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Original Studies |
The Family Federation of Finland (T.L.B., J.M.M., O.H., R.S., D.A), FIN-00100 Helsinki, Finland; the Department of Endocrinology, Lund University (T.T.), Lund, Sweden; and the Department of Medicine, Divisions of Internal Medicine (M.L., T.T.) and Obstetrics and Gynecology (C.-G.N.), Helsinki University Central Hospital, Helsinki, Finland
Address all correspondence and requests for reprints to: Tarja L. Bützow, M.D., The Family Federation of Finland, Kalevankatu 16 A, FIN-00100 Helsinki, Finland. E-mail: tarja.butzow{at}vaestoliitto.fi
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Abstract |
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Our data suggest that leptin production may be influenced by the ovarian functional state. During IVF a high relative leptin increase is associated with adiposity and a reduced ovarian response. These observations support the possibility that high leptin concentrations might reduce ovarian responsiveness to gonadotropins. Hence, leptin might explain in part why obese individuals require higher amounts of gonadotropins than lean subjects to achieve ovarian hyperstimulation.
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Introduction |
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As first reported in the 1950s, much evidence has accumulated showing that female reproductive functions in humans are disrupted when severe changes in nutritional status and energy availability take place in both over- and undernutrition. These adaptive changes are reversible when nutritional status is normalized (10, 11, 12). Leptin and leptin receptors are found in reproductive tissues (13, 14, 15, 16). In rodents, leptin action takes place via hypothalamic neuroendocrine mechanisms known to participate in the regulation of reproductive functions (17, 18, 19). Hence, leptin is considered a possible link between nutrition and reproduction. In humans, genetic leptin deficiency is associated with genital infantilism (20). Independent of adiposity, obese females have higher ob messenger ribonucleic acid (mRNA) and serum leptin levels than obese males. Serum leptin levels rise 3.4-fold more rapidly as a function of body mass index (BMI) in women than in men (3). Leptin receptors and leptin mRNA have been identified in the human hypothalamus and ovary, and leptin mRNA and protein production have been discovered in ovarian granulosa cells, oocytes, and early clea-vage stage embryos (13, 14, 15, 16, 19, 21).
Despite the fact that leptin is widely present in reproductive tissues, its relationship to reproductive hormones is still poorly understood. Controversial results have been reported during hormone replacement therapy, oral contraceptive intake, ovulatory disorders, the normal menstrual cycle, and ovulation induction. Studies on estrogen replacement therapy have failed to demonstrate an association between estrogen intake and leptin levels (22, 23). Studies on polycystic ovarian disease patients and patients suffering from anorexia have associated leptin concentrations with the functionality of adipocytes, nutritional status, and integrity of the reproductive axis, but not with ovarian steroids (9, 24, 25, 26). In vitro, however, leptin counteracts the insulin-like growth factor I-induced cogonadotropic augmentation of FSH-stimulated 17ß-estradiol production in human granulosa cells (27). Fluctuations in leptin concentrations have been found during the menstrual cycle and ovulation induction (28, 29). We, therefore, undertook the present study to determine whether leptin concentrations are influenced by ovarian hyperstimulation in in vitro fertilization (IVF) and, if so, what might be the relationship among leptin, adipose tissue, and the ovarian response.
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Subjects and Methods |
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Fifty-two women (mean ± SE age, 34 ± 0.6; range, 24–40 yr) participating in an IVF program entered the study after giving informed consent. The protocol was approved by the ethics committee of the Family Federation of Finland. All subjects underwent an examination that included measurement of height, weight, and waist to hip ratio (WHR), which was calculated as the ratio of waist to hip in centimeters. BMI was calculated as weight in kilograms divided by the square of height in meters. Fat mass, fat-free mass, and percent body fat mass (BFM%) were measured using a bioelectrical impedance method (30). All subjects underwent standard long protocol IVF treatment starting with intranasal GnRH analog treatment during the midluteal phase of the cycle before ovarian hyperstimulation using nafarelin at 800 µg/day (Synarela, G.D. Searle & Co., Morbeth, UK) or buserelin at 1200 µg/day (Suprecur Hoechst, Frankfurt am Main, Germany) for 2–3 weeks. When ovarian hyperstimulation was begun, using individualized daily doses (150–450 IU) of highly purified gonadotropin (Fertinorm-HP, Serono, Aubonne, Switzerland), the GnRH analog dosage was reduced by half. To overcome the individual FSH threshold for follicle development and effectively recruit the available follicle cohorts, a starting dose of 150 IU FSH was used for patients under 38 yr or with BMI below 27 (kg/m2) if there was no previous knowledge of a poor response or