THE INFLUENCE OF ESTRADIOLE AND TIBOLONE ADMINISTRATION ON LEPTIN LEVELS IN WOMEN WITH SURGICALLY INDUCED MENOPAUSE

Background: Several studies suggest that changes in estrogens and androgens during menopause play a role in the regulation of leptin production. Some authors present hypothesis that sex hormone replacement therapy can modulate leptin levels but up to date evidence shows that the infl uence of endogenous estrogens, androgens levels and sex hormone therapy on leptin concentration remains uncertain. Aim: To evaluate the infl uence of surgically induced menopause on serum leptin levels and the infl uence of diff erent types of hormonal therapy on serum leptin concentrations. Methods: 58 women with surgically induced menopause were divided into three groups. Women who did not receive any hormonal substitution (group 1), women who received Estradiol l mg per day (group 2) and women who received Tibolone 2,5 mg per day (group3). The levels of leptin, estradiol, testosterone, testosterone, dehydroepiandrosterone sulfate, FSH, LH and progesterone were measured in all subjects on the 5th day and after 3 months following the surgical procedure. Results: Mean serum leptin concentrations did not diff er statistically in any of the studied groups in the begining and in the end of the study. There was no correlations between serum leptin and estradiol, LH, FSH, progesterone, testosterone, free testosterone and DHEAS concentrations in any of groups before and after treatment. Conclusion: Changes in sex hormone concentrations caused by ovariectomy do not infl uence serum leptin concentrations. Also the short term administration of low dose estrogen therapy or tibolone in postmenopausal subjects does not change serum leptin levels.


INTRODUCTION
The loss of estrogens caused by menopause in women is associated with metabolical changes which cause changes in the amount and distribution of body fat 1 .Mechanisms of the infl uence of sex hormones on body fat and its distribution remain uncertain.Among the different possible mechanisms a link between sex hormones and leptin metabolism has been suggested [2][3][4][5][6][7][8][9][10] .Leptin, the adipocyte-specifi c product of the ob gene, plays an important role in food intake, fat metabolism, energy homeostasis and obesity.Leptin together with other hormones regulate eating behavior and body mass 11 .Leptin exerts its eff ects through interaction with six types of receptors.Leptin receptors are divided into secretory (ObRe), long (ObRb) and short forms (ObRa, ObRc, ObRd, ObRf).(ObRe) acts as a binding protein for leptin in the plasma in humans and mice and is important for leptin transfer into the brain.ObRb is the only receptor isoform that contains active intracellular signaling domains.This receptor is found in a number of hypothalamic nuclei where it exerts its eff ects.Action of Leptin on hypothalamic centers, decreases appetite and also probably controls the activity of the thyroid, adrenal, growth, gonadal and lactational axes.Serum leptin levels are strongly related to body fat mass and are regulated by insulin, cortisol, the adrenergic system and other hormones 12 .Several studies suggest that sex hormones, such as estrogens and androgens, may play an role in the regulation of leptin production 4,13,14 .The infl uence of endogenous estrogens, androgens levels and sex hormone replacement therapy on leptin secretion and serum leptin concentration remains uncertain.Some authors have shown that estrogens have eff ect on serum leptin levels, while others reported a lack of such infl uence 5,12,15,16 .The aim of the present study was to evaluate the infl uence of decrease of endogenous sex hormones during 3 months after surgically induced menopause on serum leptin levels and the infl uence of diff erent types of hormonal therapy (low dose estrogen therapy or tibolone administration) on serum leptin concentrations in postmenopausal subject.

Patients
The study group consisted of 57 women with surgically induced menopause.All women underwent surgical procedure -vaginal or abdominal hysterectomy with bilateral adnexectomy.The mean age of women which were taking part in the study was 47.3 years.The mean BMI of all subjects in the beginning of the study was 27.2.
Subjects were divided into three groups: 22 women did not receive any hormonal substitution (group 1), 25 women received hormonal therapy -Estradiol l mg dosis per day (group 2). 10 women received Tibolone 2.5 mg per day (group 3).Administration of medication in group 2 and 3 was started on the 5th day after surgery.All subjects gave their informed consent according to the Helsinki Declaration.

Blood collection and laboratory examination
The examination of biological material was made from venous serum samples.All parameters except leptin were determined within 120 minutes after collection.Venous blood was drawn from all individuals on the 5th day after surgery and after 3 months after surgery.For all subjects, medical data were obtained and a complete physical examination was performed.Weight and height values were recorded.The degree of obesity was described by the body mass index (BMI).Collected serum was assayed for leptin with a microplate enzyme immunoanalytical method (ELISA, BioVendor), estradiol (LEIA, SMSD), testosterone (LEIA, SMSD), free testosterone (ELISA, BioVendor), dehydroepiandrosterone sulfate (DHEAS, LEIA, SMSD), FSH (LEIA, SMSD), LH (LEIA, SMSD), progesterone (LEIA, SMSD).

