Serum Leptin Concentrations in Endometriosis

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The Journal of Clinical Endocrinology & Metabolism 87(3):1085–1087 Copyright © 2002 by The Endocrine Society

COMMENT Serum Leptin Concentrations in Endometriosis ` , EDGARDO SOMIGLIANA, ROBERTA MATRONE, ANTONELLA DUBINI, PAOLA VIGANO CARLOS BARRON, MARIO VIGNALI, AND ANNA MARIA DI BLASIO Istituto Auxologico Italiano, (P.V., A.D., A.M.D.) and II Department of Obstetrics and Gynecology (E.S., R.M., M.V.), University of Milan, 20135 Milan, Italy; and AREVIA GmbH (C.B.), 13353 Berlin, Germany It has been recently reported that serum concentrations of the adipocyte-derived hormone leptin are increased in patients affected by endometriosis. On the basis of these findings, the present study was undertaken to evaluate whether the protein may be used as a new serum marker of the disease. A consecutive series of 67 reproductive-age women who underwent laparoscopy for benign gynecological pathologies were enrolled prospectively for the study. Serum leptin concentrations, as evaluated by a conventional RIA kit, were related to baseline clinical characteristics and surgical and histologic diagnosis. Endometriosis was documented in 42 women (stage I–II in 19 patients and stage III–IV in 23 patients). Twenty-five women of similar age and body mass index, who had no laparoscopic evidence of the disease, served as control group. Se-

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EVERAL MILLION WOMEN from different ethnic and social groups suffer from pelvic endometriosis, which is a complex disorder characterized by the presence of endometrial tissue in ectopic sites outside the uterus. Endometriosis is a chronic and progressive disease that may give rise to a variety of severe and disabling symptoms. It is diagnosed in approximately 25% of women who undergo laparoscopy because of pelvic pain and in 20% of infertile women (1). To date, one of the major concerns associated with endometriosis is that a definitive diagnosis can only be obtained via laparoscopy or laparotomy (2). Indeed, ultrasonography is effective only in detecting ovarian endometriomas, but the physical examination may be helpful in the diagnosis of deeply infiltrative endometriosis of the culde-sac and rectovaginal septum. At present, reliable serum markers of the disease are lacking. Serum concentration of CA125, which is widely employed as a serum marker of endometriosis, has a limited diagnostic performance (3). As a consequence, the detection of the disease, especially at early stages, may represent a serious problem. The diagnosis of endometriosis is particularly important in stage I/II infertile women in whom laparoscopic treatment of the lesions has been reported to almost double the rate of a spontaneous pregnancy (4). For these reasons, the recent observation of a marked increase of serum leptin in women with endometriosis has been encouraging for the potential identification of a novel marker of the disease (5). The rationale to measure the adipocyte hormone leptin in endometriosis was mostly related to the recently identified link between leptin and Abbreviation: BMI, Body mass index.

rum levels of leptin resulted similar between women without and with endometriosis at any stage (mean ⴞ SEM, 12.5 ⴞ 9.4 ng/ml and 12.1 ⴞ 8.0 ng/ml, respectively). No significant association with leptin concentrations was observed in regard to stage of the disease, number of endometriotic implants, presence/absence of an endometriotic cyst or peritoneal deep endometriosis, and presence/absence of specific symptoms. Therefore, our results do not support the possibility to employ leptin measurement as a diagnostic tool for endometriosis. Further studies are needed to elucidate the relationship between leptin and endometrial system and determine the potential contribution of the molecule in implantation and early pregnancy development. (J Clin Endocrinol Metab 87: 1085–1087, 2002)

immunity (6 – 8). Leptin is well known to serve as a signal to inform the central nervous system about the state of fat stores (9). However, besides its role in obesity and metabolic disorders, leptin has lately been suggested to be involved in previously unexpected functions, specifically the regulation of angiogenesis (10) and immune response (6, 7), which are among the most important processes implicated in the development of endometriosis (11–14). In view of the potential employment of serum leptin levels as an effective indicator for the diagnosis of endometriosis, in the present study we determined concentrations of the protein in a consecutive series of patients who underwent laparoscopy for gynecological problems. Specifically, serum leptin concentrations have been measured in women with and without laparoscopic evidence of endometriosis, and values of the protein were analyzed in relation to various clinical parameters associated with the disease. Subjects and Methods Subjects Serum samples were obtained from women who attended the endoscopic surgical service of the II Department of Obstetrics and Gynecology of the University of Milan to undergo gynecological laparoscopy. During a 9-month period, from February 2000 to October 2000, 67 consecutive women were enrolled in the study. The criteria for inclusion were that: 1) the patients were in their reproductive age (range 17– 46 yr); 2) subjects were normally cycling with a menstrual cycle length of 25–35 d, with an intraindividual variation of at most 3 d and an LH/FSH ratio less than 2, as evaluated at d 5 of the menstrual cycle; 3) none had received hormones for at least 3 months before surgery; and 4) there was no evidence of endometritis or previous autoimmune, liver, endocrine, or neoplastic disorders. Exclusion criteria were the laparoscopic diag-

