The peritoneal fluid concentration of leptin is increased in women with peritoneal but not ovarian endometriosis

G. De Placido1, C. Alviggi1,5, C. Carravetta1, M.L. Pisaturo1, V. Sanna2, M. Wilding1, G.M. Lord3 and G. Matarese4

1 Dipartimento di Ginecologia e Ostetricia, Medicina della Riproduzione ed Endoscopia Ginecologica, Universita degli Studi di Napoli `Federico II', via Pansini 5, 80131, Napoli, Italy, 2 Centro di Endocrinologia e Oncologia Sperimentale -CEOS- CNR, via Pansini 5, 80131, Napoli, Italy, 3 Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK and 4 Cattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita degli Studi di Napoli `Federico II', via Pansini 5, 80131, Napoli, Italy.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was designed to measure leptin concentrations in the peritoneal fluid (PF) of women with different aspects of pelvic endometriosis. Among 36 consecutive women undergoing laparoscopy, nine were diagnosed as having minimal-mild endometriosis (stage I–II). Among nine other subjects with advanced stage (III–IV) disease, six showed one or more ovarian endometriotic cysts as the only operative finding. The remaining 18 unaffected women constituted the control group. Patients with endometriosis had significantly higher PF leptin concentrations (32.6 ± 16.2 versus 17.1 ± 6.6 ng/ml, P = 0.002); this difference remained significant when corrected for body mass index (BMI) (PF leptin/BMI ratio 1.41 ± 0.67 versus 0.76 ± 0.28, P = 0.001). Furthermore, the PF leptin/BMI ratio was significantly higher in women with peritoneal implants than in those in whom no implant was found at laparoscopy (1.6 ± 0.7 versus 0.83 ± 0.33, P = 0.007). Conversely, patients with one or more ovarian endometriomata as the only finding, had a PF leptin/BMI ratio comparable with that in women where no cyst was found (1.05 ± 0.4 versus 1.1 ± 0.65). In women with stage I–II endometriosis, a higher mean PF leptin/BMI ratio was found compared with those affected by stage III–IV (1.78 ± 0.68 versus 1.05 ± 0.43, P = 0.01). These results show that during endometriosis the presence of peritoneal disease, and not of ovarian endometriotic cysts, influences leptin concentrations in PF. The data suggest that leptin may play a role in the development of peritoneal endometriosis, and that different biochemical phenomena might be involved in the pathogenesis of the ovarian form of the disease.

Key words: angiogenesis/endometriosis/endometriotic cyst/leptin/peritoneal implants


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Endometriosis is a benign gynaecological disease characterized by the implantation and growth of endometrial tissue outside the uterine cavity. Data emerging from the literature indicate three major factors involved in the pathogenesis of the disease (Vinatier et al., 1996Go; Koninckx et al., 1998Go). First, disorders involving cell-mediated and humoral immune responses are commonly present. Second, pro-inflammatory cytokines and growth factors, such as interleukin (IL)-1ß, IL-8, tumour necrosis factor (TNF) -{alpha} and vascular endothelial growth factor (VEGF), are significantly increased in the peritoneal fluid (PF) of affected patients. Finally, the establishment of a vascular network in peritoneal implants is relevant to the development of the disease (Healy et al., 1998Go).

Leptin is a 16 kDa adipocyte-derived protein belonging to the class of helical cytokines. It is the product of the obese gene, and regulates food intake and energy expenditure (Halaas et al., 1995Go; Friedman and Halaas, 1998Go). Circulating concentrations of this hormone are correlated to body fat mass. A variety of stimuli can rapidly induce leptin expression and secretion in mice and humans, including the inflammatory mediators TNF-{alpha} and IL-1ß (Janik et al., 1997Go; Mantzoros et al., 1997Go; Zumbach et al., 1997Go). Indeed, leptin is produced during the acute phase response, and represents an early reactant together with C-reactive protein and IL-1ß during systemic inflammation and fever (Matarese, 2000Go). The long form of the leptin receptor (Ob-Rb) is expressed in the hypothalamus, but its presence has also been demonstrated in extraneural tissues such as T cells (Lord et al., 1998Go), endometrium (Kitawaki et al., 2000Go) and endothelial cells (Sierra-Honigmann et al., 1998Go).

