American Journal of Obstetrics and Gynecology
Volume 176 • Number 3 • March 1997
Copyright © 1997 Mosby-Year Book, Inc.

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Gynecology

Dr.Sinan DOĞANTÜRK

Ankara

Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis

Tasuku Harada MD

Hiroki Yoshioka MD

Souichi Yoshida MD

Tomio Iwabe MD

Yoshimasa Onohara MD

Masahiro Tanikawa MD

Naoki Terakawa MD

Yonago, Japan

OBJECTIVE: Our purpose was to investigate the relationship between the levels of interleukin-6, interleukin-6 soluble receptor, and tumor necrosis factor-alpha in peritoneal fluid and the size and number of active red endometriotic lesions.

STUDY DESIGN: In a university hospital 39 women of reproductive age underwent either laparoscopy for infertility workup or laparoscopic surgery for ovarian chocolate cysts. Peritoneal fluid was collected by laparoscopy. Active lesions, such as red flamelike lesions, glandlike lesions, and red vesicles, were scored according to the revised American Fertility Society classification system according to the size and number of active lesions. Peritoneal fluid levels of interleukin-6, interleukin-6 soluble receptor, and tumor necrosis factor-alpha levels were determined by enzyme-linked immunosorbent assays. The relationship between peritoneal fluid concentrations of interleukin-6 and tumor necrosis factor-alpha and the score of active endometriosis was investigated.

RESULTS: Peritoneal fluid levels of interleukin-6 and tumor necrosis factor-alpha were significantly higher in patients with endometriosis compared with patients without endometriosis. The concentrations in patients with active endometriosis increased as the size and the number of active lesions increased. Cyclic variations in interleukin-6 concentrations were seen in peritoneal fluid from patients with endometriosis; the concentrations in the secretary phase were significantly higher than those in the proliferative phase.

CONCLUSIONS: Increased peritoneal fluid levels of interleukin-6 in patients with active red endometriosis may relate to endometriosis-associated infertility and to the pathogenesis of endometriosis.

Key words:

Endometriosis,

interleukin-6,

tumor necrosis factor-alpha,

peritoneal fluid,

infertility,

pathogenesis

From the Department of Obstetrics and Gynecology, Tottori University School of Medicine.

Received for publication August 2, 1996; revised November 18, 1996; accepted November 22, 1996.

Reprint requests: Tasuku Harada, MD, Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683, Japan.

Endometriosis remains an enigma in spite of extensive clinical investigations and experience. The pathogenesis of endometriosis and its link to infertility is controversial. ^[1] A recently proposed theory suggests that peritoneal fluid in women with endometriosis contains an increased number of activated macrophages that secrete a variety of local products, such as growth factors and cytokines. ^[2] These factors have been implicated in the control of implantation and the growth of endometrial cells outside the uterus. ^{[3] [4] [5]} Cytokines are also believed to be related to infertility in women with endometriosis. Interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) have been detected in peritoneal fluid ^{[5] [6]} and are known to have adverse effects on embryo development. ^[7] Although there is a lack of consensus regarding whether subtle endometriotic lesions should be surgically or medically treated to achieve increased fecundity in part as a result of our lack of understanding of the pathophysiologic mechanisms of infertility in women with endometriosis. Thus to investigate the relationship of peritoneal fluid levels of cytokines to infertility in women with minimal or mild endometriosis is of clinical interest. In addition, these subtle forms of endometriosis ("red" endometriosis) represent active lesions that have been shown to invade the extracellular matrix. ^[8]

Interleukin-6 (IL-6) is a multifunctional cytokine involved in the regulation of the immune response, hematopoiesis, and inflammation. It is produced in response to infection or injury by a variety of cell types, including monocytes, lymphocytes, fibroblasts, endothelial cells, and keratinocytes. ^[9] The peritoneal fluid level of IL-6 is increased in women with pelvic disorders. ^[10] Rier et al. ^[11] showed that elevated levels of IL-6 were present in peritoneal fluid of patients with stage III-IV endometriosis. The biologic activities of IL-6 is modulated by several factors. TNF-alpha induces the production of IL-6 from endometrial stromal cells. ^[12] It was recently suggested that the IL-6 soluble receptor may regulate the ability of IL-6 to stimulate estrogen synthesis in breast cancer cells. ^[13]

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*Active endometriotic lesions were scored according to revised American Fertility Society classification system.

In this study we measured the peritoneal fluid levels of IL-6, IL-6 soluble receptor, and TNF-alpha and investigated the correlations between peritoneal fluid levels of IL-6 and TNF-alpha and the size and number of red active endometriotic lesions.

Material and methods

Subjects.

Peritoneal fluid was obtained from 38 Japanese women of reproductive age (25 to 40 years old) who were undergoing laparoscopy for infertility workup or laparoscopic cystectomy for ovarian chocolate cysts. All laparoscopic procedures were performed with the patient under general anesthesia between days 5 to 30 of the menstrual cycle. Accurate menstrual cycle dating at surgery was available for the majority of the patients. Of the 38 patients studied, 19 had pelvic endometriosis and 19 were free of endometriosis.

