Visfatin and leptin levels in women with polycystic ovaries undergoing ovarian stimulation

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1 Visfatin and leptin levels in women with polycystic ovaries undergoing ovarian stimulation Ekaterina Plati, M.D., a Evangelia Kouskouni, M.D., a Ariadne Malamitsi-Puchner, M.D., b Maria Boutsikou, M.D., b George Kaparos, Ph.D., a and Stavroula Baka, M.D. a,b a Department of Biopathology; and b Assisted Reproduction Unit, Second Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens, Greece Objective: To detect the levels of visfatin and leptin in the serum as well as in the follicular fluid (FF) of polycystic ovary syndrome (PCOS) patients undergoing controlled ovarian stimulation and to compare them with the levels found in age- and weight-matched normally ovulating women under IVF treatment. Design: Prospective study. Setting: Assisted Reproduction Unit, Second Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens, Greece. Patient(s): Forty patients with diagnosed PCOS and 40 age- and weight-matched non-pcos control women enrolled in the IVF program. Intervention(s): Blood and FF samples were collected from all subjects at oocyte retrieval. Main Outcome Measure(s): Visfatin and leptin levels were measured in serum and FF using ELISA. Result(s): Serum visfatin levels were significantly increased in women with PCOS, whereas FF visfatin levels, which were lower than serum levels, did not differ between groups. Serum leptin levels did not differ between groups and were lower than FF levels. Conclusion(s): Women with polycystic ovaries exhibit significantly increased serum visfatin and decreased FF leptin levels compared with control subjects of similar age and body mass index, indicating a probable role for visfatin in the general state of insulin resistance and a local contribution in the follicle for leptin in patients undergoing IVF treatment. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Visfatin, leptin, polycystic ovary syndrome, follicular fluid, IVF Adipose tissue, previously considered to be just an energy storage site, has emerged as a major endocrine organ secreting adipocytokines, such as visfatin and leptin, which seem to play important roles in significant aspects of metabolism (1). Leptin is a 16-kDa protein encoded by the ob gene (2). Circulating levels of leptin, mostly secreted by adipocytes, correlate with adipose tissue mass (3). The discovery of leptin in 1994 represented a strong motivation to change the scientific interest in the biology and physiopathology related to white adipose tissue. Furthermore, adipose tissue aromatizes androgens to estrogens, and its dysfunction can result in insulin resistance. Polycystic ovary syndrome (PCOS) remains a challenging clinical entity. It is the most common cause of anovulatory infertility and is associated with obesity, hyperandrogenism, and hyperinsulinemia. Obesity occurs in about half of the women affected but is not universal (4). The functional ovarian hyperandrogenism relates to insulin resistance, which is found in both lean and obese patients (5). On the other Received December 24, 2008; revised April 8, 2009; accepted April 21, 2009; published online June 12, E.P. has nothing to disclose. E.K. has nothing to disclose. A.M.-P. has nothing to disclose. M.B. has nothing to disclose. G.K. has nothing to disclose. S.B. has nothing to disclose. Reprint requests: Asst. Prof. Stavroula Baka, 104, Pindou Street, Athens, Greece (FAX: þ ivf@aretaieio.uoa.gr). hand, leptin resistance could be associated with the pathogenesis of PCOS (6). It is clear that this protein mediates between adipose tissue and the reproductive function, because granulosa cells are able to produce and store leptin, thus suggesting its local involvement in the regulation of follicular growth (7). However, its detailed role in reproduction awaits further clarification through future studies. Originally identified as pre B-cell colony-enhancing factor (PBEF) (8), visfatin is one of the most recent proteins shown to be highly expressed in adipose tissue. Fukuhara et al. (9) identified visfatin as a new protein produced in the intra-abdominal adipose tissue which shares many of the antidiabetic effects of insulin, exhibiting insulin-mimetic properties by binding to and activating the insulin receptor and thus resulting in a glucose-lowering effect. Despite numerous publications during the last 3 years, its role in metabolism remains largely unknown. There is a growing body of evidence that visfatin is increased in subjects with abdominal obesity (9, 10), type 2 diabetes mellitus, and