters including testosterone,\nandrostenedione and FAI decreased in both groups.\nHowever, no statistically significant differences between\nthe groups were reported (Table 2). Compared to the\nbaseline, SHBG showed a rising trend during the treat-\nment in both groups, but the mean score of changes in\nthe OCs was significantly higher than of the phlebotomy\ngroups (P =0 . 0 0 9 ) .\nDespite no statistically significant difference in lipid pro-\nfiles of both groups at the initiation of study (Table 1),\nthere was a statistically significant rise in triglycerides\nfrom the baseline in the OCs (114.2 (48) vs. 152 (89.9),\nP = 0.004) at the end of the 3th month of the treatment.\nAccordingly, the mean score changes of TG between the\ngroups was statistically significant (8.3 (83.9) vs. 37.7\n(65.1), P = 0.019). Changes in LDL-C, HDL-C and TC had\nno statistically significant differences within and between\nthe groups.\nAt the baseline, the mean FG scores in the phlebotomy\nand the OCs groups were 16.8 (6) and 14.3 (7), respect-\nively as decreased to 13.3 (7.4), P = 0.001 and 9.8 (7.6),\nP = 0.028, respectively, However, the changes were not\nstatistically significant between the groups (Table 2).\nAMH and PSA remained unchanged in both groups\nthroughout the study.\nSafety\nPhlebotomy were well-tolerated by all women and no\nserious side effects were reported. Dizziness and head-\naches for a few hours occurred in five women after\nphlebotomy, but they consented to undergo repeated\nphlebotomies, if needed.\nDiscussion\nThis is the first study on the endocrine, clinical and\nmetabolic effects of treatment using phlebotomy in\nTable 1 The baseline characteristics of the samples\nPhlebotomy\nN =2 7\nOCs\nN =3 0\nP-value*\nAge, (y) 24 (4.06) 24.2 (5.2) 0.878\nBMI, (kg/m2) 27.1 (5.04) 25.4 (5.3) 0.323\nWHR, 0.8 (0.06) 0.7 (0.08) 0.223\nHB, (g/dl) 14.1 (1.1) 14.3 (1.3) 0.166\nHCT 40.4 (2.1) 39.6 (2.3) 0.330\nFerritin, (ng/mL) 74.2 (54.06) 48.5 (30.9) 0.070\nSBP, (mm Hg) 102.3 (9.0) 100.2 (18.9) 0.526\nDBP, (mm Hg) 67.6 (6.4) 66.2 (7.8) 0.503\nHirsutism score, 16.8 (6) 14.3 (7) 0.063\nAcne, yes (%) 13 (74) 17 (56.6) 0.189\nFAI, median 5.2 (1.6–12.8) 4.4 (1.4 –4.7) 0.051\nTotal Testosterone, (ng/mL) 0.52 (0.3) 0.40 (0.1) 0.095\nAndrostenedione, (ng/mL) 4.7 (1.9) 4.1 (1.8) 0.350\nSHBG, (nmol/L) 35.5 (20.9) 46.6 (46.8) 0.059\nHOMA-IR, median 3.5 (1.3–4.5) 3.1 (1.4 –3.5) 0.595\nInsulin, (μU/mL) 9.5 (6.8–20) 10.8 (7.6 –14.9) 0.626\nGTT, fasting, (mg/dL) 84.3 (6.6) 83.8 (6.7) 0.813\nGTT, 2 h, (mg/dL) 101.9 (12.7) 90.2 (23.9) 0.940\nCholesterol, (mg/dL) 178.5 (37.9) 178.7 (33.3) 0.985\nTriglycerides, (mg/dL) 115.5 (107.9) 114.2 (48) 0.957\nHDL-C, (mg/dL) 45.5 (15.1) 47.5 (10.3) 0.611\nLDL-C, (mg/dL) 95.5 (26) 97.1 (22.7) 0.831\nAMH, (ng/mL) 8.1 (6) 8.7 (6) 0.471\nPSA, (ng/mL) 0.005\n(0.002–0.009)\n0.004\n(0.002–0.020)\n0.688\n*The statistical significance level was set at 0.05; Values are given as mean\n(standard deviation) or median (25 –75%), as appropriate.\nBMI Body Mass Index; WHR Waist to Hip Ratio; HB Hemoglobin; HCT\nHematocrit; SBP Systolic blood pressure; DBP Diastolic blood pressure; FAI Free\nAndrogen Index; SHBG Sex Hormone Androgen Index; HOMA-IR Homeostatic\nModel Assessment for Insulin Resistance; GTT Glucose tolerance test; HDL High\nDensity Lipoprotein; LDL Low Density Lipoprotein; AMH Anti Mullerian\nHormone; PSA Prostate Specific Antigen.; SHBG Sex hormone-binding globulin;\nOCs oral contraceptives containing cyproterone acetate\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 Page 5 of 9\n\nwomen with PCOS and comparison of the beneficial\neffects of two different treatments using the OCs con-\ntaining cyproterone acetate and phlebotomy. As a result,\nboth treatments had similar beneficial effects on insulin\nresistance as well as on androgenic profiles. However,\nOCs containing cyproterone acetate was found to be\nmore effective in the treatment of menstrual irregularity\nand phlebotomy and had less adverse effects.