Are circulating leptin and luteinizing hormone synchronized in patients with polycystic ovary syndrome?

T. Sir-Petermann1,5, V. Piwonka1, F. Pérez2, M. Maliqueo1, S.E. Recabarren3 and L. Wildt4

1 Division of Endocrinology, Department of Internal Medicine, School of Medicine, University of Chile, 2 INTA, University of Chile, Santiago, 3 Laboratory of Animal Physiology and Endocrinology, School of Veterinary Medicine, University of Concepción, Chillán, Chile and 4 Division of Gynecological Endocrinology and Reproductive Medicine, Department of Obstetrics and Gynaecology, University of Erlangen, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal and human studies suggest that leptin modulates hypothalamic–pituitary–gonadal axis functions. Leptin may stimulate gonadotrophin-releasing hormone (GnRH) release from the hypothalamus and luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from the pituitary. A synchronicity of LH and leptin pulses has been described in healthy women, suggesting that leptin probably also regulates the episodic secretion of LH. In some pathological conditions, such as polycystic ovarian syndrome (PCOS), LH–leptin interactions are not known. The aim of the present investigation was to assess the episodic fluctuations of circulating LH and leptin in PCOS patients compared to regularly menstruating women. Six PCOS patients and six normal cycling (NC) women of similar age and body mass index (BMI) were studied. To assess episodic hormone secretion, blood samples were collected at 10-min intervals for 6 h. LH and leptin concentrations were measured in all samples. For pulse analysis the cluster algorithm was used. To detect an interaction between LH and leptin pulses, an analysis of copulsatility was employed. LH concentrations were significantly higher in the PCOS group in comparison to NC women, however serum leptin concentrations and leptin pulse characteristics for PCOS patients did not differ from NC women. A strong synchronicity between LH and leptin pulses was observed in NC women; 11 coincident leptin pulses were counted with a phase shift of 0 min (P = 0.027), 18 pulses with a phase shift of –1 (P = 0.025) and 24 pulses with a phase shift of –2 (P = 0.028). PCOS patients also exhibited a synchronicity between LH and leptin pulses but weaker (only 20 of 39 pulses) and with a phase shift greater than in normal women, leptin pulses preceding LH pulses by 20 min (P = 0.0163). These results demonstrate that circulating leptin and LH are synchronized in normal women and patients with PCOS. The real significance of the apparent copulsatility between LH and leptin must be elucidated, as well as the mechanisms that account for the ultradian leptin release.

Key words: leptin/LH/polycystic ovarian syndrome/pulsatility/synchronicity


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent data in the mouse demonstrate that leptin, a protein hormone, encoded by the ob gene, which is expressed in the adipocytes (Zhang et al., 1994Go), is required for fertility (Barash et al., 1996Go). In the absence of leptin the mice become obese, diabetic and infertile; the administration of leptin reverses these defects (Ahima et al., 1996Go; Chehab et al., 1996Go).

In humans, there is also evidence that leptin plays an important role in reproduction (Rosenbaum and Leibel, 1998Go). If leptin is the signal for adequate fat stores to start and maintain ovulation and menstruation, it could account for these changes through its effect on the ovary (Cioffi et al., 1997Go; Karlsson et al., 1997Go; Spicer and Francisco, 1997Go) or on the brain (Stephens et al., 1995Go; Yu et al., 1997aGo). At the level of the central nervous system, leptin may stimulate gonadotrophin-releasing hormone (GnRH) release from the hypothalamus, and luteinizing hormone (LH) and follicle stimulating hormone (FSH) release from the pituitary, probably by acting on its own receptor and promoting nitric oxide release (Yu et al., 1997bGo).

Recently, Licinio et al. demonstrated a synchronicity of LH and leptin pulses in the mid-to-late follicular phase of the menstrual cycle of healthy women (Licinio et al., 1998Go), suggesting that leptin may regulate the minute-to-minute oscillations in plasma levels of LH.

