1 Departments of Obstetrics and Gynaecology and 2 Pharmacology, University of Thessalia, Larissa and 3 Department of Biological Chemistry, University of Ioannina, Ioannina, Greece
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Abstract |
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Key words: human/leptin/oestradiol/progesterone
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Introduction |
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Normal women have significantly higher serum leptin concentrations than normal men (Rosenbaum et al., 1996: Shimizu et al., 1997
). Also, in the luteal phase of the normal menstrual cyc1e, serum leptin concentrations are significantly higher than in the follicular phase (Hardie et al., 1997
; Mannucci et al., 1998
; Messinis et al., 1998
; Quinton et al., 1999
; Ludwig et al., 2000
), while significant positive correlations have been found between leptin and oestradiol or progesterone concentrations (Hardie et al., 1997
; Paolisso et al., 1998
; Messinis et al., 1999
). Although these studies suggest that ovarian steroids may play a role in leptin secretion in humans, no direct evidence has been provided as yet. Recently, a significant decrease in serum leptin concentrations was found following bilateral ovariectomy in normal women (Messinis et al., 1999
) and, although treatment with oestradiol was without any effect, the addition of progesterone prevented this decrease, suggesting that progesterone plays a role in the control of leptin secretion (Messinis et al., 2000
). A hypothesis was developed from these results that the increase in leptin concentrations during the second half of the menstrual cycle may be related to changes in the steroidal milieu during the periovulatory period and the luteal phase. The present study was undertaken to test this hypothesis further by examining the effect of treatment with oestradiol and progesterone on leptin concentrations in normal premenopausal women.
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Materials and methods |
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The women were investigated during the early follicular phase of three different cycles. The first two cycles were consecutive, while between the second and the third cycle there was a break of at least 1 month. However, due to inconvenience caused by the blood sampling, four of the 10 women withdrew from the study after they had completed the first and second cycles. During the first cycle (control), no treatment was given to the women (n = 10). In the second cycle (oestradiol cycle), the women (n = 10) were given oestradiol through skin patches (Estraderm TTS; Ciba-Geigy, Athens, Greece) at a dose of 100 µg/24 h on cycle day 2 (09:00 h) and 150 µg/24 h on days 3 and 4. All patches were removed on cycle day 6 (09:00 h). In the third cycle (oestradiol+progesterone cycle), the women (n = 6) received oestradiol as in the oestradiol cycle plus progesterone (Utrogestan caps; Faran, Athens, Greece) on cycle days 3, 4 and 5 at the daily dose of 400 mg i.v. (two doses of 200 mg, 12 h apart). The intention was to induce serum concentrations of oestradiol and progesterone approaching respectively those seen during the preovulatory period and the luteal phase of the normal menstrual cycle (Messinis and Templeton, 1988). Information regarding the doses of these two steroids was obtained from pilot experiments performed in two volunteers prior to the onset of the study. In each of the three cycles, blood samples were taken from days 2 to 10 (09:00 h) after an overnight fast. In the treated cycles, the samples were drawn each time before the application of treatment. Further blood samples were taken in the control cycles on day 21, and in the two treatment cycles on days 25 and 31. Concentrations of oestradiol, progesterone, leptin, FSH and LH were measured in all blood samples.
Hormone assays
Leptin was measured in all samples in duplicate using a radioimmunoassay kit (RIA; Linco Research, St Charles, MO, USA) which contained human leptin antibody prepared in rabbits and raised against highly purified human leptin, together with standards and tracer prepared with human leptin. Leptin concentrations were expressed as ng/ml, the lower limit of detection being 0.5 ng/ml. For the measurement of oestradiol, a microparticle enzyme immunoassay (MEIA) was used (AxSYM Estradiol; Abbott Laboratories, Abbott Park, IL, USA); the lower limit of detection for oestradiol was 73 pmol/l. Progesterone was measured in serum using a solid-phase, chemilluminescent enzyme immunoassay (Immulite progesterone; DPC, Los Angeles, CA, USA); the lower limit of detection for progesterone was 0.6 nmol/l. FSH and LH were measured in serum using MEIA (AxSYM FSH and AxSYM LH respectively; Abbott Laboratories); the lower limits of detection for FSH and LH were 0.37 IU/l and 0.5 IU/l respectively. The inter- and intra-assay coefficients of variation for leptin, oestradiol, progesterone, FSH and LH were 6.2 and 7.1%, 2.1 and 5.7%, 9.2 and 8.1%, 3.1 and 4.3%, and 2.4 and 4.2% respectively.
Statistical analysis
The results were analysed using a one-way analysis of variance (ANOVA) followed by Dunnet's post-hoc test. Statistical calculations for the hormone data values were carried out following log transformations, though the arithmetic means of values were presented. Correlations between various parameters were calculated using Pearson's product moment correlation coefficient analysis.