\nEthinyl estradiol and cyproterone acetate in combi-\nnation (cyproterone compound) have been widely used as\nreliable contraceptive pills in women with PCOS suffering\nfrom hyperanderogenism [ 31]. Such a combination can\nimprove various PCOS manifestations through several\npotential mechanisms including the inhibition of LH se-\ncretion from the pituitary, reduction of androgen produc-\ntion from ovaries, reduction of free androgens, increase of\ncirculating SHBG levels and thereby limiting peripheral\nandrogen exposure. Cyproterone acetate can block periph-\neral androgen receptors at target organs and can reduce\nthe ovarian androgen production and decrease the plasma\nlevels of free testosterone [32].\nHowever, OCs are known to have adverse effects on\nlipid metabolism and carbohydrate intolerance. Estrogen\nhas pro-thrombotic effects and increases the cardio-\nvascular venous thromboembolism [ 33].\nThe increased potential adverse cardio-metabolic\neffects of these drugs encourage the use of non-pharma-\ncological treatments for relieving PCOS symptoms.\nSurprisingly, a lower prevalence of diabetes has been\nobserved among blood donors [ 34]. Recently, it has been\nshown that hyperandrogenemia and insulin resistance\nare associated with ferritin [ 35, 36].\nEmerging evidence hence suggests that in the\ngeneral population, body iron stores and iron intake\nare positively associated with glucose intolerance and\ndiabetes [ 21, 37, 38]. Although, potential underlying\nmechanisms are not fully understood, iron influences\ninsulin production and sensitivity, and together they\ninfluence on iron metabolism. Therefore, iron inter-\nferes with the insulin prohibition of hepatic glucose\nsecretion, and hepatic iron stores reduce the produc-\ntion of insulin leading to systemic hyperinsulinemia\n[14]. In this respect, previous studies found the bene-\nficial effects of phlebotomy and blood donation in\npatients with type 2 diabetes [ 24, 25, 39], demonstra-\nting that iron reduction by phlebotomy improved\ninsulin sensitivity.\nWomen with PCOS may suffer from iron overload due\nto chronic infrequent menstrual bleeding, that in turn\nreduces menstrual blood loss along with reduced serum\nTable 2 Effects of the treatment in the groups\nPhlebotomy\nN =2 7\nP-value* OCs\nN =3 0\nP-value* P-value**\n3 months post-treatment Mean difference 3 months post-treatment Mean difference\nBMI 27 (4.7) −0.01 0.229 25.1 (5.3) −0.03 0.784 0.355\nWHR 0.8 (0.04) −0.01 0.346 0.7 (0.05) −0.01 0.621 0.412\nHirsutism score 13.3 (7.4) −3.7 (6.1) 0.028 9.8 (7.6) −2.1 (2.8) 0.001 0.235\nSBP, (mm Hg) 101.1 (6.5) −1.1 (10.3) 0.647 102 (8.4) 1.5 (20.1) 0.690 0.610\nDBP, (mm Hg) 65.8 (7.7) −1.7 (10.8) 0.514 65.8 (5.3) −0.5 (10) 0.780 0.701\nHOMA_IR 2.7 (1.2–4.3) −0.73 (2) 0.047 2.8 (1.8 –3.9) −0.45 (5.6) 0.032 0.516\nInsulin, (μU/mL) 8.1 (6.5–15.7) −0.5 (10.1) 0.025 9.8 (7.8 –16.1) −4.6 (23.8) 0.015 0.512\nFAI 4.1 (1.8–8) −1.1 (3.4) 0.042 3.1 (0.3 –6.8) −1.2 (2.9) 0.001 0.061\nTotal Testosterone, (ng/mL) 0.42 (0.34) −0.07 (0.1) 0.047 0.33 (0.1) −0.06 (0.1) 0.045 0.378\nAndrostenedione, (ng/mL) 3.3 (1.7) −1.3 (1.3) 0.026 2.9 (1.6) −1.1 (1.8) 0.001 0.070\nSHBG, (nmol/L) 91.1 (72.4) 55.6 (63) 0.013 163.7 (101.5) 107.06 (115.4) 0.001 0.009\nCholesterol, (mg/dL) 179.8 (67.1) 1.2 (69.4) 0.940 183.2 (41) 3.4 (32.5) 0.076 0.102\nTriglycerides, (mg/dL) 123.8 (93.4) 8.3 (83.9) 0.687 152 (89.9) 37.7 (65.1) 0.004 0.019\nHDL, (mg/dL) 43 (15.6) −2.5 (13.9) 