Journal of Clinical Endocrinology and Metabolism
Volume 85 • Number 7 • July 2000
Copyright © 2000 The Endocrine Society

 

Dr.Sinan DOĞANTÜRK

ANKARA

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Original Studies

Increased Leptin Levels in Serum and Peritoneal Fluid of Patients with Pelvic Endometriosis ^*

 

GIUSEPPE MATARESE ^{1 4}

CARLO ALVIGGI ¹

VERONICA SANNA ^{2 5}

JANE K. HOWARD ¶ A Clinical Training Fellow funded by the United Kingdom Medical Research Council. ³

GRAHAM M. LORD ¶ A Clinical Training Fellow funded by the United Kingdom Medical Research Council. ³

CARLO CARRAVETTA ¹

SILVIA FONTANA ²

ROBERT I. LECHLER ³

STEPHEN R. BLOOM ³

GIUSEPPE DE PLACIDO <sup>1

1</sup> Laboratorio di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare (G.M.) and Dipartimento di Ginecologia e Ostetricia, Area Funzionale di Medicina della Riproduzione ed Endoscopia Ginecologica (C.A., C.C., G.D.P.), Universita degli Studi di Napoli "Federico II", 80131, Napoli, Italy;
² Centro di Endocrinologia ed Oncologia Sperimentale, CNR, Napoli (V.S., S.F.), 80131, Napoli, Italy;
³ and Department of Immunology (G.M.L., R.I.L.) and Endocrine Unit (J.K.H., S.R.B.), Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, United Kingdom
⁴ A Fondo Sociale Europeo Fellow, Universita di Napoli "Federico II", Italy.
⁵ § A Consiglio Nazionale delle Ricerche Fellow, Italy.

Received November 15, 1999.
Revision received March 7, 2000.
Accepted March 29, 2000.

Address correspondence and requests for reprints to: Dr. Giuseppe Matarese, Laboratorio di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare - Universita degli Studi di Napoli "Federico II"- via S. Pansini 5, 80131, Napoli, Italy. E-mail: gmatarese@ napoli.com.

Copyright © 2000 by The Endocrine Society

 

ABSTRACT

Pelvic endometriosis is an immune-related chronic inflammatory disease, characterized by ectopic implants of endometrium in the peritoneal cavity and associated with increased secretion of proinflammatory cytokines and neoangiogenesis. Leptin, the adipocyte-derived hormone, has been shown to have a role in food intake, basal metabolism, and reproductive function. Leptin levels are dynamically regulated, being elevated by inflammatory mediators and reduced by starvation. Leptin itself can influence the proinflammatory immune responses of CD4 ⁺ T lymphocytes, and reports have also shown this hormone to be an angiogenic factor in vitro and in vivo. We investigated whether leptin concentrations in serum and peritoneal fluid (PF) differed between 13 patients with different stages of endometriosis and 15 age- and body mass index-matched controls. We found a statistically significant ( P < 0.05) increase in leptin levels in serum (30.3 ± 14.8 ng/mL) and PF (35.9 ± 17.4 ng/mL) of patients with endometriosis, compared with our control population (serum, 15.6 ± 8.4; PF, 17.5 ± 7.2 ng/mL). Regression equations, relating leptin to body mass index, were also significantly different in endometriosis patients, compared with controls. Higher levels of leptin were observed in the earlier stages of endometriosis than advanced-stage disease.

These data suggest that the proinflammatory and neoangiogenic actions of leptin may contribute to the pathogenesis of endometriosis. ( J Clin Endocrinol Metab 85: 2483-2487, 2000)

 

PELVIC ENDOMETRIOSIS is a chronic inflammatory disease characterized by implantation and growth of endometrial tissue on the pelvic peritoneum, associated with infertility and pain. In this condition, local and systemic abnormalities in immune responses have been described ^[1] . Increased production of proinflammatory cytokines, such as interleukin (IL)-1, IL-8, tumor necrosis factor-alpha (TNF-alpha), and vascular endothelial growth factor, has been shown in peritoneal fluid (PF) from these patients ^{[2] [3] [4] [5]} . Furthermore, angiogenesis is thought to be of fundamental importance in the early stages of endometriosis ^[6] . Histologically, early implants tend to display greater inflammatory and neoangiogenic appearances, compared with older endometriotic lesions ^{[4] [5] [6]} .

