Polycystic ovary syndrome (PCOS) affects approximately
6–10% of women of reproductive age
and is characterized by ovarian dysfunction, hirsutism,
hyperandrogenism, insulin resistance, and
Obesity is a very common clinical feature in
women affected by PCOS. More than 50-60% of PCOS women are obese (
Leptin, the product of the ob (obese) gene, is
a single-chain 16 kDa protein consisting of 146
amino acid residues. Leptin is produced mainly in
adipose tissue and is involved in the regulation of
energy homeostasis, reproduction, insulin action,
and lipid metabolism. The relationship between
leptin and reproductive function is complex and
not completely understood (
Leptin is a key hormone in energy homeostasis
and neuroendocrine function and has a permissive
role in the pathogenesis of reproductive dysfunction
Ghrelin, an endogenous ligand for the growth
hormone secretagogue receptor, is synthesized principally
in the stomach. It stimulates food intake and
transduces signals to hypothalamic regulatory nuclei
that control energy homeostasis and are linked to the
control of key aspects of reproduction function. The
peptide consists of 28 amino acids (
There is evidence of leptin and ghrelin operating
as endocrine-paracrine mediators, establishing a
link between energy homeostasis and reproduction
Contradictory results in studies investigating serum leptin and ghrelin encouraged us to carry out the current research. We further assessed the association between LH, FSH, testosterone, sex hormone-binding globulin (SHBG), body mass index (BMI), waist-tohip ratio (WHR), and insulin resistance, with the two above-mentioned hormones.
The present case-control study was conducted on 30 PCOS patients and 30 healthy patients matched for age, BMI, and WHR that were referred to Alzahra Hospital in Tabriz, Iran. Sampling lasted from November 2008 to February 2009. The study protocol was approved by the Ethics Committee of the Tabriz University of Medical Sciences.
After being informed of the purpose and procedures
of the study, all subjects signed an informed
consent form. The diagnosis of PCOS was made
by a gynecologist using Rotterdam criteria, which
includes clinical and/or biochemical signs of hyperandrogenism
(increased serum total testosterone
or free androgen index), oligomenorrhoea
(six or fewer menses per year) or amenorrhoea (no
menses in the last six months), and polycystic ovaries
(by ultrasonographic examination) (
Medical history, physical and pelvic examination, and complete blood tests were used to determine the healthy status of women in the control group. Exclusion criteria for all subjects included pregnancy, hypothyroidism, hyperprolactinemia, Cushing’s syndrome, congenital adrenal hyperplasia, current or previous (within the last three months) use of oral contraceptives, glucocorticoids, antiandrogens, ovulation induction agents, antidiabetic and anti-obesity drugs, or other hormonal drugs. None of the patients were affected by any neoplastic, metabolic, or other concurrent medical illness. Weight and height were measured to calculate the BMI. Body height was measured to the nearest 0.1 cm with the subject standing without shoes. Body weight in light indoor clothing was measured to the nearest 0.1 kg. The BMI was calculated using the standard formula of weight (kg)/height (m2). Waist and hip circumferences (at the level of the hipbone anterior superior iliac spines) were also measured in the standing position to calculate the WHR.
The analyses were carried out during the early follicular phase (days 3-5) in women who had menstrual cycles, and in any phase of the cycle in PCOS patients. Basal blood samples were obtained to evaluate serum leptin, ghrelin, LH, FSH, total testosterone, SHBG, fasting insulin, and glucose levels. All blood samples for each subject were assayed in duplicate and immediately centrifuged. The serum was stored at -80˚C until analysis.
In each woman, the estimate of insulin resistance
by homeostasis model assessment of insulin resistance
(HOMA-IR) was calculated with the following
formula: fasting serum insulin (mU/l) × fasting
plasma glucose (mg/dl)/405 (
All blood samples were obtained between 08:00 am and 09:00 am after an overnight fast. The serum leptin level was measured using a Human Leptin ELISA Kit (BioVendor GmbH. Im Neuenheimer Feld 583. D-69120; Heidelberg, Germany), which had an intra-assay and inter-assay coefficient of variation, 4.2-7.6%, and 4.4-6.7%, respectively and sensitivity of 0.2 ng/ml. In all subjects, plasma immunoreactive ghrelin levels were measured using a commercially available RIA that uses 125I-labeled bioactive ghrelin as a tracer and a rabbit polyclonal antibody raised against full-length octanoylated human ghrelin (Phoenix Pharmaceuticals Inc., Belmont CA, USA), which recognizes both acylated and des-acylated ghrelin. Levels of serum LH and FSH were determined by direct immunoenzymatic method (DiaMetra S.r.l; Bartolomei, Z.I Paciana, Folingo (PG), Italy). The intraassay CVs of the assays used were: 7.9% (LH) and 9.4% (FSH). The inter-assay CVs of the assays used were: 9.0% (LH) and 11.8% (FSH).