insulin resistance (11). Moreover, visfatin release may be regulated by insulin, because hyperglycemia has the effect of increasing circulating visfatin levels, but this effect is suppressed by exogenous hyperinsulinemia (10). Clearly, there are largely unknown aspects of visfatin pathophysiology that require further study. Interestingly, the insulin-mimetic effect of visfatin is mediated by a distinct binding site on the insulin /$36.00 Fertility and Sterility â Vol. 94, No. 4, September doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 receptor (9). It is quite intriguing, though, that this insulinmimetic effect is produced by the adipose tissue which relates to insulin resistance. To our knowledge, visfatin levels have never been studied in follicular fluid (FF). The present study was based on the hypothesis that because serum visfatin levels are increased in PCOS patients, this protein might be present in the FF, too. Furthermore, some differences are expected in FF visfatin levels in this group of patients who are characterized by insulin resistance. Therefore, taking into consideration the age and body mass index (BMI) of women undergoing controlled ovarian stimulation, with either normal or polycystic ovaries, we aimed to determine and compare serum and FF visfatin and leptin levels. MATERIALS AND METHODS The Institutional Review Board of our teaching hospital approved this study, and signed informed consent was obtained from each of the subjects before recruitment. The study population included 40 women with diagnosed PCOS, based on the evidence of any two of three features hyperandrogenism, menstrual irregularity, and polycystic ovary morphology (12, 13) and 40 age- and weight-matched non-pcos control women enrolled in our IVF program. At their initial visits, all subjects underwent medical evaluation and demographic data was recorded. At that point an assessment of excess terminal hair growth was performed by the same examiner, using the previously described modification of the method originally presented by Ferriman and Gallwey (mfg) (14), with a score from 0 to 4 over nine body areas (upper lip, chin, chest, upper and lower abdomen, thighs, upper and lower back, and upper arms). A score of R8 was considered to be presence of hirsutism (15). The antral follicular number was assessed in the early follicular phase by transvaginal ultrasound scans of the ovaries performed by experienced sonographers in the cycle preceding the IVF treatment. Controlled ovarian hyperstimulation was achieved in all subjects with a long protocol: daily administration of GnRH analogue from the 21st day of the pretreatment cycle (which was normalized with the administration of combined oral contraceptive) and initiation of recombinant FSH (beta-follitropin; Puregon; Organon, Oss, The Netherlands) in a daily dose of IU (depending on the patient s characteristics) when down-regulation was confirmed (serum E 2 <50 pg/ml). Follicular development was monitored with serial ultrasonographic examinations and serum E 2 measurements. When R3 follicles of 18 mm in diameter were detected on transvaginal ultrasonography, 10,000 IU hcg were administrated. Blood and FF samples were collected at oocyte retrieval, which was scheduled hours after hcg and performed transvaginally under ultrasonography. After removing the oocytes, blood-free FF samples were centrifuged at 600g for 10 minutes, and supernatants were stored at 80 C. Blood was drawn in the morning, after a 12-hour overnight fast, in pyrogen-free tubes and centrifuged immediately after clotting. Glucose and hormone levels were determined immediately after centrifugation. For the determination of leptin and visfatin levels, serum samples were stored together with the FF samples until processing. In vitro fertilization was performed with the respective male partner s spermatozoa, and all embryo transfers were performed on day 3 with a Wallace catheter under ultrasound guidance. The serum glucose (sensitivity 2.5 mg/dl, intraassay coefficient of variation [CV] 1.98%, interassay CV 0.84%) was measured by chemiluminescent immunoassay using the Abbott Architect ci-8200 autoanalyzer (Abbott Laboratories, North Chicago, IL). Serum insulin (sensitivity 1.0 mu/ml, intraassay CV 3.6%, interassay CV 4.5%), E 2 (sensitivity 10 pg/ml, intraassay CV 5.5%, interassay CV 6.7%), LH (sensitivity 0.07 miu/ml, intraassay CV 2.1%, interassay CV 3.4%), FSH (sensitivity 0.05 miu/ml, intraassay CV 2.8%, interassay CV 4.2%), and T (sensitivity 0.08 ng/ml, intraassay CV 4.0%, interassay CV 6.6%) were measured by chemiluminescent microparticle immunoassay