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International Journal of Reproductive BioMedicine
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences of Yazd
ISSN: 1680-6433 EISSN: 2008-2177
Vol. 7, Num. 2, 2009, pp. 53-58

Iranian Journal of Reproductive Medicine Vol. 7, No. 2, Spring, 2009, pp. 53-58

Relationships between free leptin and insulin resistance in women with polycystic ovary syndrome

Javad Mohiti-Ardekani1 Ph.D., Nasim Tarof1 M.Sc., Abbas Aflatonian2 M.D.  

1 Department of Biochemistry and Molecular Biology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 
Corresponding Author: Javad Mohiti-Ardekani, Department of Biochemistry and Molecular Biology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Received: 1 December 2008; accepted: 11 April 2009

Code Number: rm09010


Background: Leptin is an adipokine that circulates in a free form and bound to a soluble leptin receptor. Patients with polycystic ovary syndrome have increased insulin resistance and high incidence of obesity.
Objective: This study was carried out to evaluate levels of leptin and free leptin in women with polycystic ovarian syndrome (PCOS) and note any relationships with insulinresistance and adiposity.
Materials and Methods: We assessed the correlation of metabolic parameters with the levels of free leptin and it’s bound form in 27 PCOS women (aged 26±5.6 years) and 27 healthy women with normal menstrual cycle as controls (aged 25 ±4 years).Total leptin and insulin levels were measured using ELISA. Free leptin form was purified by Gel filtration chromatography and their collected fractions were measured by a sensitive ELISA-Kit. Insulin resistance was calculated by homeostasis model assessment (HOMA).
Results: In PCOS patients and control group a correlation between leptin and body mass index (BMI) was found. A significant difference was found between leptin and free leptin levels in PCOS subjects and controls (p<0.05). Significant correlations were found between free and total leptin with insulin resistant in PCOS subjects (r=0.78 p=0.00,r=0.84 p=0.003) and control groups respectively (r=0.86 p=0.00, r=0.69 p=0.00).
Conclusion:  Total and free leptin forms are correlated significantly with BMI in patients with PCOS and in controls. Total and free leptin forms showed significant correlations with insulin resistance but no significant difference was seen in the two groups investigated.    

Key words: Free leptin, Polycystic Ovarian Syndrome (PCOS), Insulin resistance.


Leptin,the gene product of the ob gene, is thought to provide the centralnervous system with feed-back information about fat storageof the body (1). Thus, leptin is thought to be a part of the regulationof appetite, food intake and the lipid metabolism (2).

Obese humanspresent with hyperleptinemia as an indicator of leptin resistancewhichplays a major role in thepathogenesis of obesity (3).Polycystic ovary syndrome (PCOS) is among the most common endocrinedisorders, affecting more than 5% of women of reproductive age(4, 5). An association of PCOS with peripheral insulin resistance,compensatory hyperinsulinemia and alterations in ß-cellfunction as the cause of its predisposition to develop a metabolicsyndrome (type 2 diabetes mellitus, hypertension, lipid disorders,obesity) has been established (6). According to the majority of studies, most PCOS women are insulinresistant and overweight or obese (7-11).In mice, a genetic defect in the ob gene results in severe obesityand type 2 diabetes mellitus, as well as in infertility (6).

In this mouse model, leptin injections restore fertility. Leptintreatment in normal female mice accelerates puberty (12). Inhumans, granulosa cells have been shown to secrete leptin (13)and leptin seems to influence adrenal androgen formation (14).

However, the role of leptin in ovarian function or on the reproductivesystem remains unclear. It was shown that   leptin levels did notinfluence follicular maturation and ovulation in PCOS patients(10) while Carmina et al demonstrateda negative correlation between leptin and dehydroepiandrosteronesulphate (DHEAS) levels in 21 lean PCOS women, suggesting thatandrogens may play a role in suppressing serum leptin (15).

Another study showed that leptin concentrations correlated inverselywith luteinizing hormone (LH) levels, independent of body weightand insulin resistance (16). In contrast again, Laughlin etal (17) proposed that leptin is neither involved in the hypersecretionof LH nor in the regulation of LH pulsatility. During lactationalamenorrhea Sir-Petermann et al (18) also showed that leptinsecretion is not related to LH secretion. Plasma leptin is in free and bound forms in circulation (19).