The measurement of serum SHBG was performed using an enzyme-linked immunosorbent assay (ELISA) kit (IBL Immuno-Biological Laboratories; Flughafenstrasse 52A, D-22335, Hamburg, Germany) with an intra-assay CVs of 3% and inter-assay CVs of 8.7%. Total testosterone levels were determined using a commercially available ELISA kit (Monobind Inc., 100 North Pointe Drive, Lake Forest, CA 92630, USA) which had an intra-assay CV of 5.2% and an inter-assay CV of 6 %.
All parameters studied or calculated showed normal distributions, which were confirmed by the one sample Kolmogorov-Smirnoff test. Results were expressed as mean ± standard deviation (SD). Comparisons between the two groups were made using an independent samples t test. Pearson correlation analyses were performed to define correlations between parameters. p<0.05 was regarded as statistically significant. All analyses were run using the SPSS (version 12.0, SPSS, Chicago, IL).
There were 30 women were in the PCOS group
and 30 in the control group. The anthropometric and
laboratory data of the groups are presented in
Anthropometric, metabolic, and hormonal characteristics of patients and controls
|PCOS (n=30)||Control (n=30)|
|25.83± 4.00||26.06± 4.44|
|64.40± 1 0.46||62.4± 8.82|
|160.1 ± 6.01||162.4± 6.53|
|25.00± 3.61||23.68± 3.07|
|81.01± 8.98||79.53± 6.4|
|101.00 ± 6.37||99.13± 6.35|
|0.80± 0.56||0.80± 0.60|
|21.68± 4.49**||17.96± 3.00|
|210.33 ± 58.5||216.00 ± 80.84|
|14.91± 1.78**||7.90± 1.16|
|92.6± 8.3||94.4± 8.6|
|3.47± 0.54**||1.81± 0.36|
|0.75± 0.60*||0.45± 0.26|
|31.81 ± 14.29*||52.34 ± 23.41|
|12.50± 2.33**||4.86± 2.12|
|6.03 ± 1.64||5.74 ± 1.10|
Data presented as means ± SD. BMI; body mass index, FSH; follicle-stimulating hormone, HOMA-IR; homeosta-sis model assessment of insulin resistance, LH; luteinizing hormone, PCOS; polycystic ovary syndrome, SHBG; sex hormone binding globulin and WHR; waist-to-hip ratio. *p<0.05, **p<0.001.
(A) Correlation between serum leptin levels and BMI (PCOS; r=0.85, p<0.001,Controls; r=0.93, p<0.01). (B) HOMA- IR (PCOS; r=0.67, p<0.01, Controls; r=0.77, p<0.01). (C) Total testosterone (PCOS; r=0.19, p>0.05, Controls; r= 0.02, p>0.05).
In the PCOS group, serum levels of leptin, insulin, HOMA-IR, testosterone, LH, and LH/ FSH were significantly higher than in the control group. SHBG concentration was found to be lower in the PCOS group. As for ghrelin and FSH, no significant difference was detected in either group.
Bivariate correlations (
(A) Correlation between serum Ghrelin levels and BMI (PCOS; r=-0.04, p>0.05, Controls; r=-0.22, p>0.05). (B) HOMA-IR (PCOS; r=-0.12, p>0.05, Controls; r=-0.14, p>0.05). (C) Total testosterone (PCOS; r=-0.32, p<0.05, Controls; r=-0.42, p>0.05).
Pearson correlation tests of clinical, metabolic, and hormonal parameters with leptin and ghrelin in the study groups
|PCOS (n=30)||Control (n=30)|
FSH; Follicle-stimulating hormone, HOMA-IR; Homeostasis model assessment of insulin resistance, LH; Luteinizing hormone, SHBG; Sex hormone binding globulin and WHR; Waist-to-hip ratio. * p<0.05, **p<0.01, ***p<0.001.
There was no significant correlation of ghrelin to
weight, BMI, WHR, insulin, HOMA-IR, FSH, and
LH in both groups. A significant inverse association
was found between ghrelin and testosterone
levels in both the PCOS (r=-0.32, p=0.04) and the
control group (r=-0.42, p=0.02) (
Insulin levels significantly correlated with BMI and waist circumference in both the PCOS group and the control group. BMI (r=0.78, p=0.001) and waist circumference were also positively associated with HOMA-IR (r=0.71, p=0.001).
Findings from current research show that women with PCOS had higher levels of insulin, HOMAIR, testosterone, LH, and LH/FSH and lower concentrations of SHBG.
