Circulating leptin levels during ovulation induction: relation to adiposity and ovarian morphology

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1 FERTILITY AND STERILITY VOL. 73, NO. 3, MARCH 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Circulating leptin levels during ovulation induction: relation to adiposity and ovarian morphology Steven R. Lindheim, M.D., Mark V. Sauer, M.D., Enrico Carmina, M.D., Peter L. Chang, M.D., Ralph Zimmerman, M.D., and Rogerio A. Lobo, M.D. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Columbia University, College of Physicians and Surgeons, New York, New York Received June 3, 1999; revised and accepted September 30, Presented at the 46th Annual Meeting of the Society for Gynecologic Investigation, Atlanta, Georgia, Reprint requests: Steven R. Lindheim, M.D., Columbia Presbyterian Medical Center, Department of Obstetrics and Gynecology, 622 West 168th Street, New York, New York (FAX: ) /00/$20.00 PII S (00) Objective: To assess serum leptin levels based on body habitus and ovarian morphology during controlled ovarian hyperstimulation. Design: Prospective analysis. Setting: University IVF program. Patient(s): Women undergoing IVF-ET were divided into two groups, obese ovulatory women (n 6; mean ( SD) body mass index, kg/m 2 ) and lean ovulatory women (n 20); mean ( SD) body mass index kg/m 2 ). Lean women were categorized further according to whether they had polycysticappearing ovaries (n 8) or normal-appearing ovaries (n 12). Intervention(s): Controlled ovarian hyperstimulation and IVF. Main Outcome Measure(s): Serum estradiol, testosterone, and leptin. Result(s): Mean ( SD) leptin levels were significantly higher before and after GnRH agonist downregulation in obese women ( pg/ml and pg/ml, respectively) compared with lean women ( pg/ml and pg/ml, respectively). Mean ( SD) leptin levels increased significantly in both groups ( pg/ml and pg/ml, respectively), and the mean ( SD) percentage increase was similar (55% 18% and 54.8% 17%, respectively). Mean ( SD) leptin levels were similar in women with polycystic-appearing and normal-appearing ovaries before controlled ovarian hyperstimulation, but increased significantly in women with polycystic-appearing ovaries afterward ( pg/ml and pg/ml, respectively). Conclusion(s): Significant increases in leptin levels occur during controlled ovarian hyperstimulation, suggesting that leptin plays a role in follicular growth and maturation. The exaggerated response in women with polycystic-appearing ovaries reflects either a greater number of recruited follicles or a predisposition of adipocytes to leptin production. (Fertil Steril 2000;73: by American Society for Reproductive Medicine.) Key Words: Leptin, body habitus, ovulation induction, ovarian morphology, polycystic-appearing ovaries Leptin is a hormonal product that is synthesized primarily in white adipose tissue (1). It plays a role in whole-body energy balance and body weight mediated through the sympathetic nervous system (1 3). Leptin also appears to be an important metabolic hormone, influencing processes such as insulin secretion and glucose utilization (4, 5). A role for leptin in reproduction also has been suggested (6, 7). In mice, the absence of leptin results in obesity and infertility. The administration of recombinant leptin to such mice restores fertility (7, 8). The presence of leptin and the expression of its receptors also have been identified in follicular fluid and granulosa cells (9, 10). Various hormonal factors appear to affect the production of leptin, including glucocorticoids and insulin (11 14), whereas leptin appears to inhibit in vitro insulin-like growth factor I mediated enhancement of FSH granulosa cell E 2 synthesis (15, 16). A direct relation between E 2 and leptin also has been suggested. Serum leptin levels have been reported to be higher in premenopausal women compared with postmenopausal women (17), and fluctuations during the menstrual 493