Endometriosis was staged according to the criteria of the revised American Fertility Society classification system by laparoscopic inspection of the pelvis. ^[14] Observations and surgical procedures were recorded on videotape. We assessed the size and number of all active nonpigmented lesions at laparoscopy and on viewing the videotapes. Active lesions were identified according to the criteria of Jansen and Russell ^[15] and Wiegerinck et al. ^[16] We studied lesions classified as "red," which have a vesicular or red flamelike appearance or exist as a glandular excrescence. The red flamelike lesion is an elevated blister or bleb that appears to be filled with pink to red hemorrhagic fluid and is surrounded by stroma

* p < 0.05, versus no endometriosis.

and vascularization. Submesothelial extravasation of blood beyond the limits of the vesicle is frequently observed in flamelike lesions. ^[15] The glandular lesion, or red papule, is a raised or elevated lesion that may be of a polypoid nature and is associated with a fine vascularization. Both red and pinkish vesicles were included in the category of active lesions. ^[16] Individual active nonpigmented lesions were scored on a 4-point scale according to the revised American Fertility Society scoring system: 0, no active endometriosis; 1, active lesion(s) <1 cm; 2, active lesion(s) 1 to 3 cm; 4, active lesion(s) > 3 cm. Total scores of these lesions were calculated (Table I) . Lesions were scored by examiners without knowledge of the peritoneal fluid concentrations of cytokines.

Collection and isolation of samples.

Peritoneal fluid was obtained with use of a laparoscopic cannula immediately after the introduction of the laparoscope. Aspiration was performed under direct visualization from the posterior cul-de-sac and anterior vesicouterine fold. We recorded the total fluid volume obtained. Fluid samples were centrifuged at 800 g for 10 minutes at 4° C to separate the cell pellet and the supernatant. The cell-free supernatant was then stored at -70° C until it was assayed.

Assays.

The concentrations of IL-6, IL-6 soluble receptor, and TNF-alpha were determined with specific sandwich enzyme-linked immunosorbent assays (R & D Systems, Minneapolis, MN). Microtiter plates were coated with monoclonal antibodies specific for IL-6, IL-6 soluble receptor, or TNF-alpha. The standards and samples were transferred to the wells and any IL-6, IL-6 soluble receptor, or TNF-alpha present was bound by the immobilized antibody. After unbound protein was washed away, an enzyme-linked polyclonal specific antibody was added to the wells to sandwich the IL-6, IL-6 soluble receptor, or TNF-alpha immobilized during the first incubation. An amplifier solution was added to the wells and color intensity was measured. The detection limits of the enzyme-linked immunosorbent assays were 0.1 pg/ml for IL-6, 140 pg/ml for IL-6 soluble receptor, and 0.12 pg/ml for TNF-alpha. The intraassay variations were <5%.

Statistical analysis.

The clinical and biochemical data are presented in Table II as the group mean ± SE. Because cytokine data were not normally distributed, they were analyzed with nonparametric analysis (Mann-

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Figure 1. Peritoneal fluid concentrations of IL-6 in women without and with endometriosis. Horizontal lines, Means. IL-6 levels are higher in women with endometriosis (median 28 pg/ml) than in women without endometriosis (median 20 pg/ml), p < 0.05, Mann-Whitney U test.

Whitney test). Correlation analysis with concentrations of cytokines and scores of active lesions were performed with linear regression analysis. A p value <0.05 was regarded as statistically significant.

Results

IL-6, IL-6 soluble receptor, and TNF-alpha levels.

There were significant differences in the peritoneal fluid volume and levels of IL-6 and TNF-alpha between women with and without endometriosis (Table II , Figs. 1 and 2) . Concentrations of IL-6 soluble receptor in peritoneal fluid did not differ significantly between the two groups. In patients with endometriosis a cycle-specific pattern was observed, with the concentration of IL-6 in the secretory phase significantly greater than that in the proliferative phase (Fig. 3) . This cyclic change was not evident in peritoneal fluid from women without endometriosis.

The peritoneal fluid concentration of IL-6 and TNF-alpha increased as the score of active lesions increased (Figs. 4 and 5) . There were significant positive correlations between peritoneal fluid concentrations of log-transformed IL-6 or TNF-alpha and active lesion scores ( r = 0.723, p < 0.01 for IL-6; r = 0.614, p < 0.01 for TNF-alpha).

Comment

Peritoneal fluid levels of IL-6 and TNF-alpha were increased in patients with active endometriosis in the current study. To our knowledge, the current results show for the first time the presence of a relationship between superficial red endometriotic lesions and increased

Figure 2. Peritoneal fluid concentrations of TNF-alpha in women without and with endometriosis. Horizontal lines, Means. TNF-alpha levels are higher in women with endometriosis (median 3.74 pg/ml) than in women without endometriosis (median 2.3 pg/ml), p < 0.05, Mann-Whitney U test.

