Human Reproduction Update 2000, Vol. 6, No. 5 pp.

413±418 Ó European Society of Human Reproduction and Embrology

Molecular basis for treating endometriosis with

aromatase inhibitors

Serdar E.Bulun1,*, Khaled M.Zeitoun2, Kazuto Takayama3 and Hironobu Sasano3

1
Departments of Obstetrics and Gynecology and Molecular Genetics, University of Illinois at Chicago, IL 60612, 2Department of
Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, 622 W. 168th St, New York, NY 10032±3702
and 3Departments Pathology and Obstetrics and Gynecology, Tohoku University School of Medicine, 2±1 Seiryo Machi, Sendai-Shi
980, Japan

Although treatment of one unusually aggressive case of postmenopausal endometriosis with an aromatase inhibitor
has been strikingly successful, large clinical trials are required to establish whether aromatase inhibitors will have a
signi®cant role in the medical management of endometriosis. Introduction of aromatase inhibitors into the treatment
of endometriosis underscores the importance of basic research leading to the development of novel strategies in
reproductive disorders. It was shown earlier that aromatase activity was not detectable in normal endometrium.
Aromatase, however, is expressed inappropriately in endometriosis and stimulated by prostaglandin E2. Aromatase
activity gives rise to local biosynthesis of oestrogen, which, in turn, stimulates prostaglandin E2 production, thus
establishing a positive feedback cycle. This favours accumulation of oestrogen and prostaglandins in endometriosis,
which is an in¯ammatory disorder dependent on oestrogen for growth.

Key words: aromatase/aromatase inhibitor/endometriosis/endometrium/oestrogen biosynthesis

TABLE OF CONTENTS Danazole and medroxyprogesterone acetate] or to antagonize

the effects of oestrogen on endometriotic implants (e.g. oral
Background
contraceptives, Danazole and medroxyprogesterone acetate)
Molecular aberrations in endometriosis
(Table I). There is, however, a high incidence of recurrence after
Origin of oestrogen in women
Aromatase expression in uterine tissues these medical therapies (Waller and Shaw, 1993). Eighteen
Mechanisms responsible for aromatase expression in months after completing a 6-month course of leuprolide acetate-
endometriosis depot, only 52% of patients had signi®cant relief of pain (Waller
Aberrant expression of steroidogenic factor-1 (SF1) activates and Shaw, 1993). The recurrence rate of pain in the rest of the
aromatase expression in endometriosis patients was ~5±20% per year (reaching a cumulative average rate
Clinical relevance: treatment of endometriosis with aromatase at 5 years as high as 53%). The recurrence rate at 5 years was as
inhibitors high as 75% in severe forms of endometriosis (Waller and Shaw,
Future considerations 1993). In women treated for pelvic pain, the symptoms usually
Acknowledgements return rather quickly after cessation of therapy. For a period of
References time after medical treatment, however, the intensity of symptoms
is less severe. The recurrence rates after treatment with GnRH
agonists are similar to those after Danazole, and both are similar
Background
to those obtained with surgical excision. Danazole is used less
Although current hormonal therapy of infertility associated with frequently due to its androgenic side-effects. A 6 month course of
endometriosis is not of proven value, it is somewhat successful for GnRH agonist treatment is currently the most popular regimen.
pelvic pain associated with endometriosis (Olive and Schwartz, The most serious side-effect of the GnRH agonist treatment for
1993). The relief, however, is relatively short-term (Waller and endometriosis is considered to be bone loss due to oestrogen
Shaw, 1993). Various agents used are comparable in terms of de®ciency, and oral oestrogen-progesterone preparations or
ef®cacy. Pelvic implants of endometriosis react histologically to bisphosphonates are usually `added back' to minimize bone loss
steroid hormones in a manner similar to normal endometrium. For (Surrey et al., 1995).
example, oestrogen stimulates growth of both endometriosis and As summarized above, we are still far from the cure of
eutopic endometrium. All medical treatments were designed to endometriosis, and current treatments are not satisfactory for
decrease oestrogen secretion by the ovaries [e.g. gonadotrophin effective control of pain. The radical treatment is the removal of
releasing hormone (GnRH) agonists, oral contraceptives, both ovaries, and even this was not found to be effective in a