The free leptin hasbeen shown to correlate with total serum leptin levelsand BMI in women of reproductive age. Kado et al (20)hypothesized that obese women are able to conversely increasetheir leptin activity by minimizing the bound leptin fraction,thus influencing the biological activity of leptin. In linewith this, Sinha et al (21) reported that in lean subjects,leptin circulates predominantly in the bound form, whereas inobese patients, leptin circulates mostlyin the free form. Similar results were found for Japanese menand women, showing a direct correlation between free leptin leveland BMI and parameters of insulin resistance (22).

Weight loss, following gastric restrictive surgery,leads to a reduction in circulating leptin levels and to adecrease free leptin (23). In adolescent girls with anorexia nervosa,free leptin and Free Leptin Index (FLI) but not total leptin levels were closely relatedto parameters of sexual maturation and the onset of puberty(26). Therefore, to elucidate the free leptin role in PCOS, we purified the free leptin in PCOS patients and healthy control and analyzed the interrelations between serum total leptin and free forms with insulin and insulin resistance and the other metabolic factors.

Materials and methods

Study population

The study was carried out on 54 women (27 PCOS subjects and 27 voluntary BMI, and age matched healthy women with normal menstrual cycle as controls).  Clinical diagnosis of PCOS was made from the 1990 National Institutes of Health (NIH) conference,when either oligomenorrhea(cycles lasting longer than 35 days) or amenorrhea (less thantwo menstrual cycles in the past 6 months) and either clinicalsigns of hyperandrogenism (hirsutism or obvious acne or alopeciaand/or an elevated total testosterone) were found, and other pituitary,adrenal or ovarian diseases could be excluded. PCOS as well as controlsubjects had not taken any medication known to affect carbohydratemetabolism or endocrine parameters for at least 3 months beforeentering the study.  In controls, LH, FSH and insulin levels were estimated in the early follicular phase (second or third day of menstruation) from fasting blood samples.

Data collection

In PCOS subjects and control women, clinical parameters wereassessed by physical examination, and BMI wascalculated as (weight/height2)(kg/m2). 

Insulin resistancewas calculated by homeostasis assessment model (HOMA) using the formula HOMA-IR = Fasting insulin (μU/ml) × Fasting glucose (mg/dl)/22.5 (27). Low-IR values indicate high insulin sensitivity, whereas high-HOMA values indicate low insulin sensitivity (insulin resistance). 

Biochemical assays

Leptin were measured using ELISA kits [DiagnosticSystems Laboratories (DSL), Webster, TX, USA]. The DSL ACTIVEHuman Leptin ELISA is an enzymatically amplified ‘two-step’sandwich-type immunoassay.The theoretical sensitivity or minimum detection limit as calculatedby interpolation of the mean plus two standard deviations of12 replicants of the zero standard was 0.05 ng/ml. Intra-assay variation was <5% andinterassay variation was <8% for all measured parameters.Insulin concentrations were measured by sandwich ELISA (Webster, Texas 77598-4217 USA, DSL).

Free leptin form was purified by Gel filtration methods as bellow (20).  This study was approved by the local ethical committee and each patient gave written informed consent.

Purification of free leptin form in serum

To obtain a standard curve, one vial containing 1 mg lyophilized leptin (Sigma) was dissolved in 0.5 ml HCL (1.5mM) and neutralized with 0.25 ml NaOH (7.5 mM) and applied with Marker Gel filtration Bludextran (Product Brand: Sigma  Product Number: D4772)  to Sephadex G-100 column chromatography (Amersham Biosource (1908-7101) (9/30 column).

The leptin was eluted with 0.25 mM phosphate buffer (Ph=7.4).  Fractions eluting were collected and assayed by a Sensitive ELISA Kit (Catalogue Number: kap 2281: 96 determinations. Manufactured by: Biosource Europe S-A). It showed a single bound indicating free leptin fraction.

Serum sample (0.5ml) was fractionated by Sephadex G-100 gel filtration chromatogheraphy. Fractions eluting between void and bed volumes were assayed by the ELISA kit. 

Statistical analysis

Data are presented as median plus range for non-parametric data.For better comparison,   means ±S.D. is also shown. Correlations between variables were examined by Spearman’s correlationcoefficient (rs) because analyzed data were not normally distributed.Differences between the groups were evaluated with the Mann–WhitneyU test. p values <0.05 were considered significant.