Polycystic ovarian syndrome (PCOS) is a common endocrine disorder affecting women of reproductive age. It is characterized by hyperandrogenaemia, chronic anovulation, increased LH concentrations and high incidence of obesity and insulin resistance (Dunaif et al., 1989Go; Franks, 1995Go; Poretsky and Piper, 1994Go). Thus, PCOS may serve as a useful model to study LH–leptin interactions in pathological conditions in humans. The aim of the present study was to assess the episodic fluctuations of circulating LH and leptin in PCOS patients compared to regularly menstruating women.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Six women with PCOS were selected for the study from patients attending the Unit of Endocrinology, Department of Medicine, University of Chile. Inclusion criteria were: chronic oligo- or amenorrhoea, hirsutism, plasma testosterone concentration >0.6 ng/ml or free androgen index (FAI) >5.0 and characteristic ovarian morphology on ultrasound based on the criteria described by Adams et al. (1986). All women were amenorrhoeic and anovulatory according to progesterone measurements and ultrasound examinations. Hyperprolactinaemia, androgen secreting neoplasm, Cushing's syndrome and attenuated 21-hydroxylase deficiency as well as thyroid disease were excluded by appropriate tests.

In addition, six normal cycling (NC) women of similar body mass index (BMI) acted as the control group. None of these women had taken oral contraceptives or other medication for at least 6 months before starting the study. Prior to the study, informed consent was obtained from all subjects. This study was approved by the local ethical committee.

Study protocol
The study was performed in the University Clinical Research Centre beginning at 0900 h after a 10-h overnight rest and fast.

NC women were studied in the early follicular phase of the menstrual cycle (day 3–7). In the amenorrhoeic patients, the study began whenever feasible.

For the study of episodic hormone secretion, blood samples were collected at 10 min intervals for 6 h, using a sampling device that allowed the continuous withdrawal of blood through a heparinized catheter (Sir-Petermann et al., 1995Go). Serum LH and leptin were determined in all samples. Serum total testosterone, oestradiol and sex hormone binding globulin (SHBG) were determined in samples 1, 19 and 37. The free androgen index [FAI = testosteronex100/SHBG (nmol/l)] was calculated, as the quotient of the molar concentrations of testosterone and SHBG. Serum insulin was measured in the first fasting sample. The mean plasma leptin half-life is known to be 24.9 ± 4.4 min (Klein et al., 1996Go).

Hormone assays
Serum LH was determined by electrochemiluminescense (Boehringer Mannheim, Mannheim, Germany; range: 0.1–200 IU/l), total serum leptin was measured by radioimmunoassay (Linco-Research Inc., St. Louis, MO, USA). The intra- and interassay coefficients of variation respectively were 1.1 and 2.1% for LH; 2.5 and 3.6% for leptin. Testosterone, SHBG, oestradiol and insulin were measured by radioimmunoassay using commercial kits (DPC, Los Angeles, CA, USA). The intra- and inter-assay coefficients of variation respectively were 7.0 and 11% for testosterone; 3.8 and 7.9% for SHBG; 2.7 and 8% for oestradiol and 5 and 8% for insulin.

Pulse analysis and statistical evaluation
According to Santen and Bardin, a pulse is defined as a sharp increase in hormone concentration from nadir to peak (>20%) with a progressive decline of at least two consecutive measurements of the hormone (Santen and Bardin, 1973Go). Serum leptin pulses were identified by the Veldhuis and Johnson modification of the method described by Santen and Bardin (Veldhuis and Johnson, 1986Go). A significant pulse required a nadir to peak difference >3 times the intra-assay coefficient of variation (CV) at the value of the nadir. This criterion takes into account variations of CV with hormone level and reduces the likelihood of false positive pulse detection.

For pulse analysis, the computerized version of the cluster pulse algorithm developed by Veldhuis and Johnson (1986) was used. We selected a cluster configuration of 1x2 (one sample for the test peak and two for the test nadir), and a t-value of 2.1/2.1 to constrain the likelihood of false positive pulse determination to <5%. The following mean properties of pulsatile hormone concentrations were analysed: pulse frequency (number of significant peaks/6 h), interpeak interval in min, pulse height, pulse area and pulse amplitude.

For the analysis of copulsatility, the ANCOPULS program developed by Albers was used (Albers et al., 1993Go). Additionally, the Pearson–Clopper values (Pearson and Clopper, 1934Go) were also calculated for the two combinations of hormone series to further validate the true values of P, as indicated in the report of Albers et al. (1993), computing the coincidence of peaks for all possible combinations of time window widths (0–10 samples equalling 0–100 min) and phase shifts (±4 samples equalling 40 min) and performing coincidence analyses on all possible combinations.

Comparisons between the groups were performed using the Mann–Whitney test. The significance level was set at 5%. Results are expressed as means and ranges.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table IGo shows the clinical and endocrine characteristics for PCOS women compared to NC women.