Leptin is the 16-kDa adipocyte-derived protein product of the obese gene. Like IL-2 and GH, it belongs to the class of helical cytokines and represents a link between nutritional status and energy expenditure ^{[7] [8]} . Circulating levels of this hormone are proportional to body fat mass. Recent evidence suggests that leptin may have a role as an angiogenic factor in vitro and in vivo ^{[9] [10]} . Inflammatory cytokines have been reported to stimulate leptin secretion such that an increase in serum leptin concentrations is observed after the administration of IL-1 or TNF-alpha in rodents ^{[11] [12]} . A positive correlation between leptin levels and activation of the TNF-alpha system has also been found in humans ^{[13] [14]} with increased circulating concentrations of leptin observed during fever and systemic inflammation ^{[15] [16]} . It has been demonstrated that leptin has a marked and specific effect on CD4⁺ T lymphocyte responses and their cytokine profiles, providing further links between leptin, inflammation, and immunity ^[17] .

A large amount of adipose tissue is present in the peritoneal cavity; but, to our knowledge, no data exist about its potential involvement in the regulation of local immune responses, inflammation, and angiogenesis. It has been shown that omental fat produces significant amounts of leptin with preserved sexual dimorphism, being higher in females ^[18] . Given that leptin is able to modulate angiogenesis and immune responses, we compared the levels of leptin in serum

^* Funded by Universita di Napoli "Federico II" and Consiglio Nazionale delle Ricerche.
Giuseppe Matarese and Carlo Alviggi equally contributed to this work.

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and PF of patients with different stages of endometriosis vs. an equivalent laparoscopic control group. In addition, we studied the relationship between serum and PF leptin levels and correlated these measurements with the stage of the disease.

 

Subjects and Methods

Subjects

Twenty-eight Caucasian women were entered into the study group or the control group, in a consecutive fashion, depending on laparoscopic diagnosis. Patients with clinical and ultrasonographic evidence of polycystic ovarian disease were excluded. Other comorbid conditions excluded were: diabetes; systemic, hepatic, or thyroid inflammatory disease; and any pelvic disease other than endometriosis. Subjects were not given hormonal therapy for at least 3 months before laparoscopy. Women underwent clinical examination, including measurement of height and weight, and hormonal assessment for gonadotropins and estradiol, during the early follicular phase (day 5 of the cycle) ( Table 1 ). Body mass index (BMI) was calculated as weight in kilograms divided by the square of the height in meters. Only normally cycling subjects with a menstrual cycle length of 25-35 days (intraindividual variation of, at most, 3 days) and normal LH/FSH ratio were enrolled ( Table 1 ). Ultrasonography and serum progesterone measurement, before admission to the study, confirmed ovulation in all patients. All patients underwent laparoscopy, under general anesthesia, between 0830 and 1130 h, after at least 12 h of fasting. Biopsies of ectopic endometrium were obtained in all women, and the menstrual cycle phase was defined according to the criteria of Noyes et al. ^[19] . All patients gave verbal informed consent to participation in the study.

Thirteen women had laparoscopic evidence of endometriosis. In each patient, endometriosis was staged according to the revised American Fertility Society (rAFS) classification recommended by the American Society for Reproductive Medicine. Laparoscopic diagnosis was confirmed by histology. Fifteen subjects without laparoscopic evidence of endometriosis served as the control group.

Clinical and endocrine characteristics of the study population and the

^a Mean ( SD).
^b Endometrial biopsy.
^c Normal range, 0-5 mg/L.

control individuals were comparable ( Table 1 ). Indications for laparoscopy and menstrual cycle phase of both groups are also shown in Table 1 .