2 cycle have been described (18 21). These observations suggest a role for estrogen in the regulation of leptin levels in normal physiologic states. Recent studies also have investigated serum leptin levels in women with polycystic ovary syndrome (PCOS) (22, 25). The results have been contradictory, but most studies indicate that leptin levels in women with PCOS are similar to those in matched controls, although it has been suggested that elevated leptin levels may cause follicular arrest in certain women with PCOS (15). Polycystic-appearing ovaries (PAO) have been detected on ultrasound examination in 16% 25% of normal ovulatory women without androgen excess (26 30) and may represent an incompletely expressed form of PCOS (31). Although PCOS is a heterogeneous disorder and increased body weight accentuates the metabolic disturbances in PCOS, subtle endocrine abnormalities may be uncovered in ovulatory women with PAO, including exaggerated responses to exogenous gonadotropins during ovulation induction (32). Significant changes in leptin levels have been described during controlled ovarian hyperstimulation (COH) with hmg, suggesting that E 2 may be involved in the regulation of leptin production, or vice versa (18, 19). The present study was undertaken to assess the effect of folliculogenesis on leptin levels during COH according to body habitus, and to determine whether ovarian morphology has any relation to circulating leptin levels. MATERIALS AND METHODS Two groups of women were studied: ovulatory obese women (body mass index, 27 kg/m 2 ;n 6) and ovulatory lean women (n 20) who were undergoing COH and IVF-ET cycles. The ovulatory obese subjects were undergoing IVF-ET for unexplained infertility, whereas the lean subjects were volunteers enrolled in our ovum donation program. The ovulatory lean women were subdivided further into those who had PAO (defined as an ovarian volume of 9 mlwith 10 peripherally oriented follicles of 2 8 mm in diameter in one sonographic plane with a surrounding dense stroma; n 8) and those who had normal-appearing ovaries (NAO; defined as an ovarian volume of 6 ml; n 12) (33). All patients began COH after pituitary desensitization with leuprolide acetate (TAP Pharmaceuticals, Deerfield, IL) at a dosage of 1 mg/d. Controlled ovarian hyperstimulation was initiated once the serum E 2 level was 30 pg/ml using 150 IU/d of IM hmg (Humegon; Organon Inc., West Orange, NJ). At the initiation of treatment with menotropins, the dosage of leuprolide acetate was decreased to 0.5 mg/d until the day of hcg administration. Monitoring was accomplished with serial vaginal ultrasound examinations and serum E 2 level determinations. Dosing was individualized depending on the patient s response. When 3 follicles of 18 mm in diameter and a serum E 2 level of 200 pg/ml per follicle were noted, the patient was given 10,000 IU of IM hcg (Profasi; Serono Laboratories, Inc., Randolph, MA). Serum was obtained and assayed for E 2, testosterone, and leptin the day of and the day after GnRH agonist downregulation and COH on days 5, 7, 9, or the day of hcg administration. Oocyte retrieval was performed under transvaginal ultrasound guidance hours after hcg administration. Oocytes were cultured and embryos were transferred 72 hours later. Using commercially available RIA kits (Diagnostic Products Corporation, Los Angeles, CA), serum samples were assayed for E 2 and testosterone. Intraassay and interassay coefficients of variation were 5.6% and 7.4% for E 2,and 4.3% and 6.5% for testosterone, respectively. Leptin was measured using a commercially available immunoradiometric assay kit (DSL, Webster, TX). Intraassay and interassay coefficients of variation were 4.9% and 6.6%, respectively. Statistical analysis was performed using the Student s t-test, analysis of variance, and the Bonferroni post hoc t-test. Regression analysis was performed by the method of least squares to compare the diagnostic significance of each TABLE 1 Age and body mass index of ovulatory obese and lean women. Study group Parameter Obese (n 5) Lean (n 20) Lean with PAO* (n 12) Lean with NAO (n 8) Age (y) Body mass index (kg/m 2 ) Note: Values are means SEM. *Polycystic-appearing ovaries. Normal-appearing ovaries. P.05. Lindheim. Leptin and ovulation induction. Fertil Steril Lindheim et al. Leptin and ovulation induction Vol. 73, No. 3, March 2000

3 FIGURE 1 Baseline and peak serum E 2, testosterone, and leptin levels, and number of oocytes retrieved and fertilized in obese and lean subjects. *P.05 within groups;, P.05 from baseline. Please Supply Citation Line. Fertil Steril parameter. Statistical significance was defined as P.05. Results are expressed as means SEM. RESULTS Table 1 shows the mean ages and body mass indices of the obese and lean subjects. Basal hormone profiles were similar in the two groups, except for serum leptin levels, which were significantly higher in the obese subjects (P.05). There was no correlation between leptin levels and serum E 2 levels (P.20, not significant [NS]) or serum testosterone levels (r 0.04, NS). A positive correlation was found only between serum leptin levels and body mass indices (r 0.66, P.01). Estradiol was the only hormone that was significantly reduced from baseline levels after ovarian downregulation (P.05). Serum E 2 and testosterone levels increased significantly over time in both the obese and lean subjects after COH, peaking at the time of hcg administration. There were no differences between the obese and lean subjects in mean ( SD) E 2 levels (2, pg/ml and 2, pg/ml, respectively) or testosterone levels ( ng/ml and ng/ml, respectively; P NS). Mean ( SD) serum leptin levels were significantly higher before and after the GnRH agonist was administered to obese ovulatory subjects ( pg/ml and pg/ml, respectively) compared with lean subjects ( pg/ml and , respectively; P.05) and on all days after COH. Although serum leptin levels did not vary significantly over time, significant mean ( SD) peak values were noted in both the obese and lean subjects ( pg/ml and pg/ml, respectively; P.05). However, the magnitude of the increase was similar in the two groups (54.8% 17% and 56% 18%; P NS). Mean ( SD) peak leptin levels occurred significantly earlier during COH in the lean subjects compared with the obese subjects ( days and days; respectively P.05), although the mean ( SD) number of days of COH were similar in the two groups ( days and days, respectively; P NS). The mean ( SD number of oocytes that were obtained in the two groups ( and , respectively; P NS) and the mean ( SD) FERTILITY & STERILITY 495