Patients with PCOS showed significantly higher concentrations of leptin (mean 21±9 ng/ml) vs. control (16±10 ng/ml, p=0.042), free leptin (mean 7.3±1.2) vs. control (mean 4.3±1.3 ng/ml, p=0.011) and LH (mean 76±18 IU/ml) vs. control (mean 2.87±1.93, p=0.001) in plasma (Table I).

We observed no marked difference in insulin  level between patients with PCOS (mean  26±13µIU/ml)  vs. control (mean 25±13) and FSH  level between patients with PCOS (8.69±15 IU/ml) vs. control (mean 6.56±1.46) (Table I). Furthermore, we analyzed the relationships between leptin, free leptin with BMI, insulin, insulin resistance and LH in PCOS and control groups.

Significant correlations were observed between free leptin and BMI (rs=0.78, p=0.000),insulin resistance (IR) (r=0.53, p=0.004) and insulin (r=0.93, p=0.000) in PCOS patients.  Significant correlations were also observed between free leptin and BMI (r=0.86, p=0.00), IR (r=0.57, p=0.003) and insulin (r=0.91, p=0.000) in control group (Table II).No significant correlation was observed between total and free leptin with LH. 

Table I. Characteristic of control (n=27) and PCOS patients (n=27). Values are means ±SD (median and range).

















free leptin(ng/ml)




















Table II. Correlation of leptin and free leptin levels with metabolic parameters in PCOS and control women.


Control group

PCOS patients


Leptin (ng/ml)

Free leptin


Free leptin























































Leptin levels are increased in obesity and may play a role inthe development of insulin resistance(7). Our study showed a significant high total and free leptin in PCOS patients as it is compared with controls (Table I) (p<0.05). Total leptin levels correlated significantly withBMI in both PCOS women and controls.  Leptin correlated with other metabolic parameters includinginsulin resistance and insulin level in both groups. Similar results were found in PCOS patientsfrom Australia (29), Brazil (16), Canada (10), Finland (30),Italy (15), Sweden (8), Turkey (11) and USA (17, 31). Significant correlation of total leptin with HOMA IR reflected a degree of insulin resistance in both controls and test groups (Table II). We can place our results along the large group of studies, which declare an important role of leptin in pathogenesis of insulin resistance (1, 2, 4, 6, 8, 11, 15, 20, 28, 29).

In addition, we found free leptin levels in PCOS patient and control group correlate with parameters of insulin, and insulin resistance (Table II).

Thus free leptin appears to be the appropriate form of leptin which it contributes to insulin resistance and leptin resistance.  Potential mechanisms that may mediateleptin resistance include impairment of brain leptin transportor leptin receptor internalization, and receptor post-receptorsignaling defects. Furthermore, the active hormone may be reducedby binding proteins or soluble receptors (32). Soluble leptin receptor represents the main leptin-binding compound in plasma, thusregulating its free fraction in the circulation (33).

A considerable portion of circulating leptin is free form which is affected by the degree of adiposity and nutritional state (35-37). We found a positive correlation between free leptin with BMI in PCOS patients and controls.Yannakoulia and co-workers (24) demonstrated that Free leptin index iscorrelated with total energy intake and inversely correlatedwith energy intake from dietary fat. They speculated that themacronutrient composition of the diet influences serum concentrationsof free leptin. The authors hypothesizedthat the increase of free leptin levels representsa compensatory mechanism to overcome leptin resistance.

A recently published twin study of pubertal females studyingthe genetic and environmental influences on the variations ofleptin and free form levels showed that leptin is mostly influencedby body composition (25, 34). We have demonstrated the correlation of free leptin with insulin resistance in diabetes patients (38). In conclusion, a considerable part of plasma leptin is in free form in circulation.

Although the physiological function of bound and free leptin are not well understood, it has been hypothesized that leptin is more active in its free form because this form is present in cerebrospinal fluid (CSF) (34).    We found a relationship between total leptin levels, andfree leptin with BMI, insulin resistance, and insulin levels in overweight PCOS patients and their matched control group.

However, further studiesare needed to investigate the effect of factors including food or body composition,and genetic on high level of free leptin inPCOS patients.


We thank Members of the Dept. of Biochemistry and Molecular Biology for helpful discussins. This research was supported by Shahid Sadoughi University of Medical Science and Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.


