Concentration of osteoprotegerin (OPG) in peritoneal fluid is increased in women with endometriosis

Miyuki Harada, Yutaka Osuga1, Tetsuya Hirata, Yasushi Hirota, Kaori Koga, Osamu Yoshino, Chieko Morimoto, Toshihiro Fujiwara, Mikio Momoeda, Tetsu Yano, Osamu Tsutsumi and Yuji Taketani

Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan

1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. Email: yutakaos-tky{at}umin.ac.jp


    Abstract
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 Materials and methods
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BACKGROUND: Failure of apoptosis of refluxed endometrial cells within the peritoneal cavity is a possible etiologic factor for development of endometriosis. Osteoprotegerin (OPG) is a survival factor that exerts its effect by binding to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thus preventing TRAIL from binding to the apoptosis receptors DR4 and DR5. In the present study, we addressed the possibility that the TRAIL/OPG system is involved in the pathogenesis of endometriosis. METHODS: Concentrations of OPG and TRAIL in the peritoneal fluid (PF) of women with or without endometriosis were measured using specific enzyme-linked immunoabsorbent assay. The expression of DR4 and DR5 in the endometriotic tissue was examined by reverse transcription-polymerase chain reaction. RESULTS: OPG concentrations in PF of women with endometriosis were significantly higher than those of women without endometriosis (P=0.006). With respect to the stages of the disease, the concentrations of OPG in women with stage III/IV endometriosis were significantly higher than in those without endometriosis and those with stage I/II endometriosis. On the other hand, the ratios of TRAIL/OPG concentrations were significantly lower in stage III/IV endometriosis compared to those in non-endometriosis and stage I/II endometriosis. DR5 mRNA expression was clearly detected in all the endometriotic tissues studied. CONCLUSIONS: These findings suggest that the TRAIL/OPG system is involved in the pathophysiology of endometriosis, possibly affecting the apoptosis of endometriotic cells.


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Endometriosis, defined by the presence of endometrium-like tissues outside the uterus, remains an enigmatic disease. Although the precise pathogenesis of endometriosis has not been elucidated, a widely held theory is that endometriosis originates from the uterine endometrium that is transported to the peritoneal cavity via retrograde menstruation. It has been suggested that the failure to eliminate refluxed endometrial cells, possibly due to abnormalities of the immune system, is conducive to the development of endometriosis (Lebovic et al., 2001Go). In this context, apoptosis has recently been suggested to be important in eliminating refluxed endometrial cells, and a decreased susceptibility of endometrial cells to apoptosis is proposed to be a causative factor in endometriosis (Dmowski et al., 1998Go; Gebel et al., 1998Go; Garcia-Velasco and Arici, 2003Go).

The regurgitated endometrial debris bathes in peritoneal fluid (PF), and this is a significant constituent that influences the destiny of the refluxed endometrial cells. Mounting evidence that concentrations of various substances such as prostaglandins, growth factors, cytokines and chemokines in PF are aberrant in women with endometriosis (Osuga et al., 1999Go, 2000Go, 2002Go; Koga et al., 2000Go; Yoshino et al., 2003Go) suggests that these molecules are involved in the pathogenesis of the disease. In addition, immune cells in PF in endometriosis have been shown to be defective in numbers or activities, which may indicate the association of the immune system with the pathogenesis (Berkkanoglu and Arici, 2003Go). Given that anti-apoptosis of refluxed endometrial cells is an etiological event in endometriosis, it would be reasonable to speculate that certain molecules in PF modulate apoptosis of the endometrial cells and influence the development of endometriosis.

Osteoprotegerin (OPG) has recently been shown to be a survival factor in several cell types (Malyankar et al., 2000Go; Holen et al., 2002Go; Shipman and Croucher, 2003Go;). The survival effect is mainly exerted by binding to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily (Emery et al., 1998Go). TRAIL induces the apoptosis of susceptible cells upon binding to its receptors, TRAIL receptor 1 (TRAIL-R1, DR4) (Pan et al., 1997Go) and TRAIL-R2 (DR5) (Walczak et al., 1997Go), while OPG inhibits TRAIL-induced apoptosis by antagonizing TRAIL. Despite the wide-ranging expression of OPG in human tissues and its distinctive anti-apoptotic property, the relevance of OPG in endometriosis has not yet been studied.

We hypothesized that the aberrant OPG-related cytokine system in the peritoneal cavity is related to the development of endometriosis. To address this in the present study, we measured concentrations of OPG and TRAIL in PF of women with or without endometriosis.