4 FIGURE 2 Baseline and peak serum E 2, testosterone, and leptin levels, and number of oocytes retrieved and fertilized in subjects with PAO and NAO. *P.05 within groups; P.05 from baseline. Please Supply Citation Line. Fertil Steril number that were fertilized ( and , respectively; P NS) were similar. Figure 1 shows the peak serum E 2, testosterone, and leptin levels before GnRH agonist administration, after pituitary downregulation, and after COH, as well as the number of oocytes that were retrieved and fertilized. With respect to ovarian morphology, subjects with PAO had similar basal serum FSH, E 2, and leptin levels (Fig. 2). However, mean ( SD) testosterone levels were significantly higher in subjects with PAO than in those with NAO ( ng/ml and ng/ml, respectively; P.05). In response to COH, subjects with PAO had higher mean ( SD) peak serum E 2 levels (3, pg/ml and 1, pg/ml, respectively; P NS), testosterone levels ( and ng/ml, respectively; P.05), and leptin levels ( pg/ml and pg/ml, respectively; P.05). Both E 2 and leptin levels increased significantly from baseline in both groups, but testosterone levels increased significantly from baseline (P.05) only in the subjects with PAO. The mean ( SD) number of oocytes that were obtained at aspiration was significantly higher in the subjects with PAO than in those with NAO ( and , respectively, P.05). Although the mean ( SD) number of oocytes that were fertilized was significantly higher in subjects with PAO ( ) than in subjects with NAO ( , P.05), the mean ( SD) percentage of oocytes that were fertilized was similar (70% 1.6% and 71% 2.8%, respectively; P NS). The presence of PAO (n 2) or NAO (n 4) in the obese subjects was not correlated with any differences in leptin levels during COH, but the sample size was too small to allow any definitive conclusions. DISCUSSION There is increasing evidence to suggest that leptin has an effect on ovarian function. Recent data in healthy women showed higher leptin levels during the luteal phase of spontaneous menstrual cycles that correlated with LH levels (17 20). However, a relation between estrogen and leptin also has been described during the follicular phase of both spontaneous cycles and cycles stimulated with exogenous FSH (19, 20). This suggests that leptin either has a direct effect or is regulated by gonadal steroids in the human ovary. 496 Lindheim et al. Leptin and ovulation induction Vol. 73, No. 3, March 2000

5 In contrast to previous reports, our data show that leptin production is not affected by pituitary downregulation with a GnRH agonist, and that it increases with exogenous FSH to the same degree in obese and lean women. However, lean women show this increase in serum leptin levels almost 2 days earlier than obese women. Because leptin levels do not correlate with estrogen levels, there may be other hormonal factors that affect leptin production and account for the delayed increase in serum leptin levels. Nonetheless, leptin does not seem to have an enormous clinical impact, because higher basal circulating levels and differences in leptin production with COH do not result in differences in fertilization rates or pregnancy outcomes (data not shown). The response of leptin also was evaluated in relation to ovarian morphology in ovulatory women without PCOS. It has been speculated that leptin may have an undefined role in the abnormal ovarian function of women with PCOS (34). Women with PAO are thought to represent part of the spectrum of patients with PCOS, and up to 33% of normal ovulatory women with PAO may have increased androgen secretion and lower insulin-like growth factor binding protein-1 concentrations, similar to women with PCOS and unlike women with NAO (31). Our data show that women with PAO are more hyperandrogenic than women with NAO and that they have exaggerated responses to COH that include E 2 and testosterone levels. Our data also reveal greater leptin production in women with PAO than in women with NAO, although increases in serum leptin levels did not correlate with either increasing E 2 or testosterone levels. This may represent the greater cohort of follicles recruited during COH rather than a pathophysiologic state. Alternatively, it could indicate a predisposition toward the production of leptin by adipocytes in obese subjects. In summary, leptin levels correlate with adiposity but not with increases in other sex steroid levels during COH treatment. Changes in leptin levels throughout the menstrual cycle may represent a link between normal metabolism and reproduction independent of body habitus and ovarian morphology. Endocrine abnormalities that include leptin in both obese and ovulatory women with PAO provide further insight into PCOS and suggest a wide spectrum of clinical presentation of this disorder. References 1. Zhang YY, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372: Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995;269: Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, et al. Weight reducing effects of the plasma protein encoded by the obese gene. 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Leptin concentrations in the follicular phase of spontaneous cycles and cycles superovulated with follicle stimulating hormone

Human Reproduction vol.13 no.5 pp.1152 1156, 1998 Leptin concentrations in the follicular phase of spontaneous cycles and cycles superovulated with follicle stimulating hormone I.E.Messinis 1,4, S.Milingos