Abstract
Aim: To correlate serum and follicular fluid (FF) leptin and visfatin levels with lipid lipoprotein levels in women with and without polycystic ovary syndrome (PCOS) undergoing ovarian stimulation. Materials and Methods: We studied 90 PCOS women and 94 age- and weight-matched controls, enrolled in the In Vitro Fertilization (IVF) program. Results: Total cholesterol, low-density lipoprotein (LDL)-cholesterol, triglycerides, apolipoprotein B and lipoprotein(a) levels were significantly elevated, while high-density lipoprotein (HDL)-cholesterol and apolipoprotein A1, lower in PCOS subjects. Serum and FF visfatin levels were increased in PCOS women and correlated positively with body-mass index (BMI), lipoprotein(a) and triglycerides, and negatively with apolipoprotein A1. Leptin levels were comparable between groups and positively correlated with BMI and LDL-cholesterol, and negatively with apolipoprotein B. Conclusion: Lipid lipoprotein alterations are common in reproductive-age PCOS women increasing the risk for cardiovascular diseases later in life. Leptin and visfatin play significant roles in lipid metabolism and further research is required in this area.
Over the last decades the interest in the biology and biochemistry of adipose tissue has increased considerably since this dynamic tissue can synthesize biologically-active substances, such as leptin and visfatin that regulate metabolic processes (1). Leptin, a known food regulator, has an important role in human metabolism and in the pathophysiology of polycystic ovary syndrome (PCOS) (2, 3), whereas elevated visfatin levels are found in obese subjects (4) and PCOS women (5).
PCOS represents a common endocrine disorder affecting approximately 10% of women in reproductive age, often associated with obesity, impaired lipid metabolism and increased risk for cardiovascular diseases (CVD) (6-8). In fact, PCOS has been proposed as model of lipid alterations (9) and thus, women with polycystic ovaries should assess their cardiovascular risk through a complete fasting lipoprotein lipid workout as a preventive measure (7, 10).
Since leptin and visfatin have been implicated in the pathophysiology of PCOS which is associated with dyslipidemia, we aimed to seek differences, if any, between leptin and visfatin levels in normal weight as well as overweight PCOS women and in age- and weight-matched normally-ovulating controls, and correlate them to their lipid lipoprotein profile.
Materials and Methods
Patients. In the present study we included 90 women [45 lean, body mass index (BMI) <25 kg/m2 and 45 overweight or obese, BMI >25 kg/m2] with diagnosed PCOS, according to the Rotterdam consensus criteria (11) and 94 age- and weight-matched non-PCOS controls (47 lean, BMI <25 kg/m2 and 47 overweight or obese, BMI >25 kg/m2), enrolled in our In vitro fertilization (IVF) program.
The study protocol was approved by the Institutional Review Board of our teaching hospital, and written informed consent was obtained from all participants.
Sample collection and processing. Blood and follicular fluid (FF) samples were collected at oocyte retrieval. Blood was drawn in the morning, after a 12-hour overnight fast, in pyrogen-free tubes and centrifuged immediately after clotting. Blood-free FF samples were centrifuged at 600g for 10 minutes and supernatants were stored at −80°C. Lipid and lipoprotein levels were determined immediately after centrifugation. For the determination of adipokines levels, serum samples were stored together with the FF samples until processing.
Total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, triglycerides, apolipoprotein A1, apolipoprotein B and lipoprotein(a) levels (sensitivities: 5.0 mg/dl, 2.5 mg/dl, 1.0 mg/dl, 5.0 mg/dl, 16 mg/dl, 11 mg/dl and 0.83 mg/dl, respectively; intra-assay coefficient of variation (CV): 0.8%, 1.7%, 1.4%, 0.8%, 1.9%, 4.4% and 0.4%, respectively; inter-assay CV: 0.8%, 1.1%, 2.2%, 0.6%, 1.4%, 1.8% and 1.1%, respectively), were assessed by the Abbott Architect c8000 autoanalyser (Abbott Laboratories, North Chicago, IL, USA). Leptin and visfatin levels were determined in duplicate using ELISA assays [Leptin EASIA, BioSource Europe SA, Nivelles, Belgium and Visfatin C-Terminal (Human) EIA, Phoenix Pharmaceuticals, Inc, Belmont, CA, USA, respectively]. The minimum detectable concentration was 0.1 ng/ml for both proteins. The intra-assay and inter-assay coefficients of variation were 3.6% and 5.2% for leptin and 5% and 12% for visfatin, respectively.