    Materials and methods
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 Abstract
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 Materials and methods
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A total of 64 women of reproductive age with (n=40) and without (n=24) endometriosis participated in this study. They underwent laparoscopy for pain, infertility and/or other benign gynecologic disorders. Informed consent was obtained from each woman prior to laparoscopy. All the women had regular menstrual cycles. None of the women took medications that affect hormonal and/or immunological status within 3 months before laparoscopy, or had undergone surgical treatment for endometriosis within 1 year. Endometriosis was diagnosed both laparoscopically and histologically. The stage of endometriosis was evaluated according to the revised American Society for Reproductive Medicine (r-ASRM) classification (American Society for Reproductive Medicine, 1997Go). The distribution of the stage of endometriosis was as follows: stage I, n=11; stage II, n=5; stage III, n=12; stage IV, n=12. In the non-endometriosis group, 12 women were in the proliferative phase and 12 in the secretory phase. Eight women were in each phase in stage I/II endometriosis, and 12 in each phase in stage III/IV endometriosis.

PF was collected via a laparoscopic cannula introduced into the cul-de-sac before any manipulative procedure. The fluid was centrifuged at 400 g for 10 min, and the supernatants were frozen and stored at –80°C until assay.

Peritoneum and endometriotic tissues were also collected from the women with endometriosis. Peritoneal bone marrow-derived cells (PBMC) from these women were obtained according to the method we previously described (Yoshino et al., 2003Go).

TRAIL and OPG concentrations in the PF were measured in duplicate in a blind fashion, using a specific enzyme-linked immunosorbent assay (ELISA) for TRAIL (R&D Systems Inc., Minneapolis, MN) and for OPG (Biomedica, Vienna, Austria). The minimum detectable concentration was 2.86 pg/ml for TRAIL and 0.14 pmol/l for OPG. The intra and interassay CVs were <10% for each.

Total RNA was extracted from PBMC, peritoneum and endometriotic tissues using an RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was reverse-transcribed in a 20 µl volume using a TOYOBO RT–PCR kit (TOYOBO, Osaka, Japan). Standard PCR was performed using TOYOBO Rever Tra Dash (TOYOBO) according to the manufacturer's instructions. The PCR primers for OPG, DR4 and DR5 were as follows: OPG sense primer, 5'-ATAAAGGCATGCAAACCCAG-3'; OPG antisense primer, 5'-GCCTCAAGTGCCTGAGAAAC-3' (Simonet et al., 1997Go); DR4 sense primer, 5'-ACAGCAATGGGAACATAGCC-3'; DR4 antisense primer, 5'-GTCACTCCAGGGCGTACAAT-3' (Pan et al., 1997Go); DR5 sense primer, 5'-TGCAGCCGTAGTCTTGATTG-3'; DR5 antisense primer, 5'-GCACCAAGTCTGCAAAGTCA-3' (Walczak et al., 1997Go). The PCR amplification protocols for OPG, DR4 and DR5 were all as follows: (denaturing 98°C for 10 s, annealing 60°C for 4 s, extension 74°C for 20 s) x30 cycles.

The data were described as median and interquartile range (IQR). Mann–Whitney test was used for the statistical analysis. Statistical significance was defined as P<0.05.


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The mean age of patients in the endometriosis group (32.3, SD 4.2) and the control group (32.4, SD 4.5) was virtually identical.

The concentrations of TRAIL and OPG for all PF samples were above the lower limits of the assay. The OPG concentrations in the PF of the women with endometriosis (median, 6.82 pmol/l; IQR, 5.07–11.32) were significantly higher than those of the women without endometriosis (5.12 pmol/l, 3.72–6.78; P=0.006). The women with endometriosis, then, were subdivided into those with stage I/II and those with stage III/V, for further analysis. Next, we compared the concentrations of OPG and TRAIL in the proliferative phase and those in the secretory phase. As shown in Table I, no remarkable difference in the concentrations of OPG or in those of TRAIL was observed between the phases in each group. Accordingly, we dealt with the data from both phases as one homogenous group.


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Table I. OPG and TRAIL concentrations in the peritoneal fluid of women with or without endometriosis in the proliferative and secretory phases of the menstrual cycle

 
Figure 1 depicts the concentrations of OPG and TRAIL, and the ratio of TRAIL/OPG concentrations in the women without endometriosis and those with stage I/II and stage III/V endometriosis. The concentrations of OPG in stage III/IV endometriosis (7.58 pmol/l, 5.83–12.70) were significantly higher than those in non-endometriosis (P=0.006; Figure 1A). The concentrations of OPG in stage I/II endometriosis (6.44 pmol/l, 4.42–8.16) appeared to be intermediate, while the difference compared to those in stage III/IV endometriosis was significant (P=0.021). As shown in Figure 1B, the TRAIL concentrations in stage I/II endometriosis (19.67 pg/ml, 15.67–25.34) were significantly higher than those in non-endometriosis (12.74 pg/ml, 9.72–21.07) and stage III/IV (16.83 pg/ml, 13.92–19.44) endometriosis (P=0.004, P=0.028, respectively), whereas no difference was observed between the non-endometriosis group and the stage III/IV endometriosis group.



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Figure 1. Concentrations of OPG A and TRAIL B, and TRAIL/OPG ratio C in the peritoneal fluid of women without endometriosis (n=24), women with stage I/II endometriosis (n=16), and women with stage III/IV endometriosis (n=24). Boxes represent the distance between the first (25%) and third (75%) quartiles and horizontal lines in the boxes represent medians.

