Abstract
Aim: To investigate serum and follicular fluid (FF) leptin levels in normally-ovulating women and subjects with polycystic ovary syndrome (PCOS) undergoing controlled ovarian stimulation and correlate them with their lipid lipoprotein profile. Materials and Methods: We included 70 PCOS women (35 lean and 35 overweight or obese) and 76 age- and weight-matched non-PCOS controls (39 lean and 37 overweight or obese). Results: Serum levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, apolipoprotein B, lipoprotein(a) and homocysteine in women with PCOS were significantly higher compared to control groups while levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein A1 were significantly lower. Serum leptin levels did not differ between groups and were lower than FF levels. Serum and FF leptin levels were positively correlated and were significantly decreased when apolipoprotein B levels increased. Conclusion: PCOS women exhibit lipid metabolism abnormalities putting them at increased risk of developing early atherosclerosis.
Adipose tissue represents a major endocrine organ, which secretes adipokines such as leptin with important role in human metabolism (1). Interestingly, its discovery in 1994 motivated the switch in the scientific interest regarding the biology and physiopathology of the white adipose tissue. It has been demonstrated that leptin increases tissue fat oxidation and therefore insulin sensitivity (2) while playing a significant role in the development of hyperandrogenism and infertility in women with polycystic ovary syndrome (PCOS) (1). PCOS is the most common endocrine disorder in women affecting approximately 10% of women in reproductive age (3) and is usually first diagnosed during the early reproductive years. As central obesity and insulin resistance are important factors in the pathophysiology of PCOS, increased body weight may alter insulin sensitivity with the aid of secreted adipokines, including leptin, which is a known food regulator (2).
Due to metabolic dysfunction, women with PCOS are exposed to risk for cardiovascular diseases (CVD) later in life (4), which is directly related to the progression of atherosclerosis. Atherosclerosis starts probably during adolescence or reproductive age (5) and dyslipidemia is an important determinant (3). Although dyslipidemia is a major determinant for CVD in post-menopausal women (4), the experience in women of reproductive age is rather limited. PCOS may represent an important model of lipid alterations (5) since dyslipidemia is common in young PCOS women (4). However, it has been recently recommended by both the American College of Obstetricians and Gynecologists (6) and the Androgen Excess and PCOS Society (7) that women with PCOS should have a complete fasting lipid and lipoprotein workout as part of their cardiovascular risk evaluation.
Our study was based on the hypothesis that since leptin plays an important role in human metabolism as well as in the pathophysiology of PCOS, its serum and follicular fluid (FF) levels might differ in overweight PCOS patients. Since increased Body Mass Index (BMI) and PCOS are closely-related, we investigated their effect on the lipoprotein levels in relation to the serum and FF leptin levels in different groups of PCOS women and their age- and weight-matched normally ovulating controls. We focused on differences in PCOS compared to non-PCOS control women, of both normal weight as well as overweight, in an effort to understand the burden of dyslipidemia which is associated with PCOS.
Patients and Methods
Patients. The study population included 70 women (35 lean, BMI <25 kg/m2 and 35 overweight or obese, BMI >25 kg/m2) with diagnosed PCOS, based on the evidence of any two of three features: hyperandrogenism, menstrual irregularity and polycystic ovary morphology, according to the Rotterdam consensus criteria (8) and 76 age- and weight-matched non-PCOS controls (39 lean, BMI <25 kg/m2 and 37 overweight or obese, BMI >25 kg/m2), enrolled in our in vitro fertilization (IVF) program. All women enrolled in this study were counseled to have a complete fasting lipid and lipoprotein workout as part of their cardiovascular risk assessment. The Institutional Review Board of our teaching hospital approved the study and signed informed consent was obtained from all subjects before recruitment.
