Full text of DOI:10.1093/molehr/7.5.459

Molecular Human Reproduction Vol.7, No.5 pp. 459–462, 2001
Increased concentrations of cathepsin D in peritoneal fluid
from women with endometriosis
Nobuhiro Suzumori1,3,4, Yasuhiko Ozaki2, Mayumi Ogasawara1 and Kaoru Suzumori1
2
1Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya, Japan, Perinatal Center, Institution of
3
Physiology and Pharmacology, Go¨teborg University, Go¨teborg, Sweden and Department of Pathology, Baylor College of Medicine,
Houston, TX, USA
4To whom correspondence should be addressed at: Department of Pathology, Baylor College of Medicine, One Baylor Plaza S217,
Houston, TX 77030, USA. E-mail: suzumori@bcm.tmc.edu
To assess the release of the proteolytic enzyme cathepsin D in endometriosis, concentrations in peritoneal fluid and
serum were measured by ELISA in 54 women with (n ⍧ 33) and without (n ⍧ 21) endometriosis. Surgery was
scheduled in either the proliferative or secretory phase of the menstrual cycle. The concentrations of cathepsin D
in the peritoneal fluid were markedly elevated in the endometriosis patients (median 58 ng/ml, interquartile range
0–166 ng/ml) as compared to the controls (5 ng/ml, 0–86 ng/ml), especially in women with late stage disease (n ⍧
19, stages III/IV) and in those not undergoing gonadotrophin-releasing hormone (GnRH) agonist therapy (n ⍧ 15).
No significant difference was determined in cathepsin D concentrations of the serum from women with and without
endometriosis. We conclude that cathepsin D is an important factor that may contribute to the pathogenesis of
endometriosis, possibly by promoting digestion of extracellular matrix proteins. These results have implications for
the therapeutic efficacy of GnRH agonists.
Key words: cathepsin D/endometriosis/gonadotrophin-releasing hormone/peritoneal fluid
Introduction
2000; Matarese et al., 2000). Since cathepsin D is induced by
growth factors in breast cancer cells (Cavailles et al., 1989)
and in the uterus (Maudelonde et al., 1990), we speculated
that its concentrations in peritoneal fluid might be similarly
elevated in women with endometriosis.
Cathepsin D is an aspartyl acid protease widely distributed in
animal and human cells. It is initially synthesized as an inactive
52 kDa precursor (pro-cathepsin D) and proteolytic removal of
the aminoterminal pro fragment then results in an enzymatically
active 48 kDa heterodimer consisting of two chains of 14 and
34 kDa (Capony et al., 1987; Fortenberry and Chirgwin, 1995).
Cathepsin D is induced by oestrogens in mammary cancer
cells where its concentration is correlated with a high risk of
metastasis (Spyratos et al., 1989). Its gene expression is also
differentially regulated by sex-steroid hormones in human
uterine (Moulton and Boening, 1983; Maudelonde et al., 1990)
and breast cancer cells (Cavailles et al., 1989; Brouillet
et al., 1991).
Endometriosis features progressive growth and invasion
which is oestrogen-dependent (Rock, 1992). Several lines of
evidence suggest that cytokines, chemokines, and growth
factors are involved in the maintenance and development of
endometriotic lesions. Concentrations of several molecules are
known to be elevated in the peritoneal fluid of endometriosis
patients, examples being interleukins (IL) and vascular endo-
thelial growth factor (VEGF) (Fakih et al., 1987; McLaren
et al., 1996). Recently, various elevated factors in the peritoneal
fluid, for instance insulin-like growth factors (IGF), IGF-
binding proteins and leptin have also been reported (Kim et al.,
A previous study showed that in endometriosis patients,
endometriotic tissue levels of cathepsin D are significantly
higher than those in eutopic endometrium, suggesting potential
importance for implantation and invasive growth (Bergqvist
et al., 1996). However, to our knowledge, no study of cathepsin
D levels in peritoneal fluid and serum of endometriosis patients
has been published previously. Moreover, the function of
cathepsin D under the influence of gonadotrophin-releasing
hormone (GnRH) analogues has yet to be elucidated, despite
the hypo-oestrogenism associated with the GnRH treatment,
and the suggestion (Maudelonde et al., 1990) that cathepsin
D is induced by progesterone in the human endometrium.