*To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Illinois at Chicago, 820 S. Wood St M/C 808,
Chicago, IL 60612, USA. Tel: (312) 996-8197; Fax: (312) 996-4238; E-mail: sbulun@uic.edu
414 S.E.Bulun et al.

number of cases of postmenopausal endometriosis (Metzger et al., two extraovarian sources is probably an important reason for the
1991; Takayama et al., 1998). New strategies are needed to offer high rate of treatment failures with GnRH agonists. Aromatase
women with endometriosis a reasonable chance to live without inhibitors are candidate therapeutic agents for endometriosis
suffering from chronic pelvic pain for decades. There are two (Table I). Preliminary evidence suggests that aromatase inhibitors
important caveats, which are not addressed by the GnRH agonist can eradicate unusually aggressive endometriotic lesions resistant
treatment. Firstly, we have recently shown that extremely large to other therapy (Takayama et al., 1998).
quantities of oestrogen can be produced locally within the
endometriotic cells, which represents an intracrine mechanism of
Molecular aberrations in endometriosis
oestrogen action, in contrast to ovarian secretion, which is an
endocrine means of supplying this steroid to target tissues. Local The prevalence and genetics of endometriosis is somewhat similar
oestrogen biosynthesis is not blocked by any of the currently used to those of diabetes mellitus and asthma, in that endometriosis is a
treatments for endometriosis. Secondly, oestradiol produced in common chronic disorder inherited possibly in a polygenic
peripheral tissue sites (e.g. adipose tissue and skin ®broblasts) fashion (Kennedy, 1999). Implantation of menstrual endometrium
may give rise to signi®cant circulating levels of oestradiol in a on peritoneal surfaces via retrograde menstruation is a widely
number of women. Again, GnRH agonists do not inhibit accepted mechanism for the development of endometriosis
peripheral oestrogen formation. Oestrogen production in these (Sampson, 1927). On the other hand, since retrograde menstrua-
tion occurs in nearly all women in the reproductive age group,
additional factors were postulated to contribute to the establish-
Table I. Medical treatment of endometriosis ment of endometriotic implants in pelvic peritoneum. It was
proposed that a defective immune system incapable of clearing
GnRH agonists peritoneal surfaces of menstrual debris might contribute to the
development of endometriosis (Halme et al., 1988). Additionally,
GnRH antagonists
a number of molecular aberrations were found in endometriotic
Danazol (anabolic steroid) implants, which distinguish them from the eutopic endometrium,
Oral contraceptives although both tissues appear to be histologically similar. As a
further twist, these aberrations give rise to the gain or loss of
Progestins various functions. The end result of these alterations is the
Mifepristone (RU486; progesterone receptor antagonist) enhancement of the growth and invasiveness of endometriotic
implants. For example, impaired suppression of matrix metallo-
Aromatase inhibitors
proteinases, which facilitate invasiveness, may contribute to
establishment of ectopic lesions (Bruner et al., 1997; Sharpe-
GnRH = gonadotrophin releasing hormone. Timms et al., 1998). Overproduction of the cytokine RANTES by
endometriotic implants may provide a mechanism for the
recruitment of peritoneal leukocytes (Hornung et al., 1997). The
abnormal presence of aromatase and absence of 17b-hydroxys-
teroid dehydrogenase type 2 in endometriotic implants in contrast
to eutopic endometrium may give rise to excessive local
production and impaired metabolism of oestradiol (Noble et al.,
1996; Zeitoun et al., 1998). Consequently, elevated tissue levels
of this mitogen will enhance the growth of endometriotic
implants. In the following sections, we will discuss topics that
are relevant to the use of aromatase inhibitors to treat
endometriosis. We will initially review mechanisms of oestrogen
production in humans. A discussion of the use of aromatase
inhibitors in the treatment of endometriosis will follow.