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nosis of malignant diseases. All the women underwent clinical examination, including measurement of height and weight and hormonal assessment for gonadotropins and E2 during the early follicular phase. The mean body mass index (BMI), defined as weight in kilograms divided by the square of height in meters, was calculated. Information was obtained on the presence of dyspareunia, dysmenorrhea, and/or abdominal pain (summarized as yes when visual analog score exceeded 5 in at least one of these symptoms) and infertility. Dating of the menstrual cycle phase was histologically confirmed and defined according to the criteria of Noyes et al. (15). Endometriosis was documented in 42 women. Diagnosis of ovarian endometriotic cysts and deep peritoneal lesions has always been confirmed histologically. Diagnosis of superficial peritoneal lesions was based on direct visualization when endometriotic implants presented as typical lesions (4). Lesions whose aspect was dubious were always removed to confirm their endometriotic nature by histologic examination. Three physicians active in the evaluation and treatment of endometriosis staged patients according to the revised American Society for Reproductive Medicine classification (16). Stage of the disease was found to be minimal (stage I) in 17 patients, mild (stage II) in 2 patients, moderate (stage III) in 16 patients, and severe (stage IV) in 7 patients. Other gynecological benign pathologies were present in 6 of these 42 women with endometriosis (2 patients of uterine myomas and 4 patients of nonendometriotic benign adnexal cysts). Moreover, the presence of deep endometriosis, defined as lesions infiltrating to a depth of at least 5 mm beneath the peritoneal surface, was observed in 7 of these 42 women. Twenty-five women who underwent laparoscopy and in whom no endometriosis was found served as control group. Specifically, four patients with uterine myomas, seven patients with infertility or pelvic pain of unknown origin, seven with dermoid cysts, three with serous cysts, four with mucinous cysts were enrolled. In all cases, diagnosis was histologically confirmed. Patients were informed that sera would be used for research purposes and gave written consent. Approval for this study was granted by the local Human Institutional Investigation Committee. Laparoscopies were performed between 0730 and 1000 h after at least 12 h fasting. Peripheral venous blood samples were obtained aseptically from the patients immediately before laparoscopy and successively centrifuged at 2000 rpm for 10 min. The resulting sera were then frozen at ⫺20 C until assayed. The quantitative detection of leptin levels was performed using a commercially available RIA kit (DRG Instruments GmbH, Marburg, Germany) with an intraassay coefficient of variation of 3.4 – 8.3%, an interassay coefficient of variation of 6.5%, and sensitivity of 0.5 ng/ml. The concentration of leptin was expressed as ng/ml. Data are expressed as mean plus or minus sem. Differences between groups were compared, as appropriate, by unpaired t test or ANOVA and Fisher PLSD-test as posttest. Probability less than 0.05 was considered statistically significant.

Results

Table 1 shows the age and the phase of the cycle of women with and without endometriosis. The two groups have similar characteristics including BMI values (Table 1). Moreover, no statistically significant difference in serum leptin levels was observed in relation to the phase of the menstrual phase (data not shown). Serum leptin levels did not differ between women with and without endometriosis (Table 2); no statistically significant difference could also be detected when patients with endometriosis were evaluated according to the severity of the disease (Table 2). No correlation was demonstrated between serum leptin levels and both the number of pelvic endometriotic implants and the presence/absence of deep peritoneal endometriosis or of endometriotic cysts (Table 2). Moreover, serum levels of this molecule did not seem to be related to the clinical characteristics of the disease. Indeed, levels were similar between women who did or did not report pelvic pain, dyspareunia, and/or dysmenorrhea (data not shown) or with and without infertility complaints (Table 2). Finally, no statistically significant differences were ob-

Vigano` et al. • Comments

TABLE 1. Characteristics of women with and without endometriosis Endometriosis

Controls

42

25

32.5 23– 46 165 ⫾ 6 58.6 ⫾ 8.0 21.5 ⫾ 2.6

33 17– 40 163 ⫾ 6 60.5 ⫾ 14.2 22.6 ⫾ 4.9

6 (14) 16 (38) 20 (48)

2 (8) 12 (48) 11 (44)

15 (36) 22 (52) 5 (12)

9 (36) 14 (56) 2 (8)

No. Age (yr) Median Range Height (cm)a Weight (kg)a BMI (kg/m2)a Phase of the cycle Menstrual Proliferative Secretory Main indication for surgery Infertility Adnexal mass Pelvic pain

Values in parentheses are percentages. a Height, weight, and BMI are expressed as mean ⫾

SD.