Biochemical analyses

Blood samples for hormone assays and the C-reactive protein (CRP) analysis were obtained in the operative room, before the induction of anesthesia for laparoscopy, and were immediately centrifuged. PF was collected aseptically after the insertion of the first 5 mm accessory trocar (within 20 min from the administration of anesthetic drugs), prior to any biopsy being performed, and was centrifuged to remove any cellular material. Consistent with other reports ^[20] , we demonstrated a significant rise in serum leptin levels only during the first 24-h after the operation (data not shown). All of the samples were stored at -80 C, and leptin concentrations were determined with human leptin enzyme-linked immunosorbent assay (ELISA) kits (Alexis Corporation, Laufelfingen, Switzerland) within 2 months of sampling. Leptin concentrations were calculated from standard curves generated for each assay using recombinant human leptin, according to the manufacturer's instructions. The minimum detection limit of the assay was 0.2 ng/mL. The intra- and interassay coefficients of variation were below 5%. Samples were measured in duplicate, at 450-nm wavelength, using an ELISA plate reader (Bio-Rad Laboratories, Inc., Hercules, CA).

For FSH and LH measurements, immunometric assays based on enhanced luminescence were used (Amerlite FSH and LH assay, respectively; Amersham International plc, Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). The results are expressed as IU/L. Estradiol was measured using a competitive immunoassay based on enhanced luminescence (Amerlite Estradion-60 assay, Amersham Pharmacia Biotech). The results are expressed as pmol/L. For progesterone measurement, a competitive immunoassay was used [Eastman Kodak Co. (Rochester, NY) Amerlite Progesterone assay, Amersham Pharmacia Biotech]. A progesterone rise was assessed to test ovulation for all the patients and the controls, one cycle before the study. The lower limits of detection for FSH, LH, estradiol, and progesterone were 0.5 IU/L, 0.12 IU/L, 50 pmol/L, and 0.35 nmol/L, respectively, whereas inter- and intraassay coefficients of variation were 7.5 and 6%, 9 and 6.8%, 9.1 and 8%, and 7 and 6.6%, respectively.

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For CRP measurement in serum of patients and controls, the N LATEX CRP mono kit for immunonephelometry (Bering Nephelometer Systems, Marburg, Germany) was used. The normal range for this kit was between 0-5 mg/L. The intraassay variability was less than 5%, and the interassay variability ranged from 2.6-5.7%. According to the manufacturer's instructions, the samples were diluted 1:400, and the range of detection was from 3.5-220 mg/L.

Statistical analyses

Differences between groups were analyzed with the Mann-Whitney U test for independent samples. Simple regression analysis was adopted to study the relationship between leptin levels and BMI and between serum and PF leptin concentrations in both groups. Differences between regression lines were analyzed using ANOVA. The Wilcoxon sign rank test was employed to compare the differences in paired median leptin concentrations in serum and PF from patients and controls. Data were analyzed with StatView SE+ Graphics (Abacus Concepts, Inc., Cary, NC) for Macintosh or STATA version 5.0 (Timber Lake Consultants Ltd., West Wycombe, Kent, UK) for PC. All results are reported as mean ± SD, and P < 0.05 was considered statistically significant.

 

Results

Characteristics of patients and controls are shown in Table 1 . Stage I-II endometriosis was diagnosed laparoscopically in 7 patients during investigation for infertility. Among six patients with advanced disease (stages III-IV), 5 displayed at least one ovarian endometriotic cyst (deep ovarian endometriosis) associated with peritoneal implants and/or pelvic adhesions. The sixth patient presented 3 superficial ovarian endometriomas associated with peritoneal implants. Among patients with endometriosis, 4 out of 13 had a history of mild-to-moderate pelvic pain, 2 of whom also suffered with dysmenorrhea. Two other patients had dysmenorrhea as their only symptom. We found no significant relationship between these symptoms and leptin levels nor any significant correlation between a history of pain and stage of the disease (not shown). Finally, 4 out of 15 control individuals had endoscopic evidence of Mullerian malformations. These did not have a significant impact on leptin concentrations.

Leptin and CRP levels in patients with endometriosis

Patients affected by endometriosis had significantly higher concentrations of leptin, both in serum and PF, when compared with controls ( Table 1 ). Serum leptin levels were 30.3 ± 14.8 ng/mL in patients and 15.6 ± 8.4 ng/mL in the controls ( P = 0.007); leptin concentrations in PF were 35.9 ± 17.4 ng/mL and 17.5 ± 7.2 ng/mL in patients and in the control group, respectively ( P = 0.005). CRP serum concentrations were less than 3.5 mg/L in all subjects ( Table 1 ).