endometriosis. A starting dose of 225 IU FSH was given for patients over 38 yr or with BMI above 27 or a previous history of endometriosis. Patients with previous IVFs and poor response were given a starting dose based on their previous IVF treatment responses. The daily dose was increased by 75 IU if follicular recruitment had not started in 6 days (follicles smaller than 10 mm or estradiol concentrations <0.3 nmol/L). Early morning (0800–1000 h) fasting serum samples were taken as follows: sample 1 at the time of ovarian suppression before gonadotropin administration, sample 2 at maximal ovarian hyperstimulation before the administration of 5000 IU hCG (Profasi, Serono, Aubonne, Switzerland), sample 3 at oocyte retrieval, and sample 4 16 days later after embryo transfer at the time of s-hCG test. All subjects used 200 mg progesterone (Lugesteron, Leiras, Turku, Finland) three times per day intravaginally until sample 4 and s-hCG was taken. Follicular fluid was obtained at oocyte retrieval from two representative preovulatory follicles in both ovaries. To avoid blood contamination follicular fluid was collected with a special procedure. As the needle entered the follicle, the first 1–2 mL were aspirated separately (tube 1). In the middle of the suction, a yellow, clear follicular fluid sample was taken (tube 2), and the last 1–2 mL were aspirated again separately. Only tube 2 was used in the study. The fluid was then pooled and centrifuged for 10 min at 920 x g, and the supernatant was stored at -20 C for later analysis. Follicular fluid leptin concentrations were corrected for the amount of phosphate-buffered saline used to flush the collection tubes at oocyte retrieval. Hormone concentrations were measured in all samples, except for sex hormone-binding globulin (SHBG), which was measured in sample 1 only.
Assays
Serum and follicular fluid leptin concentrations were measured using a commercial RIA (Linco Research, Inc., St. Charles, MO) with a sensitivity of 0.4 ng/mL, an intraassay coefficient of variation (CV) of 2.7–9.9%, and an interassay CV of 9.1–7.4% at levels of 4–20 ng/mL, respectively. For healthy Finnish female controls, the mean serum leptin concentration (±SD) was 13.4 ± 7.1 ng/mL (range, 2.8–32.0; n = 32; age, 20–40 yr; BMI, 23.3 ± 3.4 kg/m2; BFM%, 29.3 ± 6.3%). Fasting insulin was determined using a double antibody RIA (Phadeseph Insulin RIA, Pharmacia AB, Uppsala, Sweden) with a sensitivity of 2 mU/L, an intraassay CV less than 3%, and an interassay CV less than 10%. Well established immunofluorometric assays (Delfia, Wallac, Inc., Oy, Finland) were used for estradiol, LH, FSH, SHBG, and progesterone measurements, with sensitivities of 0.15 nmol/L, 0.3 IU/L, 0.5 IU/L, 6.0 nmol/L, and 1.0 nmol/L, and intra- and interassay CVs between 3–5% and 4–7%, respectively.
Statistical analyses
Statistical analyses were performed using a BMDP statistical software package (BMDP Statistical Software, Los Angeles, CA). Non-Gaussian-distributed variables were logarithmically transformed before analysis. The concentrations of leptin, estradiol, FSH, LH, progesterone, and insulin at different time points were compared using one-way ANOVA for repeated measures. Pairwise comparisons for variable values at different time points were conducted using Tukey’s Studentized range test. Relationships between variables were examined by calculating Pearson’s correlation coefficients, by conducting linear regression analyses and analysis of covariance. Data are expressed as the mean ± SEM. Significance was considered as P < 0.05.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The mean cumulative dose of FSH (cFSHd) administered was 31,814 ± 1,953 IU/treatment. There was a significant negative relationship between cFSHd and follicle number (r = -0.46; P < 0.001), but no significant relationship was found between cFSHd and any measure of adiposity (BMI, %BFM, or WHR), SHBG, insulin or leptin basal levels, or leptin response when analyzing all patients or leptin responders only.
A negative correlation between serum estradiol and FSH (r = -0.48; P < 0.001) and a positive correlation between estradiol and progesterone concentrations (r = 0.53; P < 0.001) were found at the time of oocyte retrieval (sample 3). Fasting insulin concentrations did not change significantly as a result of ovarian hyperstimulation. Mean insulin levels varied between 6.4 ± 2.3 and 7.3 ± 4.0 mU/L (P = NS, comparison including all time points). Throughout the study, fasting insulin concentrations were positively correlated with leptin concentrations (sample 1: r = 0.64; P < 0.001; sample 2: r = 0.43; P < 0.01; sample 3: r = 0.49; P < 0.001; sample 4: r = 0.4; P < 0.05). At the time of oocyte retrieval, there was a significant correlation between serum and follicular fluid insulin (r = 0.54; P < 0.001), serum insulin and follicular fluid leptin (r = 0.44; P < 0.01), and serum leptin and follicular fluid insulin (r = 0.50; P < 0.001) concentrations. Fasting SHBG levels at the time of ovarian suppression (65.9 ± 4.4 nmol/L) were negatively correlated to %BFM (r = -0.38; P < 0.05), leptin levels before (sample 1) and during (sample 2) ovarian hyperstimulation (r = -0.32; P < 0.05 and r = -0.4; P < 0.01, respectively), and follicular fluid leptin (r = -0.36; P < 0.05) and were positively correlated to estradiol increase (r = 0.4; P < 0.01).