Therefore, the present study was designed to assess the
menstrual cycle and endometriosis stage variations in proteo-
lytic enzyme cathepsin D levels in peritoneal fluid and serum
from women with this disease.
Materials and methods
Fifty-four women aged 21–48 years with endometriosis (n ϭ 33) and
without endometriosis (n ϭ 21) were enrolled in this study. The study
© European Society of Human Reproduction and Embryology
459

N.Suzumori et al.
Calculated values were expressed as the median and interquartile
range. Comparisons were made with Mann-Whitney and Kruskal-
Wallis non-parametric tests, with P Ͻ 0.05 being regarded as
statistically significant.
Table I. Characteristics of patients and concentrations of cathepsin D in
peritoneal fluid and serum
No. of
patients
Agea
(years)
Cathepsin D
concentrationb
(ng/ml)
Results
Peritoneal fluid
Controls
The peritoneal fluid of the women with endometriosis (n ϭ 33)
contained significantly higher (P Ͻ 0.001) concentrations of
cathepsin D (median 58 ng/ml, interquartile range 0–166 ng/
ml) than did peritoneal fluid from the controls (n ϭ 21), i.e.
women without endometriosis (5 ng/ml, 0–86 ng/ml) by the
Mann-Whitney test.
The endometriosis patients were staged from I to IV
depending on the severity of disease based on the Re-AFS
classification. The stage distribution during surgery was as
follows: total numbers (GnRH agonist-treated numbers): stage
I, 8 (3); stage II, 6 (3); stage III, 11 (5); stage IV, 8 (4). There
was significant variation (P Ͻ 0.001) in cathepsin D levels
between the control group (n ϭ 21), the endometriosis stages
I/ II group (n ϭ 14) and the endometriosis stages III/IV group
(n ϭ 19) by the Kruskal-Wallis test (Table I and Figure 1A).
In these patients, elevated cathepsin D levels in the peritoneal
fluid were noted in the stages III/ IV group (n ϭ 19) as
compared to the stages I/ II group (n ϭ 14) (Figure A,
P Ͻ 0.05).
No significant changes in cathepsin D during the menstrual
cycle were observed in any groups of the controls or endome-
triosis patients (Table I and Figure 1B). With respect to
the pre-operative GnRH agonist treatment, peritoneal fluid
concentrations of cathepsin D were lower for the GnRH
agonist-treated patients (n ϭ 15) than for the untreated cases
(n ϭ 18) (Figure 1B, P Ͻ 0.01).
Cathepsin D levels in the serum of the endometriosis patients
(n ϭ 27; median 0 ng/ml, interquartile range 0–2 ng/ml) were
not significantly different from those of the control group
(n ϭ 20; 0 ng/ml, 0–2 ng/ml) (Table I).
21
37 Ϯ 1.8
5 (0–86)c
Endometriosis
Total
Stages I and II
Stages III and IV
Proliferative phase
Controls
Endometriosis
Secretory phase
Controls
33
14
19
36 Ϯ 1.5
35 Ϯ 1.3
36 Ϯ 2.0
58 (1–166)c
21 (1–60)d
68 (1–166)d
10
19
36 Ϯ 1.9
35 Ϯ 1.6
4 (0–64)
40 (1–125)
11
14
38 Ϯ 2.9
36 Ϯ 2.0
5 (0–86)
82 (2–166)
Endometriosis
Serum
Controls
Endometriosis
20
27
35 Ϯ 1.6
34 Ϯ 1.3
0 (0–2)
0 (0–2)
aValues are means Ϯ SEM.
bValues are medians (interquartile range).
cP Ͻ 0.001, dP Ͻ 0.05 (determined by the Mann-Whitney test).
group and the control group were matched for age and menstrual
cycle phase. The study protocol was approved by the Institutional
Review Board of Nagoya City University and informed consent
was obtained from all patients. Regular ovulatory women with
endometriosis who had received GnRH analogue treatment (leuprolide
acetate depot; Takeda Seiyaku, Osaka, Japan) monthly for 4–6 months
(n ϭ 15) or no treatment before surgery (n ϭ 18), and individuals
with ovarian cystadenomas as the controls (n ϭ 21) were recruited.