0.467 51.9 (15) 4.4 (11.2) 0.053 0.072\nLDL, (mg/dL) 93.4 (27) −2.1 (22.9) 0.709 105.9 (33.4) 8.8 (32.2) 0.150 0.187\nAMH, (ng/mL) 8 (5.4) −0.1 (3.6) 0.909 6.7 (3.8) −1.9 (5) 0.043 0.191\nPSA, (ng/mL) 0.002 (0.002–0.008) 0.003 (0.01) 0.427 0.002 (0.002 –0.01) −0.005 (0.01) 0.204 0.058\nComparison of the parameters with baseline **Comparison of mean differences\nValues are given as mean (standard deviation) or median (25%-75%), as appropriate\nSBP Systolic blood pressure; DBP Diastolic blood pressure; FAI Free Androgen Index; SHBG Sex Hormone Androgen Index; HOMA-IR Homeostatic Model Assessment\nfor Insulin Resistance; HDL High Density Lipoprotein; LDL Low Density Lipoprotein; AMH Anti Mullerian Hormone; PSA Prostate Specific Antigen; OCs oral\ncontraceptives containing cyproterone acetate\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 Page 6 of 9\n\nhepcidin levels as an iron-regulatory hormone [ 40]. It is\nalso affected by insulin resistance and hyperandrogenism\nthat may improve iron absorption and reduce iron secre-\ntion from macrophages, and possibly leads to mild iron\noverload in some patients with PCOS [ 17, 35, 41]. Such\nan overload can contribute to glucose intolerance in\nwomen with PCOS [ 35] independent of chronic inflam-\nmation [ 42].\nConsistent with this hypothesis, a few studies are avail-\nable on the effects of therapeutic interventions on iron\nexess in women with PCOS. In this respect, Luque-\nRamírez et al. (2011) in a randomized clinical trial com-\npared the effect of metformin as an insulin sensitizer with\nan antiandrogenic contraceptive pill for 6 months and\nfound that patients with PCOS decreased hepcidin levels\nthat might contribute to iron overload through facilating\nthe intestinal absorption of iron [ 40].\nHowever, our study as a pioneer investigation con-\nfirmed that phlebotomy enhanced insulin sensitivity and\nimproved the state of hyperanderogenism in women\nwith PCOS. However, more studies with longer follow-\nups are still needed to confirm these thraputic effects.\nIn the present study, the side effects of phlebotomy\nwere rare. The appropriate selection of vein and careful\nprocedure of venipuncture helped us to minimize prob-\nable vasovagal reactions.\nAs the limitation of this study the short study duration\nmight not have shown the long terms effect of this treat-\nment, because the improvement of some PCOS manifesta-\ntions might need long term follow-ups. In addition, some\nPCOS confounders including the women lifestyle were\nnot assessed. No enogh power was present to performe\nthe subgroup analysis based on various PCOS phenotypes\nor obesity status. Also, the HOMA-IR has been used as a\nsurrogate marker for the assessment of IR, despite the\ngood correlation between HOMA-IR and gold standard\nclamp methods [43], which might be inaccurate in women\nwith PCOS [ 44]. Also, this study did not measure free\ntestosterone due to a lack of access to the proper method.\nIt has been shown that FAI has a good correlation with\nfree testosterone [ 45]. Since ferritin was not measured at\nthe end of the study follow-up, assessment of changes in\niron overload was impossible. Moreover, the number of\nlost to follow up subjects in the phlebotomy group was\nhigher than those in the OCs group, but our study results\nmay not be affected as there would be no statistically\nsignificant differences in basic characteristics between\nthose who lost to follow up and the remained participants\nin each group. In addition, we cross validated our data\nand found that the internal validity of the results was not\naffected by lost to follow up. Finally, the sample size was\ncalculated based on the HOMA-IR, and there was not\nadequate number of subjects for the precise comparison\nof other clinical or para-clinical manifestations of PCOS.