Sample collection and processing. Blood and FF samples were collected at oocyte retrieval. After removing the oocytes, blood-free FF samples were centrifuged at 600g for 10 min and supernatants were stored at −80°C. Blood was drawn in the morning, after a 12-h overnight fast, in pyrogen-free tubes and centrifuged immediately after clotting. Lipid levels were determined immediately after centrifugation while for the determination of leptin levels, serum samples were stored together with the FF samples until processing.
Serum lipid and lipoprotein levels, in particular total cholesterol, high-density lipoprotein (HDL)-C, low-density lipoprotein (LDL)-C, triglycerides, apolipoprotein A1, apolipoprotein B, lipoprotein(a) and homocysteine (sensitivity 5.0 mg/dl, 2.5 mg/dl, 1.0 mg/dl, 5.0 mg/dlL, 16 mg/dl, 11 mg/dl, 0.83 mg/dl and 1.0 μmol/l, respectively, intra-assay coefficient of variation (CV) 0.8 %, 1.7 %, 1.4 %, 0.8 %, 1.9 %, 4.4 %, 0.4 % and 2.0 %, respectively and inter-assay CV 0.8 %, 1.1 %, 2.2 %, 0.6 %, 1.4 %, 1.8 %, 1.1 % and 4.8 %, respectively), were assessed by the Abbott Architect c8000 autoanalyser (Abbott Laboratories, North Chicago, IL, USA).
Leptin levels were determined in duplicate using an ELISA assay (Leptin EASIA, BioSource Europe SA, Nivelles, Belgium). The minimum detectable concentration was 0.1 ng/ml while the intra-assay and inter-assay CV were 3.6% and 5.2%, respectively.
Statistical analysis. For the statistical analysis normality was examined by the Kolmogorov-Smirnov test. Data regarding FF leptin was normally distributed, while data regarding serum leptin was not normally distributed. Variables presented with normal distribution are presented as mean±SD while not normally distributed variables are presented as median (range). One-way ANOVA or Kruskal Wallis test were used to detect differences between groups on continuous variables (univariate analysis), while Pearson's χ2 test was applied to examine any possible differences between groups on categorical variables. Variables regarding serum leptin underwent a logarithmic transformation. Linear regression analysis was performed in order to examine the effect of different confounding factors on serum and FF leptin levels. Pearson's or Spearman correlation coefficient was used where appropriate in order to examine any possible correlations of serum and FF leptin levels between groups. p-Values <0.05 were considered statistically significant. Statistical analysis was performed using the SPSS 11.5 edition (Chicago, IL, USA).
Results
Subjects' demographic and biochemical data are presented in Table I. Serum levels of total cholesterol, LDL cholesterol, triglycerides, apolipoprotein B, lipoprotein(a) and homocysteine in women with PCOS were significantly higher compared to the control groups while HDL cholesterol and apolipoprotein A1 were significantly lower.
No significant differences were observed between serum and FF leptin levels in the patients included in the present study. However, FF leptin levels were higher than serum levels in all groups. The effect of group on serum leptin levels was not proven significant. Nevertheless, the effects of BMI and apolipoprotein B on serum leptin were proven statistically significant. Specifically, serum leptin was significantly increased when BMI (B=0.026, SE=0.008, p=0.001) increased, after controlling for other confounding factors. On the contrary, serum leptin levels were decreased when apolipoprotein B (B=−0.003, SE=0.001, p=0.002) levels increased. As far as the FF leptin levels were concerned, the effect of group was not proven significant. Interestingly, the effects of height and apolipoprotein B levels on FF leptin levels were statistically significant. In particular, FF leptin levels positively correlated with women's height (B=1.465, SE=0.319, p<0.001) and negatively with apolipoprotein B levels (B=−0.225, SE=0.090, p=0.014). Serum leptin levels were positively correlated with FF leptin levels when all groups were taken into consideration (r=0.953, p<0.001). The same was true when each group was studied separately: PCOS-BMI<25 (r=0.972, p<0.001), Control-BMI<25 (r=0.969, p<0.001), PCOS-BMI>25 (r=0.927, p<0.001) and Control-BMI>25 (r=0.963, p<0.001).