The variability in serum leptin levels at all four time points was
significantly accounted for by the variability in body composition. In
separate linear regression analysis, BFM% contributed to 59–64%, BMI
to 46–56%, and weight to 46–55% (all P < 0.001) of
the variability in leptin concentrations at the four time points.
Positive correlations between leptin concentration change and BFM% and
fat mass (r = 0.55 and r = 0.58; both P <
0.0005) were observed in the 43 leptin responders when they were
analyzed separately from the 9 nonresponders (Fig. 2). The percent change in leptin
concentrations from the time of suppression (sample 1) to oocyte
retrieval (sample 3) was negatively related to the number of follicles
(b = -0.28; r2 = 8.1%; P <
0.05) and the number of oocytes retrieved (b = -0.39;
r2 = 15.2%; P < 0.01), demonstrating
a negative relationship between leptin increase and ovarian response. A
positive correlation of the percentage of leptin change with the
percentage of FSH increase (r = 0.39; P < 0.01)
between samples 1 and 3 was observed.
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The percent change in leptin concentrations between suppression and
maximal stimulation was negatively related to WHR (b = -0.31;
r2 = 9.3%; P < 0.05). There was no
significant correlation with the number of follicles and BMI, %BFM, or
basal leptin or insulin concentrations. When the numbers of follicles
and oocytes were used as dependent variables, negative relationships
were demonstrated with WHR (b = -0.29 and -0.31; both
P < 0.05, respectively). Clinical and anthropometric
characteristics, together with outcome data from the IVF cycles, are
summarized in Table 1. Nine subjects of
52 (17%) received a positive pregnancy test 16 days after embryo
transfer of 1–2 embryos.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Limited data exist regarding ovarian function and leptin. The results of some studies have suggested a positive relationship between estrogen increase and leptin (29, 31), but other reports have not (32). The possible stimulatory role of estradiol on leptin secretion is also suggested by studies on estrogen supplementation after ovariectomy in mice (33). Leptin levels increase during the luteal phase (29). This is in keeping with our results showing a significant correlation between leptin and progesterone at the time of maximal stimulation. In the present study no samples were taken during the luteal phase, as all subjects were receiving exogenous progesterone treatment for luteal support. A circadian variation in leptin levels has been demonstrated (34), and leptin secretion has been shown to be pulsatile. The leptin pulse pattern is synchronized with LH secretion (35, 36). The increase in serum leptin concentrations during IVF treatment is unlikely to be due to circadian variation, because all samples were collected at the same time of day. As the pulse amplitudes described for individuals of similar BMI (37) are far lower than the 60% increase observed in our study, pulsatility is an unlikely explanation. In our study the follicular fluid leptin concentrations at the time of oocyte retrieval were similar to the serum concentrations and highly related to adiposity. Adipocytes have been shown to be a major source of leptin in the body, but leptin synthesis has also been demonstrated in ovarian granulosa cells (16, 21). Although no definitive conclusions can be drawn, it seems unlikely that the leptin increase during IVF treatment would be predominantly ovarian in origin. On the contrary, the relative leptin increase was negatively correlated with the ovarian response as measured by the number of follicles and oocytes. This relationship was further supported by a positive relationship between the percent increases in leptin and FSH concentrations. Further, the percent leptin increase was positively associated with adiposity in 43 leptin responders. Our results demonstrate in addition that the negative relationship of leptin increase and ovarian response was found when cFSHd was used as a covariate. The number of follicles retrieved was not related to the measures of body composition, adiposity, or basal leptin or insulin concentrations. Therefore, a high leptin response rather than adiposity as such was significantly related to reduced ovarian responsiveness in this study. Taken together, our findings support the possibility that increased leptin production during ovarian hyperstimulation is related to adiposity and reduced ovarian responsiveness to FSH administration.
It is not clear why obese individuals require higher doses of gonadotropins for ovarian hyperstimulation despite comparable absorption of gonadotropins from sc tissue. Leptin has recently been shown to inhibit the synergistic action of insulin-like growth factor I and FSH on granulosa cell estradiol production (27). It is, therefore, possible that leptin could act in high concentrations as an inhibitory cogonadotropin in the ovary. The present clinical data support this possibility. We speculate that the inhibitory action of leptin in the ovary might partially explain why obese individuals require higher doses of gonadotropin for ovarian hyperstimulation.
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Acknowledgments |
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Footnotes |
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Received March 10, 1999.
Revised June 7, 1999.
Accepted June 17, 1999.
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