TABLE 2. Leptin serum concentration in women with and without endometriosis No. of patients

Diagnosis Controls Endometriosis stage I–II Endometriosis stage III–IV Peritoneal endometriotic implants 0 ⱕ3 ⱖ4 Deep endometriosis Yes No Endometriotic cyst Yes No Infertility Yes Not reported

Leptin (ng/ml)

BMI (kg/m2)

P

25 20 22

12.5 ⫾ 9.4 22.6 ⫾ 4.9 NS 12.5 ⫾ 8.4 21.3 ⫾ 2.6 11.8 ⫾ 7.7 21.6 ⫾ 2.6

33a 20 14

13.9 ⫾ 9.6 22.8 ⫾ 4.4 NS 12.0 ⫾ 8.6 21.3 ⫾ 2.5 8.8 ⫾ 3.5 20.7 ⫾ 2.2

7 60

12.8 ⫾ 8.3 22.0 ⫾ 3.2 NS 12.2 ⫾ 8.6 21.9 ⫾ 3.7

22 45

11.8 ⫾ 7.7 21.6 ⫾ 2.6 NS 12.5 ⫾ 8.9 22.0 ⫾ 4.1

24 43

14.4 ⫾ 9.7 21.9 ⫾ 3.0 NS 11.1 ⫾ 7.6 21.9 ⫾ 4.0

NS, Not significant. a The number of patients without endometriotic implants might have been overestimated, because in five women with endometriotic cysts, severe pelvic adhesions did not allow adequate pelvic exploration at laparoscopy.

served when data were analyzed separately according to the different pathological entities diagnosed in the control group (data not shown). Discussion

To date, two previous studies reported on the association between serum leptin levels and endometriosis. Matalliotakis et al. (17) found similar serum levels of the protein in a limited number of nontreated endometriosis patients vs. a control group without the disease; in contrast, Matarese et al. (5) demonstrated a significant and marked increase of serum leptin concentrations in 13 patients with endometriosis, compared with 15 age- and BMI-matched controls. The results of the present study performed on a larger series indicate that no differences in serum leptin concentrations exist between women with and without endometriosis.

Vigano` et al. • Comments

The rationale to evaluate serum leptin levels in endometriosis was first based on the observation that leptin is absolutely critical for normal reproductive function (18). However, where leptin acts to exert its effect on reproduction is not yet resolved. Many data support the notion that the protein actions involve effects in the brain and, more specifically, the hypothalamus because it is important for controlling GnRH release and sexual behavior. Moreover, it has been suggested that the influence of leptin on reproduction might involve actions outside the brain as well. Interestingly, although the endometrium does not express leptin mRNA, both the full-length and the short-form variants of the leptin receptor transcript are expressed in this tissue (19). Leptin receptor mRNA expression peaked in the early secretory phase and was also present in decidual tissue of early gestation suggesting a role of the molecule, in concert with other growth factors and cytokines, in the acceptance of the fertilized egg and in early human development. Second, leptin seems to be actively involved in two of the major phenomena associated with endometriosis, specifically angiogenesis and local inflammation. Recent evidence suggest that leptin may act as angiogenic factor both in vitro and in vivo (10, 20). Furthermore, inflammatory cytokines, which are known to be activated in endometriosis, have been shown to stimulate leptin secretion (21, 22), and the protein seems to be necessary for the induction and maintenance of the proinflammatory T-helper-1 immune response (8). Based on these observations and the complete absence of a reliable marker of endometriosis, this study was aimed to investigate the possibility that leptin levels might represent an effective indicator of the disease. Indeed, from the data presented herein, it is evident that leptin measurement cannot be used for the diagnosis of endometriosis. Protein levels in serum seem to be associated with neither early stages nor severe disease. Moreover, no association was observed with specific forms of the disease that require a massive neoangiogenesis such as deep endometriosis or with specific symptoms that may reflect a peritoneal inflammatory situation. The discrepancies between our data and previous observations by Matarese et al. (5) are difficult to explain; a possible explanation may be related to the difference in the number of patients affected by infertility. Indeed, in their study, those authors included a significantly higher proportion of infertile subjects in the endometriosis group vs. controls. In contrast, our populations were homogeneous with respect to the frequencies of infertile subjects. Infertility, rather than endometriosis, may be related to the elevation of leptin levels (23). In keeping with this hypothesis, in our study, serum levels of leptin tend to be higher in patients complaining of infertility. Further studies including healthy fertile women as a control group are required to investigate this issue. In this context, it should also be noted that Mantzoros et al. (23) found lower leptin concentrations in serum taken before human CG administration in women who succeeded in becoming pregnant during assisted reproductive techniques. This finding resulted markedly evident and statistically significant when considering levels of the protein in follicular fluid, thus suggesting a direct intraovarian action of the protein, possibly interfering with the development of dominant follicles and oocyte maturation. In conclusion, the results of this study indicate that serum concentrations of the ob gene product, leptin, cannot reliably

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be used for the diagnosis of endometriosis; studies are underway to elucidate the relationship between leptin and endometrial system and to determine a possible contribution of the molecule in implantation and early pregnancy development. Acknowledgments Received May 25, 2001. Accepted November 19, 2001. Address all correspondence and requests for reprints to: Dr. Anna Maria Di Blasio, Molecular Biology Laboratory, Istituto Auxologico Italiano, Viale Monte Nero 32, 20135 Milano, Italy. E-mail: a.diblasio@ auxologico.it. This work was supported by the EndoBank program of Arevia GmbH.

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