Recent evidence suggests that leptin has immunoregulatory properties. In particular, it shows a marked and specific effect upon CD4+ T-cell responses and their cytokine profiles (Lord et al., 1998Go). Furthermore, leptin has neoangiogenic promoting activities in vivo and in vitro (Bouloumie et al., 1998Go; Sierra-Honigmann et al., 1998Go).

Given that leptin is able to promote angiogenesis and cell-mediated immune responses, the concentration of this molecule during endometriosis has recently been investigated (Matarese et al., 2000Go). The results of these studies showed that mean leptin concentration is increased in patients with endometriosis when compared with unaffected woman. The present study was designed to investigate further the relationship between leptin and pelvic endometriosis by measuring PF leptin concentrations in the presence of peritoneal implants and ovarian endometriomata, and in different revised American Fertility Society stages.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
Thirty-six consecutive woman undergoing laparoscopy for infertility (n = 15), adnexal mass (n = 4), infertility and adnexal mass (n = 5), tubal sterilization (n = 7) or congenital Müllerian malformation (n = 5) were included in the study. The study was approved by the local ethics committee, and written informed consent was obtained from all patients before the procedure.

Subjects did not receive any hormonal therapy or medication for at least 3 months before laparoscopy. All subjects underwent a clinical examination and hormonal assessment for gonadotrophins and oestradiol during the early follicular phase (day 5 of the menstrual cycle). Body mass index (BMI) was calculated as body weight (in kg) divided by the square of the height in metres. Only normally cycling and ovulating subjects with a menstrual cycle length of 25–35 days (intra-individual variation of at most 3 days), a BMI between 19 and 26 kg/m2 and normal LH/FSH ratio were enrolled.

Exclusion criteria were: clinical and/or echographic indications of polycystic ovarian disease; diabetes; systemic, hepatic or thyroid inflammatory disease; and/or any pelvic disease other than endometriosis diagnosed at the time of laparoscopy

All patients underwent laparoscopy under general anaesthesia between 08:30 and 11:30, after at least 12 h fasting. After the induction of pneumoperitoneum with 3–4 l of CO2 and the insertion of the umbilical port, PF was obtained by means of a suprapubic 1.5 mm needle. The pelvis was carefully observed by a 10 mm, 0 degree laparoscope. Endometriosis was recorded with regard to the location and size of peritoneal implants and adhesions and ovarian superficial or deep endometriomata. The revised American Fertility Society (rAFS; now American Society for Reproductive Medicine) classification (American Fertility Society, 1985Go) was used for staging.

Deep ovarian endometriomata were removed by stripping off the cyst wall, using a blunt dissection technique. Peritoneal biopsies were taken from all patients with laparoscopic evidence of the disease for the histopathological confirmation.

Recent evidence (Messinis et al., 2000Go) suggests that leptin concentrations may be conditioned by the menstrual cycle phase. During surgery, biopsies were obtained from all women for dating the eutopic endometrium, and the menstrual cycle phase was defined according to standard criteria (Noyes et al., 1950Go).

Biochemical analyses
PF was collected aseptically within 20 min of the administration of anaesthetic drugs, and immediately centrifuged for 5 min at 500 g to remove any cellular material. All 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 inter-assay coefficients of variation were <5%. Samples were measured in duplicate at 450 nm, using an ELISA plate reader (Bio-Rad Laboratories, Hercules, CA, USA).

For FSH and LH measurements, immunometric assays based on enhanced luminescence were used (Amerlite FSH and LH assay respectively; Amersham International plc, Amersham, Bucks, UK). Oestradiol was measured using a competitive immunoassay based on enhanced luminescence (Amerlite Estradion-60 assay; Amersham International). For progesterone measurement a competitive immunoassay was used (Kodak Amerlite progesterone assay; Amersham International). A progesterone increase was assessed to test ovulation for all patients and controls one cycle before the study. The lower limits of detection for FSH, LH, oestradiol and progesterone were 0.5 IU/l, 0.12 IU/l, 50 pmol/l and 0.35 nmol/l respectively, while inter- and intra-assay coefficients of variation were 6.9 and 6.1%, 8.7 and 6.6%, 9.4 and 8.0%, and 7.5 and 6.5% respectively.