Relationship between leptin levels and BMI

As expected, there was a strong positive correlation between serum and PF leptin concentrations with BMI in both control and endometriosis subjects ( Fig. 1 ). There was, however, a significant difference in regression equations describing the relationship between serum and PF leptin concentrations with BMI in the endometriosis subjects, when compared with those of the controls ( Fig. 1 ). Although the gradients of the regression lines relating leptin and BMI were similar in patients and controls, there was a significant difference in the intercepts of the regression lines ( P = 0.015,

Figure 1. Relationship between serum leptin and BMI (A) and PF leptin and BMI (B), in endometriosis patients ( solid lines and circles) and controls ( dashed lines, open circles). The regression equations relating serum or PF leptin concentrations and BMI were significantly different in endometriosis patients compared with controls. In all panels the slopes of the regression lines were similar, but the intercepts were different (A, P = 0.015; B, P = 0.005).

serum leptin vs. BMI; P = 0.005, PF leptin vs. BMI). Thus, for a given BMI, serum or PF leptin concentrations were found to be significantly higher in subjects with endometriosis than those observed in controls.

Relationship between serum and PF leptin levels

There was a strong and significant correlation between serum and PF leptin concentrations, both in endometriosis patients (r = 0.9, P = 0.0001) and controls (r = 0.8, P = 0.0003) ( Fig. 2 ).

Ten out of 13 patients with endometriosis had a higher leptin concentration in PF, compared with that of their serum. Indeed, there was a significant difference between the paired medians of serum and PF leptin concentrations in the patients ( P < 0.023, 95% CI -10.5 to -0.5 ng/mL leptin). In

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Figure 2. Relationship between PF and serum leptin in endometriosis patients ( solid lines and circles) and controls ( dashed lines, open circles).

contrast, there was no significant difference between the paired medians of serum and PF leptin concentrations in the control subjects ( P = 0.23, 95% CI -4.9 to +0.7 ng/mL leptin).

Leptin levels and rAFS endometriosis stage

Patients were divided into two categories according to the stage of endometriosis (category 1 stage I-II rAFS, category 2 stage III-IV rAFS). Leptin concentrations, both in serum and PF, were found to be significantly higher in patients with stage I-II endometriosis (n = 7), compared with those classed as stage III-IV (n = 6) (serum leptin category 1: 39.7 ± 12.5 ng/mL vs. serum leptin category 2: 19.3 ± 8.47 ng/mL, P = 0.0101; PF leptin category 1: 46.5 ± 15.6 ng/mL vs. PF leptin category 2: 23.5 ± 9.3 ng/mL, P = 0.022). These differences in leptin concentrations with stage of disease remained significant when controlled for differences in BMI by comparing the leptin/BMI ratio in serum or PF according to stage of disease (serum leptin/BMI category 1: 1.69 ± 0.55 ng/mL vs. serum leptin/BMI category 2: 0.86 ± 0.35 ng/mL, P = 0.01; PF leptin/BMI category 1: 1.98 ± 0.64 ng/mL vs. PF leptin/BMI category 2: 1.05 ± 0.38 ng/mL, P = 0.02).

Serum leptin and menstrual cycle phase

No significant differences were found in serum leptin levels between proliferative and secretive menstrual cycle phase, either in patients or controls (proliferative phase patient group: 32.9 ± 14.6 ng/mL; secretive phase patient group: 28 ± 15.7 ng/mL, P = 0.57; proliferative phase control group: 15.9 ± 8.5 ng/mL; secretive phase control group: 15.3 ± 8.8 ng/mL, P = 0.9). Patients showed higher serum leptin levels in both phases of the menstrual cycle, when compared with controls, reaching statistical significance only in the proliferative phase (proliferative phase patient group: 32.9 ± 14.6 ng/mL; proliferative phase control group: 15.9 ± 8.5 ng/mL, P = 0.028; secretive phase patient group: 28 ± 15.7 ng/mL; secretive phase control group: 15.3 ± 8.8 ng/mL, P = 0.1).