Figure 3. Distribution of IL-6 concentration in peritoneal fluid of women with endometriosis. Concentration of IL-6 in secretory phase is significantly greater than in proliferative phase ( p < 0.01).

levels of cytokines in peritoneal fluid. These results suggest that increased peritoneal fluid levels of cytokines may have roles in endometriosis-associated infertility and the pathogenesis of endometriosis.

Peritoneal endometriosis is classified into red, white, and black lesions. ^[17] Vascularization ^[17] and metabolic ^[18] activities are most pronounced in red lesions. These early

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Figure 4. Correlation between active lesion score and IL-6 concentration in peritoneal fluid from women with endometriosis. Data of IL-6 were log transformed. y = 1.024 + 0.227 x, r = 0.723, p < 0.01.

lesions invade the extracellular matrix. ^[8] We therefore classified red lesions as active lesions. Jansen and Russell ^[15] have shown that nonpigmented lesions have the histologic characteristics of endometriosis and suggested that these lesions are related to "unexplained infertility."

IL-6 is considered a major indicator of the acute-phase inflammatory response. IL-6 exhibits several biologic activities, including the induction of acute-phase proteins in hepatocytes, ^[19] the growth and differentiation of B lymphocytes, ^[20] and the activation of T lymphocytes. ^[21] Recent evidence suggests that this cytokine has important functions in reproductive physiologic mechanisms, including the regulation of ovarian steroid production, folliculogenesis, and early events related to implantation. ^{[22] [23]} It has also been suggested to be an important immunomodulator of the human endometrium. ^[12]

IL-6 and TNF-alpha in peritoneal fluid are secreted from peritoneal leukocyte components, including the monocytes and macrophages. Macrophages, which are present in the peritoneal cavity, are potent producers of IL-6, interleukin-1, and TNF-alpha. ^{[5] [6] [24]} A significantly elevated total number of macrophages is associated with endometriosis. ^[25] Increased levels of IL-6 in the secretory phase were observed in the current study. This increase may have relevance to the increased number of peritoneal fluid macrophages in the secretory phase. ^[25] Although a recent report demonstrated that peritoneal leukocytes from patients with endometriosis exhibit defects in the production of IL-6, ^[26] recent data suggest that endometrial cells present in peritoneal endometriotic implants may be another important source of IL-6. ^[11] Furthermore, Tseng et al. ^[27] reported that stromal cells cultured from endometriosis implants secrete greater concentrations of IL-6 than do stromal cells from both eutopic endometrium from women with endometriosis and endometrium

Figure 5. Correlation between active lesion score and TNF-alpha concentration in peritoneal fluid from women with endometriosis. y = 1.082 + 2.239 x, r = 0.614, p < 0.01.

from normal controls. The current results also suggest that peritoneal implants, particularly the so-called "red" lesions, may be a source of this cytokine. Immunohistochemical studies are needed to provide direct evidence of expression of IL-6 by these tissues. The results of this study support an increase in the levels of IL-6 in peritoneal fluid. Although this increase is small, it does appear to be significant. Inconsistent results have been reported on the levels of IL-6 detected in human peritoneal fluid. ^{[10] [11] [26]} It is likely that these conflicting data are related to antibody specificity of different enzyme-linked immunosorbent assay kits.

The production of IL-6 by endometriotic implants may result from stimulation by increased levels of TNF-alpha noted in peritoneal fluid of endometriosis. ^[2] In the current study the peritoneal fluid levels of TNF-alpha in women with endometriosis were greater than those in women without endometriosis. Furthermore, a significant correlation between IL-6 and TNF-alpha ( r = 0.38, p < 0.02) was observed. These data imply that TNF-alpha may have a key role in controlling cytokine synthesis in the peritoneal environment of endometriosis. Studies have shown that IL-6 soluble receptor is proteolytically cleaved from 80 kd membrane-bound IL-6 receptor ^[28] and can enhance the bioactivity of IL-6 by direct binding of the IL-6 soluble receptor/IL-6 complex to gp 130. ^{[29] [30]} IL-6 soluble receptor also may up-regulate the ability of IL-6 to produce estrogen in breast cancer cells. ^[13] The current results show that there is no difference in the peritoneal fluid levels of IL-6 soluble receptor between the women with and those without endometriosis. The results are inconsistent with those of other studies. ^[11] Evaluation of the IL-6 receptor expression in endometriotic tissues may be important for interpretation of the data. Further investigations are awaited to understand the regulation of IL-6 soluble receptor production and the role of this soluble receptor in peritoneal fluid of endometriosis.

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The revised American Fertility Society classification system is widely used to stage endometriosis. However, this system has been criticized because it does not evaluate activity of the disease. ^[12] The observations of this study suggest that a new classification system that includes a means of evaluating active lesion is needed.

In conclusion, the current results show that increased concentrations of cytokines in peritoneal fluid associated with active endometriotic lesions may relate to endometriosis-associated infertility and pathogenesis of the disease.

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