Hepatocyte growth factor concentrations are elevated in peritoneal fluid of women with endometriosis

Y. Osuga, O. Tsutsumi, R. Okagaki, Y. Takai, A. Fujimoto, A. Suenaga, M. Maruyama, M. Momoeda, T. Yano and Y. Taketani

Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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The concentrations of hepatocyte growth factor (HGF) in peritoneal fluid (PF) from women with endometriosis (n = 36) and without endometriosis (n = 40) were measured. All of the PF samples examined contained detectable concentrations of HGF. The HGF concentrations in PF from women with stage III/IV endometriosis (0.906 ng/ml, 0.561–1.185; median, interquartile range) were significantly higher (P < 0.0001) than those from women without endometriosis (0.315 ng/ml, 0.251–0.472). The HGF concentrations from women with stage I/II endometriosis (0.417 ng/ml, 0.310–1.023) appeared to be intermediate. There were no apparent variations detected among the HGF concentrations in women in the follicular or luteal phases regardless of the presence of endometriosis. Interestingly, HGF concentrations in PF from women on gonadotrophin releasing hormone analogues, independent of the presence of endometriosis, were comparable with those from untreated women. Given the known mitogenic property of HGF in human endometrial cells, these results suggest that HGF might play a role in the progression of endometriosis.

Key words: endometriosis/hepatocyte growth factor/peritoneal fluid


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hepatocyte growth factor (HGF), which is identical to scatter factor (Weidner et al., 1991Go) and tumour cytotoxic factor (Shima et al., 1991Go), is a heparin-binding glycoprotein that consists of a 60 kDa {alpha}-chain and 30 kDa ß-chain linked by disulphide bonds (Gherardi et al., 1989Go). HGF was initially discovered as a hepatotrophic factor present in the plasma of patients with fulminant hepatic failure (Gohda et al., 1986Go). Later on, several lines of evidence have implied that HGF has mitogenic, motogenic and morphogenic functions in vitro on various epithelial cells derived from rodents and humans (Nakamura et al., 1986Go; Tajima et al., 1992Go).

Recently the presence of c-MET, the receptor of HGF, on human endometrial epithelial cells has been reported (Wagatsuma et al., 1998Go). Furthermore, HGF has been shown to stimulate the proliferation, migration and morphogenesis of endometrial epithelial cells (Sugawara et al., 1997aGo). With these observations in mind, we postulated that HGF may be relevant to the pathophysiology of endometriosis, and we therefore set out to examine the presence of HGF in peritoneal fluid (PF) in women affected by endometriosis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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A total of 76 women between 25 and 38 years of age undergoing laparoscopy either for pain and/or for infertility was included in this study. In these women, 57 women had normal menstrual cycles and 19 women had been treated with gonadotrophin releasing hormone (GnRH) analogue (leuprolide acetate depot; Takeda, Tokyo, Japan) for 4–6 months. Endometriosis was diagnosed laparoscopically and histologically. The extent of the disease was staged according to the revised American Fertility Society classification (1985). Peritoneal fluids were collected from women with (n = 36) and without (n = 40) endometriosis. The distribution of the stage of endometriosis was as follows: stage I, 8; stage II, 3; stage III, 10; and stage IV, 15. The reasons for GnRH analogue treatment were the treatment of endometriosis for women with endometriosis and the pre-operative treatment of fibroma for women without endometriosis.

HGF concentrations in PF were measured in duplicate, using a specific enzyme-linked immunosorbent assay (ELISA) in a blind fashion (Institute of Immunology, Tokyo, Japan). The limit of sensitivity of this ELISA was 0.1 ng/ml per sample. The coefficients of variation were <10%.

Statistical significance was determined with the Mann–Whitney test. Results are expressed as median and interquartile range. Statistical significance was defined as P < 0.05.


    Results
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 Abstract
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 Materials and methods
 Results
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All of the PF samples examined contained detectable concentrations of HGF above the assay limit (median 0.423 ng/ml; interquartile range 0.275–0.875). Figure 1Go shows the distribution of HGF concentrations in PF of each woman, stratified according to menstrual phases, presence of endometriosis and GnRH analogue treatment. In the women without endometriosis, the concentrations of HGF were not significantly different between the subgroups of follicular phase (0.284 ng/ml, 0.226–0.404), luteal phase (0.379 ng/ml, 0.267–0.603) and GnRH analogue treatment (0.296 ng/ml, 0.214–0.408). Likewise, for the women with endometriosis, the concentrations of HGF were not significantly different between the subgroups of follicular phase (0.906 ng/ml, 0.397–1.304), luteal phase (0.536 ng/ml, 0.351–1.001) and GnRH analogue treatment (0.955 ng/ml, 0.772–1.091). Looking at corresponding subgroups with or without endometriosis, HGF concentrations from women with endometriosis either in the follicular phase or in a hypo-oestrogenic state induced by GnRH analogue were significantly higher (P < 0.05 and P < 0.01 respectively) than those from women without endometriosis. Comparing HGF concentrations in luteal phase, the concentrations from women with endometriosis were apparently higher relative to those without endometriosis but this was non-significant. In the non-endometriosis group, the HGF concentrations in PF of women with uterine fibroids and those without fibroids were not significantly different (data not shown).



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Figure 1. The distribution of hepatocyte growth factor (HGF) concentrations in peritoneal fluid from women without endometriosis and women with endometriosis in the follicular phase, luteal phase and with gonadotrophin releasing hormone (GnRH) analogue treatment. Horizontal lines represent the medians. Women with endometriosis had significantly higher concentrations of HGF than those without endometriosis in GnRH treatment (P < 0.01) and in follicular phase (P < 0.05).