Discussion
PCOS, the most common endocrine disorder in females, is usually first identified during the early reproductive years and has been associated with increased adipose tissue mass. Significant positive correlations between leptin, an adipocytokine mostly secreted by adipokines, and fat estimates (body weight, BMI and fat mass) have been reported (9). In the present study, besides a significant positive correlation between serum leptin and BMI, we found a significant positive association between FF leptin levels and women's height. To our knowledge, we are the first to report this correlation, although a positive association between height and serum leptin levels has been previously reported in adults (10) and in children (11).
To date, no definitive role for leptin in the pathophysiology of PCOS has been attributed since serum leptin levels do not differ between PCOS and age, as well as weight-matched non-PCOS subjects (12, 13), which is in accordance with our results, although higher leptin levels were reported in PCOS women compared to healthy controls (9, 14). In a recent report, investigating the independent effect of PCOS and obesity on different adipokines, Svendsen et al. (2) found a trend towards increased expression of mRNA for leptin associated with PCOS, but no effect of PCOS on plasma leptin levels.
Leptin may serve as the critical link between adipose tissue and the reproductive system (1). There is evidence that leptin is produced directly in the ovary since granulosa cells are able to produce and store leptin, thus suggesting its local involvement in the regulation of follicular growth (15). On the contrary, similar serum and FF leptin levels suggest that an autocrine secretion by the ovary itself is unlikely (12).
Demographic and biochemical data of study groups.
Women with PCOS are at increased risk for developing CVD with aging (4) through progression of atherosclerosis, and dyslipidemia is a major determinant for this. Even though experience in young women is limited, it is already known that atherosclerosis starts at a very young age since dyslipidemia is common in young adult women with PCOS (4). Our data confirm previous findings that in PCOS the most common pattern of lipid alteration is probably classic atherogenic dyslipidemia (increased total cholesterol, LDL cholesterol and triglycerides, and low HDL cholesterol) (5, 7, 16), although similar lipid levels in lean women with PCOS and in healthy women have been reported (17). As a result of increasing evidence linking PCOS women with higher LDL cholesterol levels (5), it has been recently recommended that women with PCOS should have a complete fasting lipid and lipoprotein workout as part of their cardiovascular risk assessment (6, 7). Since atherosclerotic processes start early during life, it is imperative to assess and eventually treat altered lipid values in young women with PCOS (7).
In accordance with previous studies, we noted an unfavorable lipid profile, comprising increased apolipoprotein B and lipoprotein(a) and decreased apolipoprotein A1, which are also risk factors for CVD (14, 16). Furthermore, a negative correlation between serum and FF leptin and apolipoprotein B levels was detected in our subjects, contrary to previous reports suggesting no significant correlation (18) or a positive relationship between leptin and apolipoprotein B (19). Leptin is primarily produced in adipose tissue and its circulating levels are directly proportional to the amount of body fat, fluctuating with acute changes in caloric intake. Leptin regulates lipid metabolism by preventing the accumulation of lipids in nonadipose tissue (20). Moreover, leptin decreases cholesterol biosynthesis and up-regulates cholesterol catabolism, thus decreasing plasma very-low density lipoprotein cholesterol concentrations, one of the main components of apolipoprotein B (21); this mechanism could explain the negative correlation between leptin and apolipoprotein B found in this study.
Clinical studies have suggested that increased homocysteine levels are considered as another independent risk factor for CVD, increasing the risk for atherosclerosis (14). In agreement to our results, elevated serum homocysteine levels were found in PCOS women compared to weight-matched controls (14, 16), although these findings have not been confirmed by other researchers (22).
In conclusion, our data show that women with PCOS exhibit similar serum and FF leptin levels with control subjects of similar age and BMI in this group of patients undergoing IVF treatment. Since dyslipidemia is common in PCOS women, its early identification, even during preparation for the IVF procedures, might be appropriate in terms of early preventive measures.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
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