 
We further calculated the ratios of concentrations of TRAIL to those of OPG, considering that OPG antagonizes TRAIL. As shown in Figure 1C, the TRAIL/OPG ratios in stage III/IV endometriosis (1.88, 1.08–2.92) were significantly lower than those in non-endometriosis (2.85, 1.50–4.38, P=0.032) and those in stage I/II endometriosis (3.05, 1.92–3.76, P=0.015).

When we compared the concentrations of OPG and TRAIL, and TRAIL/OPG ratios between fertile and infertile women in the non-endometriosis group, to address whether infertility has any relevance to these concentrations, the values were similar between the groups (data not shown).

Figure 2 shows the expression of OPG in the peritoneum, the endometriotic tissues and the PBMC in the PF. OPG mRNA expression was detected in all the tissues and cells examined.



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Figure 2. RT–PCR analysis of OPG expression in the peritoneum, peritoneal bone marrow-derived cells (PBMC) and endometriotic tissues. Data are representative of at least three experiments. Lane 1, peritoneum; lane 2, PBMC; lane 3, endometriotic tissues; NC, negative control; M, DNA molecular weight standards.

 
DR5 mRNA expression was clearly detected in all the endometriotic tissues studied (Figure 3), while DR4 mRNA expression was not detected in those tissues in the present study (data not shown).



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Figure 3. RT–PCR analysis of DR5 expression in endometriotic tissues. Each lane shows samples from different women (lanes 1–3). NC, negative control; M, DNA molecular weight standards.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we demonstrated that concentrations of OPG in the PF were increased in women with endometriosis as compared to those in women without endometriosis. In addition, the concentrations in stage III/IV endometriosis were higher than those in stage I/II endometriosis. The expression of DR5, a receptor that mediates the apoptotic signal of TRAIL, was detected in the endometriotic tissues.

OPG mRNA was found to be expressed in a number of tissues, including lung, heart, kidney, liver, stomach, intestine, brain, spinal cord, thyroid gland and bone (Simonet et al., 1997Go; Yasuda et al., 1998Go). In addition, the present study showed the expression of OPG mRNA in PBMC, the peritoneum and the endometriotic tissue, which could be a possible source of OPG in PF.

The anti-apoptotic effect of OPG is exerted by antagonizing TRAIL, which induces apoptosis by binding to its receptors, DR4 and DR5. The expression of DR5 in endometriotic tissues, shown in the present study, implies that the TRAIL/OPG system regulates the susceptibility to apoptosis in endometriotic cells. Therefore, our findings of an increase in OPG concentrations in PF with a decrease in TRAIL/OPG concentration ratios in stage III/IV endometriosis, as compared to those in non-endometriosis and in stage I/II endometriosis, imply that the peritoneal environment is less apoptotic for endometriotic cells and is permissive for the progress of endometriosis.

Similar to TRAIL receptors, TNF receptor (TNFR) has an intracellular death domain that mediates the signal for apoptosis. TNF{alpha} activates TNFR and induces apoptosis, while soluble TNFR (sTNFR), which is derived from the extracellular portion of TNFR, antagonizes the apoptosis-inducing effect of TNF{alpha}. Interestingly, concentrations of sTNFR in PF are elevated in women with endometriosis (Koga et al., 2000Go). As TNFR are expressed in endometriotic tissues, the increase in sTNFR concentrations in PF was suggested to promote the development of endometriosis by sparing the cells from apoptosis. Combined with the present study, these findings may underscore the idea that anti-apoptotic molecules play important roles in the pathophysiology of endometriosis.

Apart from its ability to provoke apoptosis, TRAIL exhibits immunomodulatory capacities. In animal models of autoimmunity, systemic TRAIL blockade led to exacerbation of the disease without affecting the degree of apoptosis in inflammatory cells (Song et al., 2000Go; Hilliard et al., 2001Go). In addition, TRAIL has been shown to inhibit T cell activation and proliferation without inducing T cell death (Lunemann et al., 2002Go). Given that immune dysfunction is involved in the pathophysiology of endometriosis (Nothnick, 2001Go; Berkkanoglu and Arici, 2003Go), it is feasible that aberration of the TRAIL/OPG system causes the derangement of immune homeostasis in the peritoneal cavity in women with endometriosis.

The limitation of the current study was the inability to determine whether the findings are causes or results of endometriosis. In this sense, the increase in TRAIL concentrations in stage I/II endometriosis is interesting, in that it might be due to the physiological response of the body to inhibit the progress of the disease beyond these early stages.

In summary, the present study demonstrated an increase in OPG concentrations in the PF of women with endometriosis, which may contribute to the pathophysiology of endometriosis through anti-apoptotic and immunomodulatory mechanisms.


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 Materials and methods
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Submitted on February 24, 2004; accepted on June 7, 2004.





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