Infertility is found in 10–15% of all couples 1 and defined as failure to conceive within 12 months of unprotected intercourse 2. In ∼15–17% of couples, infertility is unexplained 3. Leptin, the hormone encoded by the obesity (Ob) gene, is a 146 amino acid protein with a tertiary structure similar to that of cytokines 4. Although leptin was originally considered to be exclusively expressed in white adipose tissue, subsequent reports showed that leptin is expressed in several other areas, such as the hypothalamus 5, pituitary gland 6, fundic gastric epithelium 7, skeletal muscle, syncytiotrophoblast 8, and mammary epithelium 9. Leptin receptors (Ob-Rs) have been identified in the hypothalamus, gonadotropic cells of the anterior pituitary 6, granulosa, theca, and interstitial cells of the ovary 10, endometrium 11, and Leydig cells 12. This multifocal expression of leptin, as well as the dense presence of Ob-Rs at all levels of the hypothalamus–pituitary–gonadal axis, implies that the nutritional/leptin regulation of reproduction involves a complex network of interactions at multiple levels to regulate the hypothalamus–pituitary–gonadal axis in a paracrine and/or an endocrine manner 11. Leptin gene expression is regulated by a variety of hormones, growth factors, and cytokines. Estrogens induce leptin production 13, whereas androgens suppress leptin production 14, providing an explanation for the sexual dimorphism in serum leptin levels. Insulin increases leptin production 15, and this may contribute toward the decrease in plasma leptin levels that occurs during fasting and the hyperleptinemia that accompanies insulin resistance states 16. Proinflammatory cytokines, such as tumor necrosis factor and interleukin-1, may also directly induce leptin gene expression 17. Leptin regulates food intake and energy expenditure and participates in angiogenesis 18. In addition, it has been shown to exert direct effects on hypothalamic–pituitary gonadotropin release and follicle-stimulating hormone and 17-estradiol synthesis in female rats. This effect was associated with the increased luteinizing hormone concentrations. Leptin was also found to prevent the ovulation delay induced by starvation in female rats 19. Plasma leptin levels correlate directly with body fat mass 20. Leptin concentrations in serum increase gradually during the early follicular phase and reach a plateau at the time of midcycle gonadotropin surge and reduce to the baseline during the luteal phase in both spontaneous and gonadotropin-induced cycles 21.
Patients and methods
The current cross-sectional study was carried out at the Ain Shams University Maternity Hospital during the period between October 2009 and September 2010. The study included infertile women who were admitted from the Infertility Outpatient Clinic and planned for diagnostic or operative laparoscopy as part of infertility workup and management. Infertility was defined as inability to conceive within 12 months of unprotected intercourse 22. Indications for laparoscopy included polycystic ovarian syndrome (PCOS), pelvic endometriosis, tubal factor infertility (diagnosed by hysterosalpingogram), and unexplained infertility [defined when normal husband’s semen analysis, documented ovulation (by transvaginal ultrasonographic folliculometry or a midluteal serum progesterone ≥10 ng/ml), and normal patent tubes (by hysterosalpingogram)] were found 3. Women with uncontrolled medical disease or receiving antipsychotic drugs were excluded. The study was carried out after receiving approval from the ethical and research committee of council of the Department of Obstetrics and Gynecology, Ain Shams University. The purpose and procedures of the study were explained to all enrolled women and a written informed consent was obtained from each participant. All laparoscopy procedures were performed during the early follicular phase of the cycle (following the hospital guidelines). Venous blood samples were withdrawn from women included in the study group on day 3 of a spontaneous menstrual cycle or progestin-withdrawal bleeding to determine basal serum levels of follicle-stimulating hormone, luteinizing hormone, and leptin. On the day of laparoscopy, peritoneal fluid was aspirated through an abdominal port from the vesicouterine and Douglas pouches. The clotted blood samples and aspirated peritoneal fluid were centrifuged at 300g for 5 min; sera were stored at −70°C for hormonal assays.