Statistical analysis. Normality was examined by the Kolmogorov-Smirnov Test. Data regarding serum and FF visfatin was normally distributed, while serum and FF leptin was not normally distributed. Variables presented with normal distribution are presented as mean±Standard Deviation (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 the Pearson's x2 test was applied to examine any possible differences between groups on categorical variables. Variables regarding serum and FF 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 and visfatin levels. The Pearson's or Spearman correlation coefficient was used where appropriate in order to examine any possible correlations between serum and FF leptin and visfatin levels between groups. p-Values of <0.05 were considered statistically significant. Statistical analysis was performed using the SPSS 11.5 edition (Chicago, IL, USA).
Results
Demographic and biochemical data of subjects are presented in Table I.
In PCOS subjects we recorded significantly higher serum levels of total cholesterol, LDL-cholesterol, triglycerides, apolipoprotein B and lipoprotein(a) while HDL-cholesterol and apolipoprotein A1 were significantly lower compared to controls.
Serum and FF visfatin levels were significantly higher in PCOS women compared to controls [Beta coefficient (B)=38.643, Standard Error (SE)=4.965, p<0.001 and B=7.665, SE=2.266, p=0.001, respectively]. In contrast, serum and FF leptin levels did not differ between the groups. However, FF leptin levels were higher compared to serum levels in all groups. Furthermore, leptin and visfatin levels positively correlated to each other in both body fluids studied.
The effect of group on serum as well as FF leptin and visfatin levels was not proven to be significant. In contrast, serum and FF leptin levels correlated positively with BMI (B=0.038, SE=0.007, p<0.001 and B=0.03, SE=0.009, p=0.001, respectively) and LDL-cholesterol (B=0.002, SE=0.001, p=0.011 and B=0.002, SE=0.001, p=0.038, respectively), and negatively with triglycerides (B=−0.002, SE=0.001, p=0.016 and B=−0.009, SE=0.003, p=0.004) and apolipoprotein B (B=−0.003, SE=0.001, p=0.011 and B=−0.003, SE=0.001, p=0.009, respectively), after controlling for other confounding factors.
On the other hand, serum and FF visfatin levels correlated positively with BMI (B=2.953, SE=0.479, p<0.001 and B=1.303, SE=0.297, p<0.001, respectively) and triglycerides (B=0.139, SE=0.064, p=0.03 and B=0.09, SE=0.029, p=0.002, respectively), and negatively with apolipoprotein A1 (B=−0.206, SE=0.096, p=0.033 and B=−0.108, SE=0.044, p=0.015, respectively). Finally, we found a positive correlation between serum visfatin levels and lipoprotein(a) (B=0.261, SE=0.119, p=0.029).
Discussion
PCOS is a complex and heterogeneous endocrine disorder in female subjects, usually associated with obesity (3, 8) which is an important factor for an impaired lipid metabolism (6, 9). Furthermore, adipose tissue secretes specific proteins such as leptin and visfatin which have been implicated in the pathophysiology of PCOS (12, 13). We aimed to assess the link between leptin, visfatin and lipoprotein lipid metabolism in women with and without PCOS.
PCOS women are exposed to an increased risk for CVD later in life because they are more likely to develop hypertension and dyslipidemia (8, 9). As shown previously, and in concordance to our results, the most common pattern of lipid alteration in PCOS is classic atherogenic dyslipidemia (increased total cholesterol, LDL-cholesterol, triglycerides and decreased HDL-cholesterol), although a second mechanism might also be operative linking the androgen excess to increased LDL-cholesterol levels (2, 7, 9). In contrast, other research found comparable lipid levels in lean women with PCOS and in healthy women (14). Similar with previous reports, we found an unfavorable lipid profile, increased apolipoprotein B and lipoprotein(a) and decreased apolipoprotein A1 levels, which are also risk factors for CVD (12). Recently, a systematic review and meta-analysis (9) has demonstrated that PCOS per se increases lipid values even in BMI-matched patients, although the levels and the estimated cardiovascular risk may differ between subjects. As a result, a complete fasting lipid and lipoprotein workout in PCOS women has been recommended (7, 10).
Similar serum leptin levels have been reported in PCOS and age-, as well as weight-matched non-PCOS subjects (5, 15) which are in accordance to this study and different from other research where higher leptin levels in PCOS women compared with healthy controls were reported (12). Investigating the independent effect of PCOS and obesity on different adipokines, Svendsen et al. reported a trend toward increased expression of leptin mRNA in PCOS, but no effect on plasma leptin levels (3). We found higher FF leptin compared to serum levels. This finding is similar to a previous report (5) and can be explained by evidence that leptin is produced directly in the ovary by the granulosa cells, suggesting a probable involvement in the follicle (16).
Demographic and biochemical data of study groups.