  1. Hang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425–432.
  2. Janeckova R. The role of leptin in human physiology and pathophysiology. Physiological Research 2001; 50: 443–459.
  3. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, et al. Serum immunoreactive leptin concentrations in normal-weight and obese humans. New England Journal of Medicine 1996; 334: 292-295.
  4. Asuncion M, Calvo RM, San Millan JL, Sancho J, Avila S, Escobar-Morreale HF. A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. Journal of Clinical Endocrinology and Metabolism 2000; 85: 2434-2438.
  5. Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. Journal of Clinical Endocrinology and Metabolism 2004: 89: 2745-2749.
  6. Dunaif A, Segal KR, Futterweit W, Dobrjansky A. Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 1989; 38: 1165-1174.
  7. Loverro G, Lorusso F, Mei L, Depalo R, Cormio G, Selvaggi L. The plasma homocysteine levels are increased in polycystic ovary syndrome. Gynecologic and Obstetric Investigation 2002; 53: 157-162.
  8. Gennarelli G, Holte J, Wide L, Berne C, Lithell H. Is there a role for leptin in the endocrine and metabolic aberrations of polycystic ovary syndrome? Human Reproduction 1998; 13: 535-541.
  9. Maliqueo M, Perez-Bravo F, Calvillan M, Piwonka V, Castillo T, Sir-Petermann T. Relationship between leptin and insulin sensitivity in patients with polycystic ovary syndrome. Medicina Clinica 1999; 113: 526-530.
  10. Pirwany IR, Fleming R, Sattar N, Greer IA, Wallace AM. Circulating leptin concentrations and ovarian function in polycystic ovary syndrome. European Journal of Endocrinology 2001; 145: 289-294.
  11. Telli MH, Yildirim M, Noyan V. Serum leptin levels in patients with polycystic ovary syndrome. Fertility and Sterility 2002; 77: 932-935.
  12. Ahima RS, Dushay J, Flier SN, Prabakaran D, Flier JS. Leptin accelerates the onset of puberty in normal female mice. Journal of Clinical Investigation 1997; 99: 391-395.
  13. Agarwal SK, Vogel K, Weitsman SR, Magoffin DA. Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary. Journal of Clinical Endocrinology and Metabolism 1999; 84: 1072-1076.
  14. Biason-Lauber A, Zachmann M, Schoenle EJ. Effect of leptin on CYP17 enzymatic activities in human adrenal cells: new insight in the onset of adrenarche. Endocrinology 2000; 141: 1446-1454.
  15. Carmina E, Ferin M, Gonzalez F, Lobo RA. Evidence that insulin and androgens may participate in the regulation of serum leptin levels in women. Fertility and Sterility 1999; 72: 926-931.
  16. Spritzer PM, Poy M, Wiltgen D, Mylius LS, Capp E. Leptin concentrations in hirsute women with polycystic ovary syndrome or idiopathic hirsutism: influence on LH and relationship with hormonal, metabolic, and anthropometric measurements. Human Reproduction 2001; 16: 1340-1346.
  17. Laughlin GA, Morales AJ, Yen SS. Serum leptin levels in women with polycystic ovary syndrome: the role of insulin resistance/hyperinsulinemia. Journal of Clinical Endocrinology and Metabolism 1997; 82:1692-1696.
  18. Sir-Petermann T, Recabarren SE, Lobos A, Maliqueo M , Wildt L. Secretory pattern of leptin and LH during lactational amenorrhoea in breastfeeding normal and polycystic ovarian syndrome women. Human Reproduction 2001; 16: 244-249.
  19. Lammert A, Kiess W, Bottner A, Glasow A , Kratzsch J. Soluble leptin receptor represents the main leptin binding activity in human blood. Biochemical and Biophysical Research Communications 2001; 283: 982-988.
  20. Kado N, Kitawaki J, Koshiba H, Ishihara H, Kitaoka Y, Teramoto M, et al. Relationships between the serum levels of soluble leptin receptor and free and bound leptin in non-pregnant women of reproductive age and women undergoing controlled ovarian hyper-stimulation. Human Reproduction 2003; 18: 715-720.
  21. Sinha MK, Opentanova I, Ohannesian JP, Kolaczynski JW, Heiman ML, Hale J, et al. Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting. Journal of Clinical Investigation 1996; 98: 1277-1282.