Since there were insufficient data in normal disease-free women, data
from patients with ovarian cystadenomas were used as the controls.
The patients with GnRH agonist pre-treatment had had a 1 or 2
month(s) long wash-out period before operation. All the endometriosis
patients who gave informed consent to GnRH analogue treatment
were enrolled in the pre-treatment group; however, some patients
could not be diagnosed before surgery and some patients did not
accept pre-operative GnRH analogue treatment, being afraid of the
side-effects. We therefore had two groups of endometriosis patients
with and without GnRH analogue therapy. Basal body temperature
of patients was used to determine their cycle phase in this study. The
numbers of individuals representing proliferative and secretory phases
of the menstrual cycle are listed in Table I.
The patients with endometriosis were scored according to the
revised classification of the American Fertility Society (Re-AFS)
(American Fertility Society, 1985). Samples of peritoneal fluid were
obtained during laparoscopic surgery or laparotomy (cystectomy,
adnexectomy, or total abdominal hysterectomy). Samples of serum
were taken before surgery. The specimens of peritoneal fluid and
serum were clarified by centrifugation at 2000 g for 10 min and the
supernatants were then stored at –80°C until assayed.
Amounts of cathepsin D in the peritoneal fluid and serum were
determined with a cathepsin D enzyme-linked immunosorbent assay
(ELISA) kit (Oncogene Research Products, Cambridge, MA, USA)
following the manufacturer’s protocol. Samples from each patient
were measured in parallel and in duplicate to avoid interassay
variance. The optical density of each well was measured at dual
wavelengths of 450/595 nm. Concentrations of cathepsin D were
calculated by interpolation from the standard curve. The sensitivity
of the cathepsin D ELISA was Ͻ1 ng/ml, and the standard curve
range was 1–100 pg/ml.
Discussion
In the present study, we demonstrated that the concentration
of cathepsin D in peritoneal fluid is significantly elevated in
untreated women with endometriosis compared to the GnRH
agonist-treated patients or the controls.
Recent findings suggest that concentrations of several molec-
ules are elevated in the peritoneal fluid of endometriosis
patients. We have shown that secretory leukocyte protease
inhibitor is released from endometriotic tissue itself (Suzumori
et al., 1999) and it is reported that this is also the case for
IL-6 (Tseng et al., 1996). Previously it has been found
that cathepsin D levels (median 460 pmol/mg DNA) in
endometriotic tissues are significantly higher than in eutopic
endometrium (Bergqvist et al., 1996). Our present findings
suggest that cathepsin D production from spreading endometri-
otic lesions may be high so that an elevated level in the
peritoneal fluid becomes recognized in late stages of endome-
triosis, e.g. stages III and IV based on the Re-AFS classification.
Thus, the proteolytic enzyme cathepsin D could be involved
in the spreading and development of endometriosis and its
460

Cathepsin D in peritoneal fluid of endometriosis
endometriosis, indicates that hormonal induction of cathepsin D
may not be a major factor. Several lines of evidence suggest
that tissue cathepsin D concentrations appear to be correlated
more with tumour invasiveness than with hormonal respons-
iveness, independent of regulation by oestrogen (Spyratos
et al., 1989). Therefore, we suggest that invasion with endome-
triosis may occur locally with some contribution of cathepsin
D regardless of ovarian steroids. However, since peritoneal
fluid concentrations of cathepsin D in the GnRH agonist-
treated patients were relatively low, it is likely that induction
of hypo-oestrogenism may therefore reduce the local invas-
iveness.