Origin of oestrogen in women

Oestrogen is produced in several human tissues that contain the
enzyme named aromatase, which catalyses the conversion of C19
steroids to oestrogens (Simpson et al., 1994). In premenopausal
women, granulosa cells of the Graa®an follicle in the ovary
Figure 1. Origin of oestrogen in women. In ovulatory women, oestradiol (E2)
is secreted by the ovary in a cyclic manner. During treatment with a represent the primary site of aromatase expression. Therefore,
gonadotrophin releasing hormone analogue or postmenopausal period, ovarian secretion in a cyclic manner accounts for the largest
however, peripheral tissues (e.g. adipose tissue and skin), represent the major portion of oestradiol production in women in the reproductive age
source of oestradiol. Aromatase activity in these peripheral tissues gives rise to group (Simpson et al., 1994). Aromatase expression in ovarian
the conversion of androstenedione (A) of adrenal and ovarian origins to
oestrone (E1). Oestrone is further converted to the potent oestrogen oestradiol
granulosa cells is under the control of FSH, which activates a
in peripheral tissues. Therefore, two important sources of circulating oestradiol signalling pathway involving cyclic adenosine monophosphate
in women are ovarian secretion and peripheral aromatization. (cAMP), steroidogenic factor-1 (SF-1), and cAMP response
 Aromatase and endometriosis 415

element binding protein (CREB). Binding of the two latter factors biosynthesis does not take place in healthy uterine tissues (Bulun
to the promoter of the CYP19 (aromatase P450) gene gives rise to et al., 1994a). On the other hand, we demonstrated CYP19 gene
the events leading to the production of aromatase protein expression and aromatase activity in uterine leiomyomas,
(Michael et al., 1995, 1997). endometrial cancer and endometriosis (Bulun et al., 1994a,b;
Extraovarian tissues become the major source of oestrogen Noble et al., 1996, 1997) (Figure 2). It is intriguing that all these
production after the cessation of ovarian function in postmeno- pathological tissues use primarily the ovarian type promoter for
pausal women (Figure 1). In particular, markedly high levels of aromatase expression. This promoter is stimulated via a cAMP-
aromatase activity are present in adipose tissue and skin dependent signalling pathway in both leiomyomas and endome-
®broblasts (Ackerman et al., 1981). Since adipose tissue triosis (Bulun et al., 1994; Noble et al., 1996, 1997; Zeitoun et al.,
comprises a signi®cant portion of the human body, aromatase 1999). Thus, aromatase expression is inappropriately activated in
activity in this tissue probably accounts for the largest part of oestrogen-dependent disorders of the uterus, whereas aromatase
oestrogen production in postmenopausal women (MacDonald et activity is absent in the disease-free counterparts of these tissues.
al., 1978). Additionally, oestrogen produced locally by aromatase In these particular instances, aberrant aromatase expression may
activity in breast adipose tissue promotes the development and be analogous to activation of an oncogene, since the end-product
growth of oestrogen-dependent breast malignancies (Bulun et al., oestrogen is a potent mitogen for uterine leiomyomas, endome-
1993; Yue et al., 1998). Aromatase expression in adipose tissue is trial cancer and endometriosis (Figure 2).
limited to undifferentiated ®broblasts and is not present in
signi®cant quantities in lipid-®lled mature adipocytes (Ackerman
Mechanisms responsible for aromatase expression
et al., 1981; Price et al., 1992). Glucocorticoids together with
in endometriosis
members of the IL-6 cytokine family regulate aromatase
expression in adipose ®broblasts via an alternative promoter that Upon demonstration of relatively high quantities of aromatase
is located ~20 000 base pairs upstream of the ovarian promoter P450 (P450arom) transcripts in endometriosis (much higher than
(Zhao et al., 1995). Androstenedione of adrenal origin is the those found in the adipose tissue), we next used endometriosis-
major substrate for aromatase in extragonadal tissues (Simpson et derived stromal cells in monolayer culture as a model system to
al., 1994). Androstenedione is aromatized to become oestrone, study the regulation of aromatase (Noble et al., 1996, 1997).
which is further reduced to the potent oestrogen oestradiol in Endometriotic stromal cells cultured by this method have been
these tissues (MacDonald et al., 1978) (Figure 1). previously characterized in terms of vimentin and cytokeratin
expression and were reported to retain oestrogen receptors and
oestrogen responsiveness (Ryan et al., 1994). We characterized
Aromatase expression in uterine tissues
these endometriotic stromal cells further by demonstrating
Both myometrium and endometrium undergo signi®cant histolo- prolactin mRNA expression in response to treatment with
gical and biochemical changes under the in¯uence of oestrogen. medroxyprogesterone acetate plus dibutyryl cAMP (our unpub-
Oestrogen action in uterine tissues is of paramount physiological lished observations). This veri®es the presence of endometrial-
importance in terms of preparation for implantation. Oestrogen type cells in culture, which are responsive to hormonal treatment.
Prolactin transcripts were also detected in cultured stromal cells
from eutopic endometrium but not in ovarian granulosa and theca
cells subjected to the same treatments (our unpublished observa-
tions). Baseline aromatase activity in endometriosis-derived
stromal cells ranged from 0.65 to 6 pmol/mg protein/4 h. No
signi®cant stimulation of aromatase activity was observed by
various cytokines [interleukin (IL)-1b, IL-2, IL-6, IL-11,
oncostatin M, IL-15, tumour necrosis factor (TNF)-b] or steroids
(oestradiol, progesterone agonist R5020, dexamethasone).
Dibutyryl cAMP induced aromatase activity in these cells by
26-60-fold the baseline values, whereas the addition of phorbol
acetate neither potentiated nor diminished this response (Noble et
al., 1997). Because of the in¯ammatory nature of endometriosis,
we treated these stromal cells with various prostanoids. Whereas
treatments with prostaglandin (PG) I2, PGF2a, PGJ2 failed to elicit
a response, PGE2 treatment gave rise to a dose-dependent
induction of aromatase activity by up to 19- to 44-fold in
Figure 2. Aberrant aromatase expression in endometriosis. Androstenedione endometriosis-derived cells from different patients (Noble et al.,
(A) of adrenal and ovarian origins become converted to oestrone (E1) in 1997). These changes in aromatase activity were accompanied by
endometriotic tissue. Oestrone itself is only weakly oestrogenic and should be comparable changes in the amounts of P450arom mRNA. A
converted to oestradiol (E2) for full oestrogenic action. Endometriotic tissue modi®ed rapid ampli®cation of 5¢-cDNA ends (5¢-RACE)/
expresses the enzyme, 17b-hydroxysteroid dehydrogenase type 1, which Southern hybridization of the promoter-speci®c sequences in
catalyses this conversion (Zeitoun et al., 1998). While gonadotrophin releasing
hormone analogues (GnRHa) suppress oestradiol secretion from the ovary, P450arom transcripts revealed almost exclusive use of the ovarian
only aromatase inhibitors are capable of eliminating oestrogen formation in type promoter for aromatase expression in PGE2- and dibutyryl
endometriotic tissue in the presence of adrenal function. cAMP-treated endometriotic cells.
416 S.E.Bulun et al.