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Table I. Clinical and endocrine characteristics for normal cycling (NC) and polycystic ovary syndrome (PCOS) women
 
The mean age of the two groups was not significantly different. The BMI was similar in both groups. As expected, testosterone concentrations and FAI were significantly higher and SHBG was significantly lower in PCOS women compared to NC women. Oestradiol and insulin concentrations were not significantly different between the two groups.

Pulse analysis
Table IIGo shows the LH and leptin pulse characteristics in NC and PCOS women. In both groups, LH and leptin pulse frequencies were similar and they did not differ between groups. LH secretion, expressed as the transverse mean of LH concentrations and LH pulse amplitude, were significantly higher in the PCOS group in comparison to NC women, however serum leptin levels for PCOS patients did not differ from NC women. Moreover an apparent coincidence between LH and leptin pulses was observed in NC and PCOS women, a representative case for each group being shown in Figure 1Go.


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Table II. Luteinizing hormone (LH), leptin concentration and pulse characteristics in normal cycling (NC) and polycystic ovary syndrome (PCOS) women
 


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Figure 1. Representative pulsatile pattern of luteinizing hormone (LH) and leptin in one normal cycling woman (A), and one PCOS patient (B), during 6 h. Arrows indicate pulses identified by cluster analysis.

 
Coincidence of pulses
All combinations of time window widths and phase shifts analysed for LH–leptin interaction in NC and PCOS women are shown in Table IIIGo. For each set of parameters, the probability of event by chance was calculated. In NC women, for the 36 LH pulses found, 11 coincident leptin pulses were counted with a phase shift = 0. The probability of finding this high rate of coincident pulses by chance was P(x) = 0.027. A highly significant interaction between LH and leptin pulses can be assumed. For 11 coincidences, the Pearson–Clopper values of the confidence interval confirmed the rejection of the null hypothesis. For the 36 LH pulses found, 18 coincident leptin pulses were counted with a phase shift = –1. P(x) was 0.026. For these 18 coincidences, the Pearson–Clopper values of the confidence interval confirmed the P value. Finally, 24 coincident leptin pulses with a phase shift = –2 were counted. P(x) was 0.028. The Pearson–Clopper values of the confidence interval confirmed the rejection of the null hypothesis again. In summary, of the 36 LH pulses found in NC women, 54 coincident leptin pulses were counted with a phase shift = 0 to –2, corresponding to a time window of 0–30 min.


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Table III. Probabilities of coincident pulsatility for LH and leptin in normal cycling (NC) and polycystic ovary syndrome (PCOS) women
 
In PCOS patients, for 39 LH pulses found, 20 coincident leptin pulses were counted with a phase shift = –2. The probability of finding this high rate of coincident pulses by chance was P(x) = 0.016. Thus, the null hypothesis was rejected, assuming a highly significant interaction between LH and leptin pulses also in this group. For 20 coincidences, the Pearson–Clopper values of the confidence interval confirmed the rejection of the null hypothesis again. In summary, of the 39 LH pulses found in the PCOS group, only 20 coincident leptin pulses were counted with a time shift = –2, corresponding to a time window of 20 min.

Hormone concentrations
Statistical analysis demonstrated a normal distribution for the leptin data. PCOS women showed leptin concentrations comparable to NC women with a similar BMI. Therefore, a positive linear correlation was obtained between serum leptin concentrations and BMI in both groups (Figure 2Go).



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Figure 2. Correlation between body mass index (BMI) and leptin concentrations in normally cycling (NC) (A) and polycystic ovarian syndrome (PCOS) women (B).

 
In a simple linear regression analysis, serum leptin was also positively correlated with body weight (r = 0.833, P < 0.001) and serum insulin (r = 0.749, P < 0.005). Serum leptin was inversely correlated with serum SHBG concentrations (r = –0.645, P < 0.024). When PCOS and NC women were analysed separately, the correlations for serum leptin with body weight and insulin were significant in both groups. There were no significant correlations between leptin and FAI (r = 0.234) and between leptin and oestradiol (r = 0.299) or testosterone (r = –0.266) concentrations.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we evaluated the episodic fluctuations of circulating LH and leptin in patients with PCOS compared to NC women studied in the early follicular phase of the menstrual cycle (day 3–7). Circulating leptin levels in patients with PCOS did not differ from those in age and weight matched controls. Moreover, the pulsatile patterns of leptin in hyperandrogenaemic and normal cycling women were similar and resemble the pulsatile pattern of LH.