Inclusion and exclusion criteria
Inclusion criteria -women who underwent surgical procedure -hysterectomy and bilateral adnexectomy for benign indication with regular menstruation cycle before surgery.
Exclusion criteria included elevation of CRP and diabetes treated with insulin on insulinotherapy.

Statistical data processing
Data were processed by means of the Medcalc software.Associated constants are expressed as mean ± standard deviation and median, unless indicated otherwise.The levels of Reg-Iα in the subgroups were compared by variance analysis (ANOVA, Kruskal-Wallis according to distribution type) and by means of ROC analysis.The leptin concentration as well as other quantities were mutually correlated using the Spearmann correlation coeffi cient.Category data were compared by the χ 2 test.Value of p < 0.05 was considered as statistically signifi cant.Normality was evaluated by the χ 2 test.

RESULTS
Mean serum leptin concentrations did not diff er statistically in any of the studied groups in the begining and also in the end of the study (Tables 1, 2, and 3), (Graph1).Statistical analysis showed no correlations between serum leptin and estradiol, LH, FSH, progesterone, testosterone, free testosterone and DHEAS concentrations in any of groups before and after treatment.

DISCUSSION
Leptin is involved in body-weight regulation and energy balance 11,17,18 .As an endocrine mediator, leptin also fulfi ls other tasks, all intreactions of this hormone are still not completely understood.Some studies have reported that estrogens can contribute in regulation of leptin production and its serum concentration 19 .Results of many authors support this theory, they have reported genderrelated diff erences in leptin concentration between men and women 20,21 .These diff erences may be partly explained by variability in the amounts of subcutaneous adipose tissue 22 .
Women in general have more body fat than men, and diff erent fat distribution.The serum leptin concentration is closely positively related to fat mass.Women have more subcutaneous fat than visceral fat, while the opposite condition is present in men.It has been shown that subcutaneous fat expresses more leptin mRNA than abdominal fat, this may partially explain the gender diff erences in leptin levels between the sexes 23 .Despite this evidence it has also been suggested that serum leptin concentrations may be related to diff erences in estrogen concentrations.Physiological sex hormones fl uctuations are present during menstruation cycle.Results published on serum leptin levels caused by hormonal changes during the physiological menstrual cycle vary considerably.Some studies showed signifi cant increase in the late follicular phase, and some did so on the day of the onset of the luteinising hormone (LH) 24,25 .Other investigations described signifi cant increase in serum leptin levels in the late luteal phase [26][27][28][29][30][31] .Other authors reported only small, not statistically signifi cant variations during the menstrual cycle [32][33][34] .Menopausal transition is marked by a fall in the level of estrogen and a rise in levels of serum follicle-stimulating hormone (FSH) and luteinizing hormone(LH).
The infl uence of menopause on leptin levels is poorly understood.Various studies have shown diff erences in leptin values in pre-and postmenopausal women.Rosenbaum and associates 20 suggested that the leptin concentration is lower in postmenopausal than in premenopausal women, also Ayub N, et al. showed a highly signifi cant diference in comparison of the mean serum leptin concentrations between pre -menopausal and postmenopausal non-obese women 16 .Di Carlo and co-workers observed an increased serum leptin level in untreated postmenopausal women compared with premenopausal values 25 .
On the other hand some investigators didn't show any diference in leptin levels in postmenopausal women a decrease of endogenous estradiole levels did not exert any eff ect on serum leptin levels 5,12,16,17 .They hypothesize that circulating levels of leptin are not related to the estrogen levels.Our results are in accordance with their conclusions.We did not observe any correlations between serum leptin levels and E2, progesterone, LH and FSH in any of the study groups.
The infl uence of hormone replacement therapy in postmenopausal women on serum leptin concentration is still poorly understood.Some authors such as Di Carlo and co-workers suggested that sex hormone replacement thera-The infl uence of estradiole and tibolone administration on leptin levels in women with surgically induced menopause py can modulate leptin levels.In their study, an increased serum leptin level in untreated postmenopausal women decreased to premenopausal values after hormonal treatment 35 .In study of Elbers et al. transdermal oestrogen replacement therapy during 2 months in postmenopausal women slightly increased total serum leptin levels 36 .The study of Cagnaci et al. presented diff erent results.Low doses of transdermal estradiol did not exert any infl uence on fasting leptin levels 37 .Some investigators take the possibility into account that diff erences