Statistical analyses
For the analysis of variance (ANOVA), PF concentrations of leptin expressed in ng/ml were analysed as PF leptin/BMI ratio. A two-within, one-between design was adopted, having the presence of implants and the presence of endometriotic cysts as within factors, and the presence of endometriosis as between factor. A one-way ANOVA was performed to determine the effect of the menstrual cycle phase and of stage I–II and III–IV rAFS on PF leptin concentrations. Simple regression analysis was used in order to study the relationship between leptin concentrations and BMI in both groups. Data were analysed with StatViewTM SE+ Graphics (Abacus Concepts, Inc., Cary, NC, USA) for Macintosh. All results are reported as mean ± SD, and a P-value < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Eighteen women had laparoscopic evidence and histological confirmation of endometriosis. Nine were diagnosed to have a minimal-to-mild disease (stage I–II), and nine were affected by advanced disease (stage III–IV). In the latter group, six showed at least one ovarian endometriotic cyst as the only operative finding, associated with adnexal filmy adhesions in two cases. The remaining three subjects displayed peritoneal implants associated with one or more ovarian endometriotic cyst. Eighteen consecutive subjects without laparoscopic evidence of endometriosis represented the control group. The characteristics and indications for laparoscopy of patients and controls are shown in Table IGo.


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Table I. Baseline characteristics and peritoneal fluid (PF) leptin concentrations of patients and controls
 
Patients affected by endometriosis were found to have significantly higher PF leptin concentration and PF leptin/BMI ratio when compared with controls (32.6 ± 16.2 versus 17.1 ± 6.6 ng/ml, P = 0.002; 1.41 ± 0.67 versus 0.76 ± 0.28, P = 0.001) (Table IGo). Single regression analysis revealed a positive correlation between PF leptin concentrations and BMI in both control (r = 0.53, P = 0.02) and endometriosis subjects (r = 0.52, P = 0.03).

In the study population, no significant effect of menstrual cycle phase on PF leptin/BMI ratio was observed (data not shown). Clear laparoscopic evidence of corpus luteum was observed in four out of nine endometriotic patients, and in three out of eight controls in the secretive phase of the menstrual cycle. A one-way ANOVA did not show any significant main effect of corpus luteum on PF leptin concentrations. A two-way ANOVA showed a significant main effect of peritoneal endometriotic implants (P = 0.007), but not of endometriotic cysts on PF leptin/BMI ratio (Table IIGo). A statistically significant interaction between these two main factors was observed (P = 0.01).


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Table II. Effects of endometriotic implants and endometriotic ovarian cysts on peritoneal fluid (PF) leptin concentrations in the 36 subjects studied (two-way analysis of variance)
 
When women with endometriosis were divided according to the rAFS stage, a significantly higher mean PF leptin/BMI ratio was found in patients with stage I–II disease compared with those affected by stage III–IV (PF leptin stage I–II 41.7 ± 16.7 ng/ml versus PF leptin stage III–IV 23.6 ± 9.9 ng/ml, P = 0.024). These data were confirmed when controlled for BMI (PF leptin/BMI stage I–II 1.78 ± 0.68 versus PF leptin/BMI stage III–IV 1.05 ± 0.43, P = 0.01).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, concentrations of leptin—a molecule with angiogenic and immunoregulatory properties—were measured in the PF of patients with pelvic endometriosis. In a previous report, a significant increase in the concentration of leptin in affected women was shown. Although mean serum and PF concentrations of leptin were significantly increased during the disease, not all affected patients had increased leptin concentrations (Matarese et al., 2000Go). It can be postulated that there exists a specific subgroup of endometriotic subjects with an increase in leptin concentration. This observation could explain the discrepancy between our previous data and those reported by others (Metalliotakis et al., 2000Go) which showed no significant differences in serum leptin between affected women and subjects without laparoscopic evidence of the disease.