using the Abbott Architect i-1000 SR autoanalyzer (Abbott Laboratories). and serum SHBG (sensitivity 0.35 nmol/l, intraassay CV 2.1%, interassay CV 2.7%) was measured by electrochemiluminescence immunoassay with the Roche Elecsys 2010 autoanalyzer (Roche Diagnostics, Indianapolis, IN). Free androgen index (FAI) was calculated as serum T (nmol/l) 100/SHBG (nmol/l) (16), and insulin resistance was estimated according to the homeostasis model-resistance index (HOMA-IR) as fasting insulin (mu/ml) fasting glucose (mmol/l)/22.5, as previously reported (17). Levels of leptin and visfatin were determined in duplicate using ELISA assays [Leptin EASIA; BioSource Europe, Nivelles, Belgium; and Visfatin C-Terminal (Human) EIA; Phoenix Pharmaceuticals, Belmont, CA]. The minimum detectable concentration was 0.1 ng/ml for both proteins. The intraassay and interassay CVs were, respectively, 3.6% and 5.2% for leptin and 5% and 12% for visfatin. Statistical Analysis Data regarding FF leptin and serum and FF visfatin levels were normally distributed (Kolmogorov-Smirnov test); therefore, linear regression analysis was used to examine the effect of group, age, number of oocytes, and BMI on FF leptin and serum and FF visfatin levels. Serum leptin was not normally distributed; therefore, nonparametric Mann-Whitney U test was used to detect any differences between the two groups. Independent-samples t test was used to detect differences between the PCOS and control groups when the examined variables followed normal distribution. Otherwise, Mann-Whitney U test was applied. Pearson or Spearman correlation coefficients were used to 1452 Plati et al. Visfatin and leptin in PCOS IVF patients Vol. 94, No. 4, September 2010

3 detect positive or negative correlations, where appropriate. P values of <.05 were considered to be significant. RESULTS Demographic, clinical, and biochemical data of normally ovulating women and women with PCOS undergoing controlled ovarian stimulation are presented in Table 1. The two groups were similar in age and BMI. In the PCOS group there was a significantly higher number of retrieved oocytes (P<.001) and fertilized oocytes (P<.001), and the number of pregnancies was higher without reaching statistical significance. Serum leptin levels did not differ between groups. In the PCOS group the effect of BMI on FF leptin levels was found to be significant, and the effect of age was only indicative. For every year increase in women s age, FF leptin was decreased by 1.31 ng/ml on average when adjusted for BMI. In contrast, FF leptin levels were elevated by ng/ml on average for every unit increase in BMI when adjusted for age. The effect of age, number of oocytes, and BMI on serum and FF visfatin levels were not statistically significant. In the control group, the effect of age and BMI on FF leptin levels were found to be significant. For every year increase in women s age, FF leptin levels were significantly decreased by 1.77 ng/ml on average when adjusted for BMI. In contrast, FF leptin levels were elevated by 9.93 ng/ml on average for every unit increase in BMI when adjusted for age. The effect of age on serum visfatin levels was indicative, and the effect of BMI was not significant. For every year increase in women s age serum visfatin levels were decreased by 2.18 ng/ml on average. The effect of BMI on FF visfatin levels was found to be significant, and the effect of age was indicative. For every year increase in women s age, FF visfatin levels were decreased by 0.99 ng/ml on average when adjusted for BMI. Finally, FF visfatin was elevated by 4.32 ng/ml on average for every unit increase in BMI when adjusted for age (Table 2). In Table 3, significant correlations between leptin and visfatin levels (both in serum and in FF), age, and BMI are presented. TABLE 1 Demographic, clinical, and biochemical data of women with normal (control) and polycystic (PCOS) ovaries undergoing controlled ovarian stimulation. Control PCOS P value a Age (y) NS BMI (kg/m 2 ) NS Cycle length (d) 29 (27 31) 47.5 (32 60) <.001 Clinical hirsutism, n (%) 0 32 (80) <.001 Antral follicle count <.001 No. oocytes <.001 No. fertilized oocytes <.001 No. embryos transferred NS No. of pregnancies (%) 11 (27.5) 16 (40) NS Glucose (mg/dl) <.001 Insulin (miu/ml) <.001 HOMA-IR (miu/ml) <.001 Insulin resistance, n (%) 0 23 (57.5) <.001 FSH (miu/ml) LH (miu/ml) 6.6 ( ) 15.9 ( ) <.001 E 2 (pg/ml) <.001 Total T (ng/ml) 0.4 ( ) 0.8 ( ) <.001 SHBG (nmol/l) <.001 Free androgen index (%) 2.2 ( ) 8.9 ( ) <.001 Serum leptin (ng/ml) 24.5 ( ) 22.6 ( ) NS FF leptin (ng/ml) Serum visfatin (ng/ml) FF visfatin (ng/ml) NS Note: Values are mean SE or median (range) unless otherwise indicated. BMI ¼ body mass index; FF ¼ follicular fluid; HOMA-IR ¼ homeostatis model assessment of insulin resistance. a P values between.05 and.1 are indicative. Plati. Visfatin and leptin in PCOS IVF patients. Fertil Steril Fertility and Sterility â 1453