Serum leptin assay
Serum leptin was assayed using enzyme-linked immunosorbent assay (Human Leptin RIA Kit; Linco Research, St Charles, Illinois, USA) on the basis of the ‘sandwich principle’. The microtiter wells were coated with a monoclonal antibody directed toward a unique antigenic site on the leptin molecule. An aliquot of patient sample containing endogenous leptin was incubated in the coated well with a specific rabbit antileptin antibody. A sandwich complex was formed. After incubation, the unbound material was washed off and an anti-rabbit peroxidase conjugate was added for detection of the bound leptin. After addition of the substrate solution, the intensity of color that developed was proportionate to the concentration of leptin in the patient sample. Calibration curves were constructed for the assay by plotting the absorbance value versus the concentration for each calibrator. Leptin concentrations of patients were then read from the calibration curve.
Sample size justification
Sample size was calculated using EpiInfo version 6.0, [Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA] setting the power (1−β) at 0.8, significance level (α) at 0.05. Data from a previous study 22 showed that the mean peritoneal fluid leptin concentration was 44.2±12.9 ng/ml in women with unexplained infertility and 4.6±1.2 ng/ml in women with PCOS (P<0.001). Calculation according to these values produced a minimal sample size of 11 cases in each group. Therefore, the total sample size was calculated to be 44 cases.
Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS for Windows) version 15.0 (IBM Corporation, New York, USA). Measured data were described as range, mean, and SD. Comparison between more than two metric groups was performed using one-way analysis of variance. Comparison between more than two categorical groups was performed using the χ2-test. The correlation between two metric variables was estimated using Pearson’s correlation coefficient. Significance level was set at 0.05.
A total of 45 women were included in the study. The mean age of the included women was 29.02±3.93 years (range: 20–39 years). The mean BMI was 25.49±2.76 kg/m2 (range: 21–31 kg/m2). The mean age of menarche was 13.04±1.49 years (range: 11–16 years). The mean duration of infertility was 4.48±1.76 years (range: 2–9 years). Of the 45 women included, 12 (26.7%) had PCOS, eight (17.8%) had pelvic endometriosis, 15 (33.3%) had tubal factor infertility, whereas 10 (22.2%) had unexplained infertility. Of the 45 women included, 25 (55.6%) had primary infertility, whereas 20 (44.4%) had secondary infertility. The median parity was 1 (range: 0–2). The median number of previous abortions was 0 (range: 0–3). There was no significant difference between women with different etiologies of infertility regarding age. Women with PCOS had significantly higher mean BMI and age of menarche when compared with women with other etiologies. The mean duration of infertility was significantly higher in women with endometriosis and unexplained infertility when compared with women with other etiologies. The proportion of women who had primary infertility was significantly higher in women with PCOS and endometriosis, whereas secondary infertility was significantly more prevalent among women with tubal factor and unexplained infertility (Table 1).
The mean serum level of leptin in all included women was 16.85±3.81 ng/ml (range: 11–24.2 ng/ml). The mean peritoneal fluid level of leptin in all included women was 30.09±5.47 ng/ml (range: 21.9–45 ng/ml). Women with PCOS had a significantly higher mean serum leptin level when compared with women with other etiologies, whereas women with endometriosis had a significantly higher mean peritoneal fluid leptin level when compared with other etiologies (Table 2).
There was a nonsignificant poor correlation between serum and peritoneal levels of leptin (r=−0.08, P>0.05). There was a significant positive correlation between serum leptin concentration and each of BMI (r=0.69, P<0.001) and age of menarche (r=0.42, P=0.004), and a significant negative correlation between serum leptin concentration and duration of infertility (r=−0.37, P=0.01). There was a significant negative correlation between peritoneal fluid leptin concentration and BMI (r=−0.32, P=0.03). There was a significant positive correlation between peritoneal fluid leptin concentration and duration of infertility (r=0.34, P=0.02). There was no significant correlation between either serum or peritoneal fluid leptin concentration and age (Table 3).
As leptin exerts significant effects on the female reproductive system, it might be related to infertility, which affects ∼10% of individuals at reproductive age and 15% of couples 20. Although the roles of leptin in the pathogenesis of PCOS and endometriosis have been investigated in a number of studies, there is still ambiguity and lack of evidence. The present study was designed to investigate the leptin concentrations in peritoneal fluid and serum samples of 45 women diagnosed with different etiologies of infertility. Tubal factor, PCOS, and endometriosis were determined as three major causes of infertility, and the remaining women for whom any valid pathology could not be established were considered to have unexplained infertility after laparoscopic evaluation. Women detected to have bilateral tubal occlusion by laparoscopic chromotubation were included in the tubal factor group. Ciofiet et al.  reported that preovulatory follicles released leptin into the peritoneal fluid and suggested that PCOS reduced leptin levels by interrupting the development of follicles. Mantzoros and colleagues supported the former finding by showing that amenorrheic and oligomenorrheic women had lower levels of leptin in peritoneal fluid 24. Gogacz and colleagues found that eight women with PCOS had significantly lower peritoneal fluid leptin concentrations compared with eight women with endometriosis and 15 women with unexplained infertility 25.