Higher leptin levels may have a role in the
pathophysiology of PCOS. Leptin has a dual effect
on reproduction. The positive effect of leptin
is its role as a trigger of puberty on hypothalamicpituitary
axis by stimulating estrogen secretion.
The negative impact of leptin, in conditions like
hyperleptinemia is the inhibition of the ovarian response
to gonadotrophin stimulation (
Studies of leptin levels in PCOS women have
yielded conflicting results. Similar to the findings
of Mitkov et al. (
A correlation between serum leptin and BMI has
been shown in PCOS women (
SHBG is a glycoprotein produced in the liver
acting as a carrier for different sexual steroid hormones.
It shows a higher affinity for testosterone
Insulin resistance and subsequent hyperinsulinemia
are found in 50-70% of PCOS patients. High
insulin levels are associated with hyperandrogenism
and anovulation (
The steady-state basal serum glucose and insulin
concentrations are determined by their interaction
in a feedback loop. A computer model is used to
predict the homeostatic concentrations that result
from varying degrees of insulin resistance and
β-cell deficiency. Comparison of a patient’s fasting
levels with the model’s predictions provides
a quantitative assessment of the contributions of
deficient β-cell function and insulin resistance to
the fasting hyperglycemia (homeostasis model assessment;
In the present study the PCOS group showed higher insulin levels, HOMA-IR, elevated testosterone, and decreased SHBG levels than the healthy controls.
Peripheral (hepatic and skeletal muscle) insulin
sensitivity and pancreatic β-cell function is improved
via leptin action in these sites (
Current research has revealed that in the PCOS
and control group leptin was positively correlated
with insulin levels and HOMA-IR. There is evidence
of leptin ability in stimulating GnRH from
the hypothalamus and LH/FSH release from the
Studies in which 24 hour LH pulses were observed
in PCOS patients (
Studies investigating the association of leptin with LH that measured 24 hours LH and leptin pulses provide a more precise, sensitive, and objective index of alterations in bi-hormonal linkage than studies using a single measurement of serum concentration of these two hormones, as used in our study. However, the involvement of leptin in modulating LH and FSH via its pulsatile secretory characteristics has yet to be elucidated in either healthy or PCOS subjects.
It has been shown that most obese individuals
need higher doses of gonadotropins for ovary
hyper-stimulation, despite comparable absorption
of gonadotropins from subcutaneous tissue (
However, similar to Rouru et al. (
In hypogonadal men, testosterone supplementation
has been shown to normalize elevated leptin
concentrations without any changes in body fat or
Lower levels of SHBG probably mirror a higher
testosterone to estrogen ratio (
In our study, ghrelin levels did not show a significant
difference between the two groups. In the
literature, obese PCOS compared to obese healthy
subjects have been found to have lower ghrelin
levels, but when lean and obese PCOS groups
were taken as a whole and compared to BMImatched
controls, ghrelin levels were found to
be similar between both groups (
Similar to the findings of our study, Schofl et
It has been shown that ghrelin administration to
healthy humans at pharmacological doses reduces
insulin secretion (
In humans the specific effects of ghrelin on LH secretion have not been indicated. It is feasible that more comprehensive analyses involving precise assessment of LH pulsatility after ghrelin administration might reveal a subtle regulatory role of ghrelin in the control of gonadotropin secretion in humans. We have found no correlation between ghrelin levels and serum LH, FSH, or LH/FSH ratio in PCOS and control groups. These finding do not support the idea that ghrelin might alter gonadotropin levels.
The ghrelin receptor is found not only in the
CNS but also in the ovarian tissues, suggesting
a possible reproductive function (
In the current research, we further examined the association of testosterone with BMI and waist circumference. As the BMI and waist circumference increased, the serum testosterone levels showed a significant elevation. These findings have demonstrated that the increase of body weight and fat tissue is associated with abnormalities in sex steroid balance.
Some limitations of the present study were the relatively low sample size, narrow range of BMI, and the measuring of leptin and ghrelin only in a fasting state. Matching subjects for age and anthropometric indices have been considered as strengths of our study. However, to reach a better understanding of PCOS pathophysiology, more studies are warranted in which PCOS patients are grouped based on their BMI, insulin and androgen levels, presence of clinical features of hyperandrogenism, and severity of polycystic ovaries. Ghrelin and leptin or other hormones should be precisely measured in both fasting and postprandial states in relation to endocrine parameters.
The findings of this study have suggested that indices of adiposity (BMI and WHR) are responsible for elevated leptin, insulin resistance, and testosterone levels in PCOS patients. The role of leptin and ghrelin in the pathogenesis of PCOS may occur by ways other than the simple concentration of these hormones in circulation, particularly as leptin inserts its endocrine effects mostly through modulating insulin levels.