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.
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Abstract |
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Key words: angiogenesis/endometriosis/endometriotic cyst/leptin/peritoneal implants
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Introduction |
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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., 1995; Friedman and Halaas, 1998
). 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-
and IL-1ß (Janik et al., 1997
; Mantzoros et al., 1997
; Zumbach et al., 1997
). 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, 2000
). 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., 1998
), endometrium (Kitawaki et al., 2000
) and endothelial cells (Sierra-Honigmann et al., 1998
).
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., 1998). Furthermore, leptin has neoangiogenic promoting activities in vivo and in vitro (Bouloumie et al., 1998
; Sierra-Honigmann et al., 1998
).
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., 2000). 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.
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Materials and methods |
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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 2535 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 34 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, 1985) 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., 2000) 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., 1950
).
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 III and IIIIV 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.
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Results |
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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 II). A statistically significant interaction between these two main factors was observed (P = 0.01).
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Discussion |
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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 III 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, 1997) 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., 2000), 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., 2000
) 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., 2000). 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., 1993). Indeed, the concentration of VEGF is significantly higher in the PF from women with endometriosis (McLaren et al., 1996a
) and this seems to correlate with the severity of the disease (Shifren et al., 1996
). 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., 1996b
). 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., 1998
). Concentrations of IL-8 are also correlated with the extent of active endometriosis (Iwabe et al., 1998
). 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on November 16, 2000; accepted on March 9, 2001.