4 TABLE 2 Results of linear regression analysis. Variable B a P value a 95% CI b PCOS c group (n ¼ 40) FF d leptin Age BMI e < Control group (n ¼ 40) FF leptin Age BMI 9.93 < S f visfatin Age FF visfatin Age BMI Note: B ¼ Linear regression b coefficient; CI ¼ confidence interval; PCOS ¼ polycystic ovary syndrome; S ¼ serum; other abbreviations as in Table 1. a P values between.05 and.1 are indicative. The effect of age and BMI on S visfatin and FF visfatin in PCOS group were not statistically significant. Plati. Visfatin and leptin in PCOS IVF patients. Fertil Steril TABLE 3 Serum and FF leptin and visfatin, age, and BMI correlations. Variable r P value PCOS group (n ¼ 40) S leptin vs. FF leptin <.001 S leptin vs. BMI <.001 FF leptin vs. BMI <.001 FF visfatin vs. S visfatin <.001 Control group (n ¼ 40) S leptin vs. FF leptin <.001 S leptin vs. S Visfatin S leptin vs. FF visfatin S leptin vs. BMI FF leptin vs. FF visfatin FF leptin vs. BMI <.001 FF leptin vs. age S visfatin vs. FF visfatin <.001 FF visfatin vs. BMI Note: BMI ¼ body mass index; other abbreviations as in Tables 1 and 2. Plati. Visfatin and leptin in PCOS IVF patients. Fertil Steril In the PCOS group, serum visfatin was not significantly correlated with total T levels or with FAI. On the other hand, serum visfatin was significantly elevated in PCOS women with insulin resistance compared with those without insulin resistance (P¼.043). Follicular fluid visfatin was indicatively increased in women with insulin resistance (P¼.052). No significant correlations were observed between serum visfatin and serum insulin levels. Additionally, serum leptin was negatively correlated with E 2 levels (r ¼ 0.351; P¼.027), and no significant correlations were observed between serum leptin and total T, LH, and FAI. Finally, no significant differences were observed between serum and FF leptin levels in PCOS patients with or without clinical and/or biologic hyperandrogenism. DISCUSSION Recently it has been shown that a variety of factors are produced in the adipose tissue, thus contributing and modulating peripheral metabolic processes. Polycystic ovary syndrome is a complex disorder characterized by hyperandrogenism, insulin resistance, and an abnormal secretory pattern of GnRH resulting in a high LH/FSH ratio (6). There is considerable evidence about the role of leptin in the regulation of the hypothalamic-pituitary-gonadotropin axis by accelerating GnRH pulsatility via indirect mechanisms (6). Furthermore, it may stimulate LH and FSH release (18). To date, no definitive role for leptin in the pathophysiology of PCOS has been attributed, because serum leptin levels do not differ between PCOS and age- and weight-matched non-pcos subjects (19, 20), results which have been confirmed by the present study. Leptin may act as the critical link between adipose tissue and the reproductive system (6), and its expression is regulated by different hormones. It is already known that estrogens induce (21), whereas androgens suppress, leptin production (22), providing an explanation for the decreased levels we found in the FF of PCOS subjects. On the other hand, insulin increases leptin production (23), contributing to the hyperleptinemia that accompanies the insulin resistance state, as in PCOS (24). However, the present data contradict other studies (21, 25, 26), where, in women with PCOS, serum leptin was negatively correlated with E 2 levels, and no significant correlations were observed between serum leptin and LH, total T, and FAI, as previously reported (25). Given the pulsatile way of leptin secretion, an alteration of its normal pattern might be expected in PCOS women (27). There is evidence that leptin is produced directly in the ovary, because bilateral ovariectomy induced decreased serum leptin levels (28). In contrast, similar serum and FF leptin levels suggest that an autocrine secretion by the ovary itself is unlikely (20, 29). Abbas et al. (30) found no leptin in the supernatants of cumulus cells cultures or in the cell lysates, concluding that leptin may not be produced in significant levels by cumulus cells and is not related to oocyte competency. In the literature, data regarding FF leptin levels are contradictory. There are studies reporting a beneficial effect of higher FF leptin levels on the oocyte maturation rate (31) and fertilization rate (7). In contrast, lower FF leptin levels were associated with successful outcome in IVF (32 34), or high serum and FF leptin levels may account for decreased fertilization and pregnancy rates in PCOS 1454 Plati et al. Visfatin and leptin in PCOS IVF patients Vol. 94, No. 4, September 2010