 
Because there were no significant variations in HGF concentrations during the menstrual cycle and no apparent changes in HGF concentrations with or without GnRH analogue treatment in both the non-endometriosis and endometriosis groups, we pooled the data to analyse HGF concentrations in relation to the severity of endometriosis. The HGF concentrations in women with different stages of endometriosis are depicted in Figure 2Go. The HGF concentrations in women with stage III/IV endometriosis (0.906 ng/ml, 0.561–1.185) were significantly higher (P < 0.0001) than those without endometriosis (0.315 ng/ml, 0.251–0.472). The HGF concentrations from women with stage I/II endometriosis (0.417 ng/ml, 0.310–1.023) appeared to be intermediate, though no statistical significance was found when compared with either non-endometriosis or stage III/IV endometriosis.



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Figure 2. The hepatocyte growth factor (HGF) concentrations in peritoneal fluid from women without endometriosis (n = 40), women with stage I/II endometriosis (n = 11) and women with stage III/IV endometriosis (n = 25). Boxes represent the distance between the first (25%) and third (75%) quartiles and horizontal lines in the boxes represent medians. Women with stage III/IV endometriosis had significantly higher concentrations of HGF than women without endometriosis (P < 0.0001).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study demonstrated that the concentrations of HGF in PF in women with endometriosis are elevated compared to women without endometriosis. At present, the mechanism of the elevation in HGF concentrations has yet to be clarified. One possibility is that an increase in interleukin-6 (IL-6) concentrations in PF in women with endometriosis (Punnonen et al., 1996Go; Harada et al., 1997Go) may be relevant to the present findings in light of the fact that IL-6 induces HGF promoter activity (Liu et al., 1994Go). In a recent study, IL-6 concentrations in PF were shown to be positively correlated with active lesion scores in women with endometriosis (Harada et al., 1997Go). In our study, active lesion scores of endometriosis were, on average, far higher in stage III/IV relative to stage I/II endometriosis (data not shown here). Higher HGF concentrations in stage III/IV endometriosis, therefore, may be consistent with the notion that IL-6 may increase the production of HGF. Furthermore, HGF has been shown to be produced by eutopic endometrial stromal cells (Sugawara et al., 1997bGo). Thus, it is likely that endometriotic stromal tissues are capable of producing HGF. If this is the case, an increase in the concentrations of IL-6 in PF may enhance the production of HGF from endometriotic tissues.

Another finding of the present study is that the elevated concentrations of HGF in non-treated women were comparable with those in women treated with GnRH analogue in the endometriosis group. Given the fact that HGF concentrations in PF from non-endometriotic women also did not differ with or without GnRH analogue treatment, it may be that HGF concentrations in PF are not affected by ovarian functions. Whatever the mechanism, it is conceivable that HGF may make a sharp contrast with IL-1 and tumour necrosis factor, their higher concentrations in PF with endometriosis decreasing after GnRH analogue treatment (Taketani et al., 1992Go).

Serum HGF concentration has been reported to show cyclic variation during the menstrual cycle (Negami et al., 1995Go). In our study, no particular variation during the menstrual cycle was detected in HGF concentrations in PF. At this time, it is difficult to explain the disparity between HGF concentrations in serum and those in PF in relation to the menstrual cycle. Although the sources of HGF present in serum are not clarified, HGF coming from endometrial tissue seems to constitute a substantial percentage, given the cyclic change in its serum concentrations during the menstrual cycle. Endometriotic implants can respond to gonadal hormones. However, the majority of the implants do not demonstrate synchronous histological changes with the comparable uterine endometrium. More specifically, about one-half of the implants demonstrate no morphological changes induced by cyclic secretion of ovarian steroids (Ochs and Schweppe, 1995Go). On the assumption that HGF in PF derives largely from endometriotic implants, differential hormonal responses of the implants may, in part, explain the lack of apparent changes in HGF concentrations in PF during the menstrual cycle.

HGF has been shown to be mitogenic in endometrial epithelial cells in vitro (Sugawara et al., 1997aGo). In addition, recent studies provided evidence suggesting that HGF stimulates angiogenesis (Moriyama et al., 1998Go; Van Belle et al., 1998Go). Assuming that these properties of HGF are at play in endometriotic tissues, an elevation of HGF concentration in PF may work to accelerate the growth of endometriosis. A recent study has suggested that HGF produced by peritoneal fibroblasts may modulate mesothelial cell morphology and thereby promote peritoneal dissemination of cancer cells (Yashiro et al., 1996Go). Given this possibility, it is intriguing to speculate that HGF might play a role in facilitating the implantation of endometrial cells onto the peritoneal surface in addition to its potential growth-promoting effect.

Like HGF, epidermal growth factor (EGF) and fibroblast growth factor (FGF) are thought to be mitogenic to endometrial cells (Smith, 1994Go). Furthermore, EGF has been shown to have a mitogenic effect on endometriotic cells of endometrial cyst (Taketani and Mizuno, 1992Go). However, EGF and FGF concentrations in PF do not differ between women with endometriosis and without endometriosis (Huang et al., 1996Go). In this sense, HGF is unique among growth factors mitogenic to endometrial cells, and therefore, may be a possible central player in the proliferation of endometriotic cells.


    Acknowledgments
 
We would like to thank Dr R.Matsuoka and Dr K.Naritaka for their co-operation in the collection of specimens.


    Notes
 
1 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on December 7, 1998; accepted on March 1, 1999.