In summary, PGE2 induction of aromatase activity in reporter gene were transfected into endometriotic stromal cells.
endometrial stromal cells is mediated through increased intracel- Two critical regulatory regions for cAMP induction of promoter
lular levels of cAMP. The basis for markedly high levels of activity were identi®ed: a ±214/-100 bp proximal region
aromatase expression in endometriosis in contrast to absent or responsible for a 3.7-fold induction and a ±517/-214 distal region
barely detectable quantities in the eutopic endometrium may be responsible for potentiation of cAMP response up to 13-fold. In
due to the transformation of endometrial stromal cells after the proximal region, we studied eutopic endometrial and
implantation in the pelvic peritoneum and ovary in response to endometriotic nuclear protein binding to a nuclear receptor half-
locally produced paracrine factors. The potential aromatization site and an imperfect cAMP response element (CRE). CRE-
capability of eutopic endometrial cells from women with genetic binding activity in nuclear proteins from both endometriotic and
predisposition to develop endometriosis may facilitate the eutopic endometrial cells gave rise to formation of identical
implantation process and growth in pelvic peritoneum by DNA±protein complexes (Figure 3). The nuclear receptor half-
increasing local oestradiol concentrations by the activities of site probe, on the other hand, formed a distinct complex with
aromatase and 17b-hydroxysteroid dehydrogenase (HSD) type 1 nuclear proteins from endometriotic cells, which migrated at a
(Noble et al., 1996; Zeitoun et al., 1998). Oestradiol, in turn, will much faster rate compared with the complex formed with nuclear
induce the activity of cyclo-oxygenase type 2 (COX-2), the rate- proteins from eutopic endometrial cells. Employing recombinant
limiting enzyme for PGE2 biosynthesis (Huang et al., 1996). The proteins and antibodies against SF-1 and chicken ovalbumin
in¯ammatory process in endometriotic tissues giving rise to upstream promoter-transcription factor (COUP-TF), we demon-
increased production of cytokines (e.g. IL-1b, TNFa) by strated that COUP-TF, but not SF-1, bound to nuclear receptor
monocytes and macrophages will also promote PGE2 production half-site in eutopic endometrial cells, whereas SF-1 was the
in this tissue (Huang et al., 1998). Thus a positive feedback cycle primary nuclear receptor half-site-binding protein in endometrio-
is established, whereby local productions of oestrogen and PGE2 tic cells (Figure 3). In fact, COUP-TF mRNA was present in both
are enhanced by complex molecular interactions. eutopic endometrial and endometriotic tissues, whereas SF-1
mRNA was detected in all endometriotic tissues, but in only three
out of 15 eutopic endometrial tissues. Moreover, we demonstrated
Aberrant expression of steroidogenic factor-1 (SF-1)
a dose-dependent direct competition between SF-1 and COUP-TF
activates aromatase expression in endometriosis
for occupancy of the nuclear receptor half-site, to which SF-1
An intriguing observation during the previous studies was the bound with a higher af®nity. Finally, ectopic expression of SF-1
unresponsiveness of eutopic endometrial stromal cells to cAMP in eutopic endometrial and endometriotic cells strikingly
analogues in contrast to drastic cAMP induction of aromatase potentiated baseline and cAMP-induced activities of the ±517
expression in endometriosis-derived cells (Figure 3). Thus, we promoter construct, whereas ectopic expression of COUP-TF
decided to determine whether differential binding of transcription almost completely abolished these activities. In conclusion,
factors to the CYP19 (aromatase P450) promoter in response to COUP-TF is a factor responsible for the inhibition of aromatase
cAMP is a mechanism involved in this process. Deletion mutants expression in eutopic endometrial stromal cells, which lack SF-1
of the 5¢-¯anking region of this promoter fused to Luciferase expression in the majority of the samples; whereas aberrant SF-1
expression in endometriotic stromal cells can override this
inhibition by competing for the same DNA binding site, which
is likely to account for high levels of baseline and cAMP-induced
aromatase activity (Zeitoun et al., 1999) (Figure 3).