5 women (35). The present results are in agreement with these observations, because we detected significantly decreased FF leptin levels in PCOS patients together with an increased, but not statistically significant, pregnancy rate in these patients. Interestingly, Hiromitsu et al. (19) suggested that high FF leptin levels do not contribute to the pathogenesis of PCOS but characterize an unbalanced state of serum and FF levels in PCOS, concluding that PCOS ovarian granulosa cells may produce less leptin than normal or that the leptin transport capacity may be decreased in women with PCOS. It has long been recognized that women with PCOS display a greater frequency of both hyperinsulinemia and insulin resistance than normally ovulating patients, presenting a defect in insulin-stimulated glucose utilization in peripheral tissues (36). Visfatin, a recently recognized adipocytokine, exerts insulin-mimetic effects by binding to and activating the insulin receptor in a distinct manner from insulin (9). However, the available information about visfatin is limited, and the present study is the first to detect visfatin levels in the FF of patients undergoing IVF. Women with PCOS have an android fat distribution pattern which may result in changed adipose tissue function and adipocytokine levels (37). Interestingly, it has been shown that visfatin is released from fat cells during lipolysis rather than being secreted (38) and that norepinephrine is able to stimulate lipolysis in visceral fat cells to a higher extent in PCOS compared with non-pcos patients (39). Furthermore, in PCOS subjects, the lipolytic effect is augmented because of a selective increase in the function of protein kinase A hormone-sensitive lipase complex in visceral fat cells (39). In the present study, serum visfatin levels were significantly elevated in women with PCOS, probably as result of increased lipolysis, contributing to the general state of insulin resistance in this category of patients. No significant correlations were observed between serum visfatin and serum insulin levels, although serum visfatin levels were significantly elevated in PCOS women with insulin resistance compared with those without insulin resistance. This observation is in agreement with previously published studies regarding PCOS patients (40) as well as other insulin resistant conditions, such as type 2 diabetes (11) or morbid obesity (10). Moreover, FF visfatin levels were only indicatively increased in the present PCOS subjects with insulin resistance. On the other hand, in contrast to the study performed on women with unstimulated ovaries by Kowalska et al. (40), in the present PCOS group serum visfatin was not significantly correlated with total T levels or FAI. The explanation for a possible association between visfatin and markers of hyperandrogenism is at present unknown. We can only speculate a probable influence of the IVF treatment on our subjects ovaries, because women enrolled in the present study were undergoing controlled ovarian stimulation, where the ovaries have to support primarily the estrogen production. Nevertheless, the observation that FF visfatin levels did not differ between PCOS and normally ovulating women under IVF treatment, and because these levels were lower than serum visfatin levels, any direct involvement of this molecule in the follicle probably cannot be supported. The exact relationship between visfatin levels, age, and BMI needs further clarification, because controversies arise from different studies with statements in favor (41) and against (11). Both of the present study groups comprised lean women which, according to a previously published study (40), constitutes an important parameter when attempting to understand the metabolic role of visfatin in vivo. In their study, Chan et al. (42) reported that plasma visfatin levels were significantly correlated with BMI in simple regression analysis but not in multiple regression analysis. Furthermore, age was significantly and negatively associated with plasma visfatin levels (42). In unstimulated cycles, patients with PCOS had higher plasma visfatin levels compared with controls, and these levels were positively correlated with BMI but not with age (43). 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