Leptin was the first protein identified as an endocrine product of adipose tissue that regulates appetite, food intake and thus body weight (1). It has been suggested that leptin is an independent risk factor for CVD and is likely to be an important link in the development of cardiovascular risk through promotion of atherosclerosis and obesity (17). In our study serum and FF leptin levels were negatively-correlated with triglycerides and apolipoprotein B, results which are contradictory to other research (15, 17). The relationship between leptin and lipid lipoprotein metabolites in PCOS patients is complex and unclear (12). However, extreme heterogeneity of the PCOS (altered levels of all lipids or only some) could explain the diversity of data (9). Interestingly, Erel et al. (2) found a poor correlation between serum leptin levels and lipid metabolic products in PCOS and normal women. Leptin levels fluctuate proportionally to changes in caloric intake to decrease cholesterol biosynthesis and increase cholesterol catabolism, thus decreasing plasma very-low density lipoprotein cholesterol level, one of the main components of apolipoprotein B (18); this mechanism could offer an explanation for the negative correlation between leptin and apolipoprotein B found in our study. In contrast, a positive correlation was observed between leptin and BMI, a finding which is similar to other research (2, 13, 17).
We herein report increased visfatin levels in women with PCOS, results which are confirmed by previous publications (5, 14, 15, 19-21), although similar serum visfatin levels in controls and PCOS patients have been documented (22). Visfatin has been found in both visceral and subcutaneous adipose tissue (21) whereas its levels have been correlated with BMI and/or the percentage of body fat, with controversial findings. Similar to our results, Berndt et al. (4) found a significant positive association between visfatin and BMI, whereas Chan et al. (19) reported that visfatin levels were significantly correlated with BMI in simple regression analysis but not in multiple regression analysis. In obese women serum visfatin levels were significantly higher compared to controls (4) or even significantly lower (23). The reason for the divergent results published in the literature is at present unclear. Kowalska et al. (20) reported increased serum visfatin levels only in lean PCOS subjects, suggesting that obesity can alter visfatin secretion and its metabolic role. PCOS is a complex and heterogeneous clinical entity and a diversity of factors can influence its characteristic features in different population groups. We found lower visfatin levels in FF compared to serum in PCOS and normally ovulating women under IVF treatment, which probably suggest that this protein has no direct involvement in the follicle. However, serum and FF visfatin levels correlated positively in all our groups studied. The same was true for leptin and visfatin levels in both body fluids studied.
The relationship between visfatin levels, anthropometric parameters and lipoprotein metabolites in PCOS patients remains controversial. In our study, correlation analysis regarding visfatin showed a positive correlation with BMI, triglycerides and lipoprotein(a) and a negative one with apolipoprotein A1. Recently, triglyceride levels were positively associated with visfatin gene expression in adipose tissue, which in turn increased the free fatty acids levels (21). In other reports, visfatin levels were positively associated with other parameters of lipid metabolism such as total cholesterol and HDL-cholesterol (14, 20) or total cholesterol and LDL-cholesterol (24). In contrast, visfatin levels did not correlate with any fat parameters (15) or correlated negatively with total cholesterol, LDL-cholesterol, triglycerides and lipoprotein(a) and positively with HDL-cholesterol levels (6, 22). The contradictory data found in the literature may suggest that visfatin has a probable role in lipoprotein lipid metabolism in female subjects with an, as yet, unknown mechanism. However, the human visfatin gene has been identified in a linkage region for metabolic syndrome-related phenotypes, BMI, HDL-cholesterol and triglycerides, with a possible influence on lipid homeostasis, thus explaining the variations in the lipid lipoprotein profiles in different populations through the detected genetic polymorphisms (25). As for the associations between FF visfatin levels and lipid metabolic products in PCOS and in normal women, it is possible that any correlations could be a result of the role that visfatin plays in the lipoprotein lipid metabolism in general.
The contradictory results in the literature regarding leptin and visfatin relationship to lipid lipoprotein profile could be explained by the different recruitment criteria and confounding factors in the published studies (15, 24), ethnic heterogeneity and genetic background with different genetic polymorphisms (25), the inability to distinguish between the ovulatory and anovulatory PCOS phenotypes and discrepancies in the laboratory measurements (15).
In conclusion, lipid lipoprotein profile alterations are common in infertile Greek reproductive-age PCOS women, increasing the risk for cardiovascular diseases later in life. Leptin and visfatin have significant roles in lipid metabolism, although further studies are required to explore this unclear topic with possible new clinical and therapeutical implications.
- Received April 17, 2014.
- Revision received June 27, 2014.
- Accepted June 30, 2014.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
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