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Original Studies |
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.), Università degli Studi di Napoli "Federico II", 80131, Napoli, Italy; Centro di Endocrinologia ed Oncologia Sperimentale, CNR, Napoli (V.S., S.F.), 80131, Napoli, Italy; 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
Address correspondence and requests for reprints to: Dr. Giuseppe Matarese, Laboratorio di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare - Università degli Studi di Napoli "Federico II"- via S. Pansini 5, 80131, Napoli, Italy. E-mail: gmatarese{at}napoli.com
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Abstract |
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These data suggest that the proinflammatory and neoangiogenic actions of leptin may contribute to the pathogenesis of endometriosis.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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(TNF-), 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-
in rodents (11, 12). A positive correlation between leptin levels and
activation of the TNF- 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 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.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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.
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Clinical and endocrine characteristics of the study population and the
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, Laüfelfingen, 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.
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.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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 Müllerian 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, 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.
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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).
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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).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, 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 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- 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.
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Acknowledgments |
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Footnotes |
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2 Giuseppe Matarese and Carlo Alviggi equally contributed to this
work.
3 A Fondo Sociale Europeo Fellow, Università di Napoli
"Federico II", Italy.
4 A Consiglio Nazionale delle Ricerche Fellow, Italy.
5 A Clinical Training Fellow funded by the United Kingdom
Medical Research Council.
Received November 15, 1999.
Revised March 7, 2000.
Accepted March 29, 2000.
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References |
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system in humans. J Clin Endocrinol Metab. 82:3408–3413.This article has been cited by other articles:
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