In clinical oncology, serum and tissue levels of cathepsin D
have been widely studied with regard to a potential prognostic
value, particularly in breast cancer patients (Westley and May,
1996; Pujol et al., 1999). Overexpression of cathepsin D in
tumour tissue was thereby observed to be an independent
marker for identifying patients at risk of disease progression.
However, elevated cathepsin D serum values have only been
recognized in pancreatitis or liver cirrhosis patients, and not
in individuals with breast cancer (Brouillet et at., 1991; Leto
et al., 1997). Recent reports have in fact suggested that serum
cathepsin D concentrations are not elevated in most cancer
patients, regardless of the clinical stage or the tumour size
(Leto et al., 1997; Westhoff et al., 1998). In spite of high
cathepsin D levels in endometriotic tissue (Bergqvist et al.,
1996) and our present findings for peritoneal fluid, our data
also indicate that there is no potential for diagnostic use of
serum cathepsin D levels for patients with endometriosis.
In malignant cells, cathepsin D has been found to be
overexpressed or abnormally processed, resulting in its cyto-
plasmic accumulation and excessive secretion (Strojan et al.,
1998). Several findings suggest that it may be related to local
invasion and metastasis of tumour cells, and it is able to
initiate proteolytic events resulting in degradation of basement
membrane and extracellular matrix components (Scambia et al.,
1991; Strojan et al., 1998). In endometrial adenocarcinomas,
the levels of cathepsin D are higher than in normal endometrium
and correlate positively with loss of differentiation and the
degree of myometrial invasion (Nazeer et at., 1992). Our data
point to elevated cathepsin D levels in the peritoneum due to
proteolytic invasion of endometriotic lesions.
Figure 1. Concentrations of cathepsin D (CD) in peritoneal fluid
determined by enzyme-linked immunosorbent assay. (A) There was
significant variation (P Ͻ 0.001) in cathepsin D levels among the
control group (n ϭ 21), the endometriosis stages I/II group (n ϭ
14) and the endometriosis stages III/IV group (n ϭ 19). In the
endometriosis patients, elevated cathepsin D levels (P Ͻ 0.05) were
noted in the stages III/IV group as compared to the stages I/II
group. (B) No significant variation in cathepsin D levels within the
menstrual cycle was noted (P: the proliferative phase; S: the
secretory phase) in any groups marked **. No significant difference
was observed between the controls and the GnRH agonist-treated
patients. However, the cathepsin D levels of the untreated patients
(n ϭ 18; n ϭ 10, the proliferative phase; n ϭ 8, the secretory
phase) were significantly higher than those from patients receiving
GnRH agonist treatment (n ϭ 15; n ϭ 9, the proliferative phase;
n ϭ 6, the secretory phase, P Ͻ 0.01).
We conclude that cathepsin D is an important factor that
may contribute to the pathogenesis of endometriosis, possibly
by digesting extracellular matrix proteins. This information
has implications for the therapeutic efficacy of GnRH agonists.
measurement in the peritoneal fluid could be a good marker
for the severity of the disease.
Acknowledgements
There is currently considerable interest in optimal timing of
surgery according to the phase of the menstrual cycle in terms
of tumour biology (Badwe et al., 1995; Holdaway et al., 1997)
and prognosis (Veronesi et al., 1994; Mohr et al., 1996). Since
endometriotic lesions are influenced by cyclic changes in
ovarian steroids, we examined whether the cathepsin D concen-
tration varies with the phase of the menstrual cycle. Although
cathepsin D is induced by oestrogens in mammary cancer
cells, the lack of correlation between peritoneal fluid cathepsin
D levels and the menstrual phase, regardless of the presence of
We would like to thank the following: Drs Katsuo Ikuta, Kinue
Katano and Noritaka Suzuki (Department of Obstetrics and
Gynecology, Nagoya City University, Nagoya), Drs Shoji Kajiura
and Masahiko Mizuno (Nagoya City Johoku Hospital, Nagoya) and
Dr Koichi Koike (Nagoya City Moriyama Hospital, Nagoya) for their
advice and help with collecting samples.
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Received on October 30, 2000; accepted on February 27, 2001
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