Clinical relevance: treatment of endometriosis with

aromatase inhibitors
Aromatase inhibitors have been widely used to treat postmeno-
pausal breast cancer (Santen, 1991). The therapeutic potential of
aromatase inhibitors in breast cancer is comparable to that of
tamoxifen (Santen, 1993). Two possible effects of these
Figure 3. Regulation of CYP19 (aromatase P450) gene expression via the
ovarian-type promoter in stromal cells from the eutopic endometrium and medications in breast cancer were postulated: Aromatase
endometriotic tissue (endometriosis). Aromatase P450 mRNA in the eutopic inhibitors suppress oestrogen production in peripheral tissues
endometrium is absent or barely detectable. Thus, signi®cant transcription of such as fat and decrease circulating oestrogen levels considerably
the CYP19 gene is blocked in this tissue. In endometriosis-derived stromal (Iveson et al., 1993). Additionally, it was proposed that inhibition
cells, on the other hand, levels of aromatase P450 mRNA can be stimulated to
of local aromatase activity in breast tissue proximal to malignant
extremely high levels by cAMP analogues. Cyclic AMP response element
binding protein (CREB) binds to this promoter in both cell types but does not cells is a key mechanism responsible for the therapeutic effects of
determine by itself the absence or presence of promoter activity. The these inhibitors (Sourdaine et al., 1996; Yue et al., 1998). Very
inhibition of aromatase expression in eutopic endometrium is regulated by little is known about possible hormonal and reproductive
binding of the inhibitory transcription factor, chicken ovalbumin upstream alterations caused by aromatase inhibitors in women in the
promoter-transcription factor (COUP-TF) to the CYP19 gene promoter. The
stimulatory transcription factor, steroidogenic factor-1 (SF-1), is aberrantly
reproductive age group. One study on adult female bonnet
expressed in endometriotic tissue and competes with COUP-TF to occupy the monkeys revealed that ovulation continued to occur despite
same response element. Binding of SF-1 to the CYP19 gene promoter together markedly reduced levels of oestradiol (Selvaraj et al., 1995).
with CREB gives rise to the activation of aromatase expression. Nonetheless, the successful use of aromatase inhibitors in the
 Aromatase and endometriosis 417