\nTherefore, observiation of no significant differences on\nthese items could be due to the small sample size, which\nshould be interpreted with cautious.\nConclusion\nThis study suggest that the phlebotomy therapy is asso-\nciated with a tendency to decrease insulin resistance and\nhyperanderogenism, that merits further investigations. In\naddition, large prospective, randomized controlled trials\nwith longer treatment periods are recommended to\nconfirm the study findings.\nAbbreviations\nAMH: Anti-mullerian hormone; BMI: body mass index;\nDHEAS: Dehydroepiandrosterone sulfate; FAI: Free androgen index;\nFG: Ferriman-Gallwey; HC: Hip circumference; HDL-C: high density\nlipoprotein-cholesterol; HOMA-IR: Homeostatic Model Assessment for Insulin\nResistance; LDL-C: low density lipoprotein-cholesterol; OCPs: Combined oral\ncontraceptive pills; OCs: oral contraceptive pills; PCOS: Polycystic ovarian\nsyndrome; PSA: prostate specific antigen; SD: standard deviation; SHBG: Sex\nhormone-binding globulin; TC: Total cholesterol; TT: total testosterone;\nWC: waist circumference; WHR: waist to hip ratio\nAcknowledgements\nThe authors would like to thank all the women with PCIS for their sincere\ncooperation with the research project.\nAuthors’ contributions\nSBG was involved in the project development, data analysis, manuscript\nwriting, and critical discussion. HA was involved in conceptualizing the study,\ndata collection, and critical discussions. NS was involved in conceptualizing\nthe study and critical discussions. MT was involved in the project\ndevelopment, laboratory analysis, and critical discussions. FRT was involved\nin conceptualizing the study, the project development, data analysis, critical\ndiscussions, and manuscript writing. All authors read and approved the final\nmanuscript to be published.\nFunding\nNone.\nAvailability of data and materials\nThe datasets used and analyzed during the current study are available via\nsending email to the corresponding author in case of request for future\nresearch.\nEthics approval and consent to participate\nThis study was approved by the ethics committee affiliated with the\nResearch Institute for Endocrine Sciences. It also was registered and\napproved on the Iranian Registry of Clinical Trials under the code of\nIRCT2013080514277N1. A written informed consent was obtained from the\nwomen with PCOS before the initiation of the study.\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthor details\n1Reproductive Endocrinology Research Center, Research Institute for\nEndocrine Sciences, Shahid Beheshti University of Medical Sciences, No 24,\nParvane Street, Yaman Street, Velenjak, P.O.Box: 19395-4763, Tehran, Iran.\n2Endocrine Research Center, Research Institute for Endocrine Sciences,\nShahid Beheshti University of Medical Sciences, Tehran, Iran. 3Prevention of\nMetabolic Disorders Research Center, Research Institute for Endocrine\nSciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 Page 7 of 9\n\nReceived: 23 December 2018 Accepted: 15 August 2019\nReferences\n1. Tehrani FR, Simbar M, Tohidi M, Hosseinpanah F, Azizi F. 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Position statement: utility,\nlimitations, and pitfalls in measuring testosterone: an Endocrine Society\nposition statement. J Clin Endocrinol Metab. 2007;92:405 –13.\nPublisher’sN o t e\nSpringer Nature remains neutral with regard to jurisdictional claims in\npublished maps and institutional affiliations.\nBehboudi-Gandevani et al. Journal of Ovarian Research           (2019) 12:78 Page 9 of 9","source_license":"CC0","license_restricted":false}