The present study was designed to identify which determinants influence leptin concentration in affected women by finding a correlation with different laparoscopic appearances of the disease. Here, the main finding is that patients displaying peritoneal implants at all stages of endometriosis showed higher PF leptin concentrations than women in whom no implant was observed. Furthermore, the presence of ovarian endometriomata had no significant main effect on leptin concentration. These data suggest that the presence of peritoneal disease, and not of ovarian endometriotic cysts, is the factor that influences the concentration of leptin in PF during endometriosis. Although the current findings demonstrate significantly higher PF concentrations of leptin in stage I–II endometriosis, this may be because, in the population studied, six out of nine women affected by advanced stage endometriosis did not display any peritoneal lesion except endometriotic cysts at laparoscopy.

Previous studies (Nisolle and Donnez, 1997Go) reported morphological, morphometric and histochemical evidence suggesting that peritoneal and ovarian endometriosis are to be considered different entities. It appears that this is the first report to show a correlation of a biochemical variable with different appearances of pelvic endometriosis. The difference in PF leptin concentration among patients with peritoneal implants and ovarian endometriomata may be due to alternative patterns of secretion between the two conditions, thus corroborating the hypothesis of different pathogenesis. However, it is also possible that leptin is free to diffuse into the PF during peritoneal disease, whereas it is sequestrated into the cystic fluid of endometriomata.

In our previous study (Matarese et al., 2000Go), it was suggested that the production of leptin during endometriosis was intra-abdominal. The differences observed among the diverse phenotypes of the disease (peritoneal versus ovarian) further support this observation. In addition, the discrepancy concerning serum leptin between our previous evidence and findings by others (Metalliotakis et al., 2000Go) may also be due to different laparoscopic appearance of pelvic endometriosis in the two series (the studies are not comparable from this point of view).

Following confirmation of our data, it is necessary to clarify whether leptin is released by endometriotic foci and/or by peritoneal fat deposits. Furthermore, the Ob-Rb receptor has recently been shown to be expressed in human endometrium (Kitawaki et al., 2000Go). The possible role of this receptor in normal endometrium remains unclear. However, it is possible to speculate that increased production of leptin during endometriosis may also sustain and promote ectopic endometrial tissue growth and inflammation.

Other angiogenic factors have been shown to be increased during endometriosis (Oosterlynck et al., 1993Go). Indeed, the concentration of VEGF is significantly higher in the PF from women with endometriosis (McLaren et al., 1996aGo) and this seems to correlate with the severity of the disease (Shifren et al., 1996Go). Peritoneal macrophages are probably the main source of VEGF, and the production of VEGF from these cells seems to be regulated by ovarian steroids (McLaren et al., 1996bGo). IL-8, a chemoattractant and angiogenic cytokine, is also increased in the PF of patients with endometriosis, especially in the early stages of the disease (Gazvani et al., 1998Go). Concentrations of IL-8 are also correlated with the extent of active endometriosis (Iwabe et al., 1998Go). Further studies are currently being performed in order to investigate the relationship between leptin and these angiogenic factors during endometriosis.

In conclusion, the results of this study confirmed that leptin concentrations in PF are higher than expected when controlled for BMI in patients with endometriosis. However, the increase in leptin concentrations is not observed in patients lacking peritoneal implants. The data suggest that the development of ovarian endometriotic cysts and peritoneal implants may be caused by different pathogenetic mechanisms.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Universita di Napoli `Federico II' and Consiglio Nazionale delle Ricerche for financial support of this study. They also thank Gianluca Ficca for assistance with the statistical analysis, Giuseppina Ruggiero for assistance with ELISA analysis, and Renato Franco for his contribution in the histological analysis. G.De Placido and C.Alviggi contributed equally to this study.


    Notes
 
5 To whom correspondence should be addressed at: 132, via Caldieri, 80138, Naples, Italy. E-mail: calviggi{at}hotmail.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 16, 2000; accepted on March 9, 2001.