The present study confirmed that women with PCOS had significantly lower peritoneal fluid levels of leptin. However, it is difficult to clarify whether reduced follicular development decreases leptin levels in peritoneal environments of PCOS patients or a ‘primary’ leptin deficiency in the peritoneal environment alters ovarian functions in PCOS. Leptin is exclusively produced by adipose tissue; thus, plasma leptin levels correlate directly with BMI 20. As PCOS tends to cause weight gain or obesity, it might be related to elevated concentrations of leptin in plasma. In the study carried out by Larsen 22, PCOS patients had plasma leptin concentrations that were statistically similar to those of patients with endometriosis and unexplained infertility, but the correlation of BMI values with plasma leptin levels was unspecified. In the present study, PCOS patients were also found to have significantly higher BMI values and relevantly elevated plasma leptin levels. As both the leptin receptor and molecule are expressed in human endometrium, endometriosis implants may be considered as potential sources of leptin 11. Leptin was shown to induce the expression of matrix metalloproteinases, vascular permeability, and angiogenesis, which are believed to play an important role in the pathogenesis of endometriosis 26. Moreover, high doses of leptin are known to enhance mitogenic activity in cultured eutopic and ectopic endometrial stromal cells 27. De Placido et al.26 and Mahutte et al.28 reported that leptin concentrations in peritoneal fluid were significantly elevated in women with endometriosis compared with the control group. In a study carried out by Gogacz et al.25, eight women with endometriosis were found to have significantly higher peritoneal fluid leptin concentrations compared with eight women with PCOS and 15 women with unexplained infertility. Bedaiwy et al.29 compared 60 patients with endometriosis with 48 women (38 women undergoing tubal ligation/reanastomosis and 10 women with idiopathic infertility), and claimed that endometriosis resulted in significantly elevated levels of leptin in peritoneal fluid.
The present study confirmed the former findings, indicating significantly higher leptin levels in peritoneal environments of endometriosis patients compared with patients with unexplained infertility, PCOS, or tubal factor. Therefore, leptin might be accepted as a factor responsible for acute inflammation, angiogenesis, and endometrial invasion, which participate in the pathogenesis of endometriosis. Wu et al.27 have indicated that the expressions of leptin molecules and receptors are significantly increased in endometriosis implants compared with normal endometrium. However, mRNA levels for the receptor were found to be decreased in association with the advancing stage of endometriosis. Several studies clinically confirmed that peritoneal fluid leptin levels were inversely correlated with the stage of endometriosis 28. In contrast, Bedaiwy et al. 29 documented a positive correlation between peritoneal fluid leptin levels and the endometriosis stage. The present study is in agreement with the latter finding that peritoneal fluid leptin levels correlate directly with the extent of endometriosis.
The contradictory results of these investigations might be attributed to the extent and severity of peritoneal involvement in the selected patients diagnosed with endometriosis. In the studies carried out by De Placido et al.26 and Mahutte et al.28, the majority of the endometriosis patients were affected by advanced-stage endometriosis and had ovarian endometriomas instead of peritoneal implants. However, the majority of endometriosis patients in the present study and those of Bedaiwy and colleagues had peritoneal implants. Consequently, it might be hypothesized that leptin may be an active factor in the pathogenesis of PCOS and endometriosis, which are two major causes of primary infertility. A mild leptin deficiency in the peritoneal environment may interrupt follicular development and ultimately lead to PCOS. Leptin has angiogenic and mitogenic properties, which trigger inflammatory cytokines and eventually result in the development of endometriosis implants. Significantly higher levels of leptin in peritoneal environments of endometriosis patients strongly imply the important role of this common pathology. Further evaluation is required to shed light on the role of leptin in primary infertility and specifically in endometriosis.
The authors thank Dr Rania Abo Shady, assistant professor of Clinical Pathology, Ain Shams University, for her contribution in performing the laboratory work.
Conflicts of interest
There are no conflicts of interest.
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