  22. Ogawa T, Hirose H, Yamamoto Y, Nishikai K, Miyashita K, Nakamura H, et al. Relationships between serum soluble leptin receptor level and serum leptin and adiponectin levels, insulin resistance index, lipid profile, and leptin receptor gene polymorphisms in the Japanese population. Metabolism 2004; 53: 879-885.
  23. Van Dielen FM, van’t Veer C, Buurman WA, Greve JW. Leptin and soluble leptin receptor levels in obese and weight-losing individuals. Journal of Clinical Endocrinology and Metabolism 2002; 87: 1708-1716.
  24. Yannakoulia M, Yiannakouris N, Bluher S, Matalas AL, Klimis-Zacas D , Mantzoros CS. Body fat mass and macronutrient intake in relation to circulating soluble leptin receptor, free leptin index, adiponectin, and resistin concentrations in healthy humans. Journal of Clinical Endocrinology and Metabolism 2003; 88: 1730-1736.
  25. Misra M, Miller KK, Almazan C, Ramaswamy K, Aggarwal A, Herzog DB, et al. Hormonal and body composition predictors of soluble leptin receptor, leptin, and free leptin index in adolescent girls with anorexia nervosa and controls and relation to insulin sensitivity. Journal of Clinical Endocrinology and Metabolism 2004; 89: 3486-3495.
  26. Li HJ, Ji CY, Wang W, Hu YH. A twin study for serum leptin, soluble leptin receptor and free insulin-like growth factor-1 in pub-ertal females. Journal of Clinical Endocrinology and Metabolism 2005; 90: 3659-3664.
  27. Matthews D, Hosker J, Rudenski A, Naylor B, Treacher D, Turner R. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: 412-419.
  28. Sepilian VP, Crochet JR, Nagamani M. Serum soluble leptin receptor levels and free leptin index in woman with polycystic ovary syndrome: relationship to insulin resistance and androgens.  Fertile sterile 2006; 85: 1441-1447.
  29. Chapman IM, Wittert GA, Norman RJ. Circulating leptin concentrations in polycystic ovary syndrome: relation to anthropometric and metabolic parameters. Clinical Endocrinology 1997; 46: 175-181.
  30. Rouru J, Anttila L, Koskinen P, Penttila TA, Irjala K, Huupponen R, et al. Serum leptin concentrations in women with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 1997; 82: 1697-1700.
  31. Mantzorous CS, Dunaif A, Fiiler S. Leptin concentration in the polysistic ovary syndroume. Hounal of Clinical Endocrinology and Metabolism 1997; 82: 1697-1700.
  32. Houseknecht KL, Baile CA, Matteri RL, Spurlock ME. The biology of leptin: a review. Journal of Animal Science 1998; 76: 1405-1420.
  33. Landt M, Parvin CA, Wong M. Leptin in cerebrospinal fluid from children: correlation with plasma leptin, sexual dimorphism, and lack of protein binding. Clinical Chemistry 2000; 46: 854-858.
  34. Shimizu H, Shimomura K, Negishi M, Masunaga M, Uehara Y, Sato N, et al. Circulating concentrations of soluble leptin receptor: influence of menstrual cycle and diet therapy. Nutrition 2002; 18: 309-312.
  35. Kado N, Kitawaki J, Koshiba H, Ishihara H, Kitaoka Y, Teramoto M, et al. Relationships between the serum levels of soluble leptin receptor and free and bound leptin in non-pregnant women of reproductive age and women undergoing controlled ovarian hyperstimulation. Human Reproduction 2003; 18: 715-720.
  36. Jordan J, Brabant G, Brinsuk M, Tank J, Horn R, Luft FC, et al.  Heritability of free and receptor-bound leptin in normal twins. Am J Physiol Regul Integr Comp Physiol 2005; 288: 411- 416.
  37. Nobili V, Manco M, Ciampalini P, Diciommo V, Devito R, Piemonte F, et al.  Leptin, free leptin index, insulin resistance and liver fibrosis in children with non-alcoholic   fatty   liver   disease.    European   Journal   of Endocrinolog 2006; 155: 735-743.
  38. Mohiti-Ardekani J, Talebi F, Afkhami M.  Free leptin in diabetic patients and its correlation to insulin level. Pakestan J of Biological Sci 2009; 12: 397-400.

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