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Results |
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All women in the oestradiol and the oestradiol+progesterone cycles had a menstrual-like bleeding 23 days after the end of the treatment. The duration (mean ± SEM) of the oestradiol cycles (33.3 ± 1.0 days) and the oestradiol+progesterone cycles (36.3 ± 1.6 days) was significantly longer than the duration of the control cycles (28.5 ± 0.8 days) (P < 0.01). This was due to the delayed ovulation which was confirmed in all cycles by an increase in progesterone concentration (control cycles day 21, 44.3 ± 4.8 nmol/1; oestradiol cycles day 25, 47.9 ± 3.6 nmo1/1; oestradiol+progesterone cycles day 31, 40.7 ± 6.8 nmol/l).
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Discussion |
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Previous studies have shown that oestradiol can stimulate the secretion of leptin from fat tissue cultures in animals and women (Murakami et al., 1995; Casabiell et al., 1998
). A recent study has shown that serum leptin concentrations increased significantly in postmenopausal women after 2 months of treatment with oestradiol at a dose of 2 mg/day (Elbers et al., 1999
). That such an effect of oestradiol was not seen in the present study might be related to differences in the protocol used, in which treatment of the women with this steroid was for a much shorter period. Clearly, this finding does not exclude the possibility that oestradiol plays a role in the control of leptin secretion. It is possible that oestradiol exerted a priming effect on fat cells that subsequently responded to progesterone. The effect of combinations of oestradiol and progesterone on leptin concentrations has also been investigated in previous studies in which normal women were given either the oral contraceptive pill or hormone replacement therapy (HRT), but circulating concentrations of leptin were not affected (Kohrt et al., 1996
; Castracane et al., 1998
; Cella et al., 2000
). There are, however, differences between the present and the previous studies that are mainly related to the type and dosage of the steroids used. In particular, the `pill' contains synthetic steroids that may act differently from natural steroids, while in HRT formulas the daily dosages used for replacement create circulating concentrations of steroids that may be insufficient to stimulate the fat cells. In the current study, during treatment with oestradiol and progesterone, serum concentrations of oestradiol showed a pattern of increase similar to that seen during the preovulatory period of the normal menstrual cycle and, although progesterone concentrations showed an abrupt increase, the concentrations were similar to those seen in the luteal phase of the cycle (Messinis and Templeton, 1988
). It is possible, therefore, that in terms of a stimulating effect of oestradiol and progesterone on leptin secretion, doses of these steroids inducing a rapid increase in blood levelseven for a short period of timeare required rather than the prolonged administration of a low-dose regimen. Whether a threshold mechanism is involved remains to be clarified.
The possibility that in the current study oestradiol plus progesterone treatment stimulated leptin secretion through an effect on pituitary gonadotrophin secretion is not likely. Although in normal women the pulses of leptin are synchronous with the pulses of LH and oestradiol (Licinio et al., 1998), in the current study increments of LH and FSH, in the form of a surge, occurred only during treatment with oestradiol, i.e. when leptin concentrations remained unchanged. In addition, although LH and FSH concentrations declined up to day 5 in both treatment cycles, an increase in serum leptin concentration was seen only during the oestradiol plus progesterone treatment. Furthermore, no significant correlations were found between leptin and LH or FSH concentrations in either treatment group. It is evident, therefore, that the stimulating effect of oestradiol plus progesterone on leptin secretion was not mediated by FSH or LH. The current results are consistent with those of a recent study, according to which treatment with oestradiol plus progesterone prevented the decrease in leptin concentration that was induced by ovariectomy in women (Messinis et al., 2000
). From a physiological point of view, the present results support the hypothesis that the increasing concentrations of oestradiol and progesterone during the periovulatory period and the luteal phase of the menstrual cycle are probably responsible for the increased concentrations of leptin during the luteal phase. The fact that in the current study leptin concentrations increased significantly even after a relatively short rise of oestradiol and progesterone concentrations indicates that this process of leptin stimulation by these steroids is rather sensitive. A more sustained elevation of oestradiol and progesterone, therefore, as occurs during the luteal phase of the normal menstrual cycle, would be expected to result in a longer increase in leptin concentrations.
The importance of the increased serum leptin concentrations during the luteal phase of the cycle is unclear. It has been speculated that leptin at that stage may play a role in embryo implantation, possibly through a mechanism that affects the invasion phase (Gonzalez et al., 2000). Alternatively, increased leptin concentrations may help the body to meet the metabolic demands of a pregnancy (Messinis and Milingos, 1999
). In terms of the possible impact that a progesterone-induced increase in leptin concentrations might have in clinical practice, the use of micronized progesterone might be considered in HRT regimens, though this possibility needs to be investigated.
In conclusion, the current study shows for the first time that preovulatory concentrations of oestradiol induced in the early follicular phase of the cycle by the administration of this steroid to normal women did not affect leptin secretion. However, induction of luteal phase concentrations of progesterone, in addition to the increased concentrations of oestradiol, can stimulate leptin secretion. These findings may provide an explanation for the previously reported increased leptin concentrations that occur during the second half of the normal menstrual cycle.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 10, 2000; accepted on May 4, 2001.