in leptin level may rather depend on the dose and type of hormonal therapy used 38 .They suggest that only a supraphysiological estro-gen or gestagen concentration can act on adipocytes and modulate leptin production.But the conclusions of other authors do not support this theory.In study of Castello branco et al. women were randomly allocated to either one of three diff erent doses of norethisterone (50 microg/ day, 175 microg/day, or 550 microg/day) continuously combined with a fi xed dose of 17beta-estradiol (350 microg/day) for nasal administration, or 17beta-estradiol at 2 mg/day combined with oral norethisterone acetate at 1 mg/day both intranasal and oral therapy had the same eff ect of increasing the levels of leptin after 24 weeks of administration 39 .Laivuori and co-workers in a 1-year com-parative study did not fi nd any diference between oral or transdermal therapy in postmenopausal women treated with oral or transdermal E^sub 2^ + NETA.Neither oral nor transdermal E^sub 2^ + NETA caused any signifi cant change in plasma leptin concentrations or BMI after 2, 6 and 12 months of treatment 40 .In our study, hormonal changes during surgically induced menopause, administration of low dose estrogen therapy or tibolone did not exert any eff ect on serum leptin levels.These results indicate that sex hormone replacement therapy has no eff ect on serum leptin concentration.
Several investigators performed studies in postmenopausal women in which tibolone (a selective tissue estrogenic activity regulator) was administered.Tibolone administration relieves climacteric complaints and prevents bone loss without stimulating the endometrium and breast.Tibolone is metabolized in the liver and intestine into 3α-and 3β-hydroxy metabolites and the 3-keto-Δ 4 metabolite.The 3-hydroxy metabolites activate only the estrogen receptor (ER), whereas the 3-keto-Δ 4 metabolite stimulates both progesterone and androgen receptors, but not the ER.Tibolone does not stimulate the endometrium because it is converted locally into its metabolically stable 3-keto-Δ 4 metabolite, which shows progestogenic activity.We did not observe any eff ect of tibolone administration on leptin concentration.Our results are in agreement with results of Tommaselli GA.He compared leptin levels in untreated postmenopausal women and postmenopausal women treated with tibolone.No signifi cant change in serum leptin levels was found in subjects receiving tibolone 41 .Also Lambrinoudaki et al. evaluated the eff ect of tibolone and also estrogen replacement therapy (ERT) and continuous combined hormone replacement therapy (HRT) on serum leptin levels in healthy postmenopausal Neither ERT/HRT or tibolone exerted any eff ect on serum leptin after 6 months of treatment 42 .Odabasi et al. studied the eff ects of tibolone on leptin levels in a 6-month, prospective, randomized, double-blind, placebo-controlled study.Tibolone decreased leptin levels, total fat percentage, and total fat mass 43 .Deogou et al. compared the changes in body composition and in leptin levels in postmenopausal women receiving hormone therapy (HT) or tibolone for 6 months.Women in the tibolone group had a signifi cant decrease in leptin levels accompanied by decreased total fat mass, fat percentage, and increased total lean mass 44 .In our study during 3 months of tibolone administration we did not observe any eff ect of tibolone on serum leptin levels.
Up to date the infl uence of endogenous estrogens and androgens levels changes during menopause and the infl uence of sex hormone replacement therapy on leptin secretion and serum leptin concentration remains uncertain.Based on our result we can conclude that in our study changes in sex hormone concentrations caused by ovariectomy did not infl uence serum leptin concentrations.We did not observe any changes in leptin levels during 3 months after surgically induced menopause.We did not fi nd any link between sex hormone levels changes in postmenopausal women and leptin concentration.Also the administration of short term low dose estrogen therapy or administration of tibolone in postmenopausal subjects did not change serum leptin levels.Our results are in conclusion with results of authors who also did not fi nd any relation between leptin level and menopausal status.The diff erences between our results and results of authors who have found diff erent leptin values between pre-and postmenopausal women can be explained by specifi c composition of our sample (relatively low age of patients who underwent surgical procedure).Also most of the subjects were non-obese women with low BMI, which is resulting in low leptin levels and low incidence of leptin resistance.Our results also refl ect metabolic and hormonal changes only in a short period of 3 months after surgically induced menopause.

Table 1 .
Mean values of measured parameters on the 5th day after surgery and 3 months after surgery -Group 1.

Table 2 .
Mean values of measured parameters on the 5th day after surgery and 3 months after surgery -Group 2.

Table 3 .
Mean values of measured parameters on the 5th day after surgery and 3 months after surgery -Group 3.