 

Discussion

To our knowledge, this is the first study to examine serum and PF leptin concentrations in patients with pelvic endometriosis. We have found that leptin levels were significantly increased in the serum and PF of subjects with pelvic endometriosis, when compared with age and BMI-matched controls. In our population, this increase was independent of the menstrual cycle phase. Importantly, regression lines of serum and PF leptin levels vs. BMI were significantly different in controls and patients ( Fig. 1, A and B ); such that subjects with pelvic endometriosis had significantly higher concentrations of leptin, both in their serum and PF, than would be expected, given their BMI. In this study, BMI was taken as a surrogate marker of body fat mass. To our knowledge, there is no reported significant difference in total body fat mass in endometriosis patients, when compared to control subjects ^[21] . Therefore it is unlikely that the difference in leptin levels observed in our study, between endometriosis patients and control subjects, is attributable to differences in body fat mass.

In addition, this is the first study measuring leptin levels in PF in nondialysed subjects ^{[22] [23]} . It should be noticed that, in our control population, PF leptin was similar to serum levels, in contrast to published findings obtained from cerebrospinal and amniotic fluid ^{[24] [25]} .

CRP, positively correlated with leptin increase during systemic inflammation ^{[22] [23]} , was within the normal range in all subjects. The observation, that neither the control subjects nor the endometriosis patients in this study had an elevation of the serum levels of this acute phase reactant, suggests that the increase in serum and PF leptin concentrations seen in our endometriosis patients may not be attributable to an ongoing systemic inflammatory process. Whether the high leptin concentrations found in endometriosis are the result of increased production in the peritoneal cavity, or from extra-abdominal fat depots, remains to be determined. Our finding that there was a small, but significantly higher, leptin concentration in PF, compared with serum in patients with pelvic endometriosis, raises the question of whether the source of the high leptin levels in these subjects could indeed be intraabdominal. Increased omental fat production of leptin in the peritoneal cavity has been previously observed in some pathologic conditions, such as women with chronic renal failure but not in men ^[23] . Another possibility could be that the catabolic rate of PF leptin may be slower in some endometriosis patients than in controls. Further studies would be required to address these issues and also to determine whether the high leptin concentrations found in these patients is casual or consequent to the pathogenesis of pelvic endometriosis. Interestingly, when we compared leptin levels in patients with endometriosis according to the stage of their disease, we found that, when evaluated according to the rAFS classification, serum and PF leptin levels were higher in women with endometriosis stages I-II (see Results) than stages III-IV. This difference was still present when leptin/BMI ratios were compared (see Results).

Recent evidence indicates that neoangiogenesis plays a key role in the early stages of development of endometriotic implants ^[4] . It has been demonstrated that an important

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vascular network surrounds more active implants, suggesting that angiogenic factors present in the PF would regulate growth and progression of peritoneal lesions. Leptin has recently been reported to stimulate angiogenesis ^{[9] [10]} . Whether this property of leptin could contribute to the pathogenesis of endometriosis warrants further investigation. In addition, the increase in IL-1 and TNF-alpha in PF of women with endometriosis ^{[26] [2]} may be related to the present findings, given that these inflammatory cytokines can stimulate leptin secretion ^{[11] [12] [13] [14] [15] [16]} .

In conclusion, we have found that leptin levels in both serum and PF are higher than expected, when controlled for BMI, in patients with endometriosis. In addition, there is a slight, but significant, increase in PF leptin levels, compared with serum levels in patients and those levels are significantly higher in early stage disease when compared with advanced stage endometriosis. Leptin is now recognized to have immunoregulatory ^[17] , proinflammatory ^[16] , and angiogenic ^{[9] [10]} properties, all of which have been considered to play a role in the pathogenesis of endometriosis. Our findings suggest a potential novel role for leptin in the etiology of this disease.

Acknowledgments

We thank Antonio Mollo and Maria Teresa Varricchio for helpful discussion of the manuscript, Antonio Molis for the CRP measurements, and Giuseppina Ruggiero for assistance with ELISA analysis.

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