treatment of postmenopausal breast cancer, which is an oestrogen- concentrations of oestradiol, a known mitogen for endometriosis.
dependent disease, and aberrant expression of aromatase in Aberrant expression of the transcription factor, SF-1 in endome-
endometriotic implants encouraged us to use these medications to triotic tissue gives rise to the inappropriate presence of aromatase
treat endometriosis. expression leading to local oestrogen biosynthesis. This may play
The woman in the ®rst published report was referred to us due a signi®cant role in the aetiology of postmenopausal endome-
to vaginal cuff endometriosis resistant to all existing treatments triosis as exempli®ed by a remarkable response of this disorder to
including bilateral oophorectomy followed by multiple laparo- treatment with an aromatase inhibitor. Although both SF-1 and
tomies for resection of lesions (Takayama et al., 1998). We aromatase are expressed in endometriotic implants from pre-
followed the size of this vaginal lesion by direct visualization. menopausal women, the clinical signi®cance of these ®ndings in
Brie¯y, this 57 year old woman weighing 217 lbs underwent this group is yet to be determined. It is tempting to postulate that
hysterectomy and bilateral oophorectomy 20 years prior to our addition of aromatase inhibitors to GnRH analogues may increase
evaluation. After surgical menopause, endometriosis recurred the disease-free interval signi®cantly. It is further tempting to
twice causing bilateral blockage of ureters and complete loss of postulate that the use of an aromatase inhibitor as a single agent
left kidney function. She had two laparotomies for resection of may suppress endometriosis, while permitting a woman to
retroperitoneal endometriosis and in®ltrated segments of ureters ovulate, since aromatase inhibitors at therapeutic doses for breast
followed by bilateral ureteral reimplantation. A year before the cancer and endometriosis do not block ovulation (Selvaraj et al.,
initiation of treatment with an aromatase inhibitor, endometriosis 1995). Thus, aromatase inhibitors may potentially be used in the
recurred for the third time at her vaginal cuff and did not respond treatment of infertility associated with endometriosis. These
to treatment with megesterol acetate for 4 months. At this point, issues are yet to be clari®ed by future studies. Finally, the most
she was taking large doses of hydrocodone, methadone and non- serious side-effect of aromatase inhibitors appears to be bone loss
steroidal anti-in¯ammatory medications. Her serum FSH level (Takayama et al., 1998). There are, however, no large-scale
was in the postmenopausal range (61 IU/l), whereas oestradiol studies to assess the magnitude of this potential side-effect. Since
level was higher than expected (46 pg/ml). High oestradiol level we are entering an era of use of aromatase inhibitors to treat non-
might be explicable in terms of obesity, which is known be malignant disorders such as endometriosis, these will be
associated with increased oestrogen formation (MacDonald et al., extremely important questions. The roles of various add-back
1978). High levels of aromatase P450 mRNA were detected in a regimens to prevent this potential side-effect remain to be seen.
biopsy of the vaginal endometriotic implant. After the aromatase
inhibitor anastrozole, 1 mg/day (plus alendronate 10 mg/day and
calcium supplement) was initiated, pelvic pain rapidly decreased Acknowledgements
and disappeared within 2 months, and she discontinued all pain We thank Dee Alexander and Rosemary Bell for providing expert editorial
medications. Oestradiol level was reduced to 27 pg/ml. assistance.
Endometriotic implant at the vaginal apex decreased from a 30
mm red polypoid mass to a 3 mm scar tissue within 9 months. A
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