There are no previous studies dealing with the pulsatile release of leptin in PCOS women. In healthy women, Licinio et al. demonstrated a synchronicity of LH and leptin pulses in the mid-to-late follicular phase of the menstrual cycle, with a stronger pattern of coupling at night than during daytime (Lucinio et al., 1998). In this study, a strong synchronicity between LH and leptin pulses was also observed in NC women during the early follicular phase of the menstrual cycle. PCOS patients also exhibited a synchronicity between LH and leptin pulses. However, this synchronicity was weaker and had a phase shift or lag greater than in normal women. This could be explained by a greater LH pulse area in PCOS compared to normal women, which will increase the time lag between both hormones. There are several ways to explain the phenomenon of coupling of LH and leptin release. The first is that LH regulates leptin secretion. The second is that leptin regulates LH secretion and the third is that both hormones are driven by a common oscillator whose nature and location are not currently known. For most hormones, blood sampling at 10-min intervals enables the major secretory episodes to be detected as has been shown in this study. The pattern of pituitary hormone secretion may be viewed as being the consequence of the activity of an oscillator modulated by internal or external factors, as could account for the modulatory effect of leptin on GnRH-LH secretion, as has been demonstrated in experimental studies (Yu et al., 1997aGo). In this respect, it appears to be more likely that leptin regulates GnRH-LH secretion than vice versa. This assumption is based on a study in girls with precocious puberty treated with GnRH analogues in which leptin pulsatility persists despite the inhibition of the gonadal axis (Palmert et al., 1998Go), as well as on the observation that GnRH administration to these two groups of women in the present study was able to stimulate LH release without an increase in leptin release (data not shown), and finally on the dynamics of hormone secretion. Leptin concentrations increased in blood before or concomitant with, but seldom after, LH peaks.

In regard to mean hormone levels, the results of this study confirm those of recent investigations (Chapman et al., 1997Go; Gennarelli et al 1998Go), which reported no differences in serum leptin concentrations between women with PCOS and their controls by measuring single samples. As observed in these studies, BMI exhibited a strong correlation with leptin, suggesting that BMI, as an indirect reflection of body fat mass, is the main predictor of elevated leptin concentrations in these patients. Gender differences in leptin concentrations also suggest that sex steroids could be involved in the control of leptin production (Rosenbaum et al., 1996Go; Saad et al., 1997Go); however, in this study, hyperandrogenaemic patients showed plasma leptin levels similar to normal women, which suggests that leptin levels seem not to be affected by hyperandrogenaemia, thus supporting previous observations (Chapman et al., 1997Go; Laughlin et al., 1997Go; Mantzoros et al., 1997Go; Rouru et al., 1997). During the normal menstrual cycle, changes in circulating leptin levels were associated with changes in oestradiol and progesterone (Hardie et al., 1997Go; Messinis et al., 1998Go). This study was performed at the early follicular phase of the menstrual cycle in the NC women when the levels of oestradiol were low and comparable to those presented by the anovulatory PCOS women. Therefore, oestradiol levels are probably not involved in the lack of differences in leptin levels between both study groups.

In the present study, a clear association between fasting insulin concentrations and serum leptin was found in both groups of women, suggesting that insulin could also be involved in the regulation of leptin concentrations, as has been suggested (Laughlin et al., 1997Go). All of these observations are in agreement with those of Geisthövel et al. who concluded that hyperandrogenaemia does not have predictive value for leptin concentrations in premenopausal subjects (Geisthövel et al., 1998Go), but hyperinsulinaemia exerts an effect independent of obesity, which is the strongest predictor for elevation of leptin concentrations.

In summary, this study demonstrates that for a given BMI, leptin concentration is not different in normal compared to PCOS women, that there are no differences between normal and PCOS women in the pulsatile characteristics of circulating leptin, and finally that circulating leptin and LH are synchronized in patients with PCOS, but less strongly and with a more pronounced phase shift than in normal women. The real significance of the apparent copulsatility between LH and leptin must be elucidated, as well as the mechanisms that account for the ultradian leptin release.


    Acknowledgments
 
The authors thank Dr N.Albers from the University of Hannover for providing us the Ancopuls program and Dr S.Hass and Dr M.Meyer from the University of Erlangen for their advice with the statistical analysis. This work was supported by Fondecyt 1970291 grant and Alexander von Humboldt Foundation.


    Notes
 
5 To whom correspondence should be addressed at: Las Palmeras 299, Interior Quinta Normal, Casilla 33052, Correo 33, Santiago, Chile Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on August 17, 1998; accepted on February 15, 1999.