estrogen metabolizing enzymes in endometrium and endometriosis

Estrogen metabolizing enzymes in endometriumand endometriosis

H. Dassen1,2, C. Punyadeera1,2,6, R. Kamps1,3, B. Delvoux3, A. Van Langendonckt4, J. Donnez4,B. Husen5, H. Thole5, G. Dunselman1,3,8 and P. Groothuis1,3,7

1Research Institute GROW, University Hospital Maastricht/University Maastricht, Peter Debyelaan, The Netherlands; 2Department of

Pathology, University Hospital Maastricht/University Maastricht, Peter Debyelaan, The Netherlands; 3Department of Obstetrics and

Gynaecology, University Hospital Maastricht/University Maastricht, Peter Debyelaan 25 HX 6229, The Netherlands; 4Department

of Gynaecology, Universite de Catholique de Louvain, Brussels, Belgium; 5Solvay Pharmaceuticals Research Laboratories,

Hannover, Germany

8Correspondence address. E-mail [email protected]

BACKGROUND: Estradiol (E2) is an important promoter of the growth of both eutopic and ectopic endometrium.The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endo-metrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS: In this study, we havelooked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17b-hydroxy-steroid dehydrogenases (17b-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] ineutopic and ectopic endometrium of patients (n 5 14) with deep-infiltrative endometriosis as well as in disease-freeendometrium (n 5 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrualcycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS:Aromatase and 17b-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAsfor types 2 and 4 17b-HSD, EST and STS were readily detectable. Only 17b-HSD type 2 and EST genes showed sen-sitivity to progesterone in normal endometrium. Types 1 and 2 17b-HSD and STS protein was detected in normalendometrium using new polyclonal antibodies. CONCLUSIONS: In endometriosis lesions, the balance is tilted infavor of enzymes producing E2. This is due to a suppression of types 2 and 4 17b-HSD, and an increased expressionof aromatase and type 1 17b-HSD in ectopic endometrium.

Keywords: endometriosis; steroidogenic enzymes; aromatase; estrogen metabolism; endometrium

Introduction

An estimated six percent of women suffer from endometriosis

and have complaints such as chronic pelvic pain and some are

even infertile because of this disease (Olive and Schwartz,

1993; Vessey et al., 1993; Kjerulff et al., 1996). Endometriosis

is defined as the presence of endometrial glands and stroma in

extra-uterine sites, mostly the pelvic peritoneum, ovaries and

rectovaginal space (Olive and Schwartz, 1993; Zeitoun et al.,

1999; Bulun et al., 2000, 2002a).

Endometriosis is an estrogen-dependent disease (Dizerega

et al., 1980; Zeitoun et al., 1998). The primary source of estra-

diol (E2) is the ovary, and therapies aimed at suppressing

ovarian function or antagonizing the actions of estrogens,

have proven to be effective. It has long been known that the

endometrium itself is a source of E2, estrone, estrone sulfate

and estrone-3-sulfate (Tseng et al., 1982; Tseng, 1984b) and

that elevated aromatase activity is associated with malignancies

of the endometrium (Tseng et al., 1982; Yamaki et al., 1985). It

took however, another 10 years for investigators to suspect that

endometriotic lesions may be self-supporting as evidenced by

aromatase overexpression (Yamamoto et al., 1993; Noble

et al., 1996,1997). Aromatase overexpression has now indeed

been confirmed in numerous reports at the transcript level

(Noble et al., 1996; Bulun et al., 1997; Matsuzaki et al.,

2006; Smuc et al., 2007), at the protein level (Kitawaki et al.,

1997,1999; Murakami et al., 2006; Velasco et al., 2006), as

well as at the activity level (Kitawaki et al., 1997; Noble

et al., 1997; Murakami et al., 2006). The clinical relevance of

these observations was illustrated by the fact that aromatase

inhibitors were effective in the treatment of endometriosis,

particularly in those cases where GnRH agonist treatment

6Present address: Department of Molecular Diagnostics, Philips Research,

High Tech Campus 11, 5656 AE Eindhoven, The Netherlands7Present address: Department of Pharmacology, N.V. Organon, Postbus 20,

5340 BH Oss, The Netherlands

# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

All rights reserved. For Permissions, please email: [email protected]

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failed (Takayama et al., 1998; Ailawadi et al., 2004), support-

ing the notion that significant estrogen production continues

at extraovarian sites, including adrenals, adipose tissue, skin

and endometriotic lesions.

The net production of 17b-oestradiol is the result of a

delicate balance between the synthesis and the inactivation of

E2. Next to aromatase, the production of E2 is also mediated

through the 17b-hydroxysteroid dehydrogenases (HSD) type

1, 3, 5, 7 and 12, as well as steroid sulfatase (STS), which con-

verts the sulfated estrogens to biologically active estrogens

(Matsuoka et al., 2002; Moeller and Adamski, 2006). Gene

transcripts for types 1 and 7 17b-HSD and estrogen sulfatase

(Fusi et al., 2005; Smuc et al., 2007) were found to be over-

expressed in endometriotic tissues when compared with

normal endometrium. In the human, aromatase produces

mainly estrone and not E2, which would support a key role

for the type 1 17b-HSD as well.

Another key protein, which is believed to be involved in a

rate-limiting step in steroid biosynthesis, is the steroidogenic

acute regulatory protein (StAR). Even though StAR is not an

enzyme, it is responsible for the transport of the substrate for

steroid synthesis, cholesterol, across the mitchondrial mem-

brane. StAR is highly overexpressed in endometriotic lesions

(Tsai et al., 2001).

The conversion of E2 into less active metabolites in endo-

metrium tissue is believed to be mediated for a large part by

17b-HSDs types 2, 4 and 8, which form by an oxidative reac-

tion androstenedione and estrone (Miettinen et al., 1996; Husen

et al., 2001; Luu-The, 2001; Mensah-Nyagan et al., 2001;

Yang et al., 2001), and by the estrogen sulphotransferase

(EST), which conjugates sulfate groups to the 3-hydroxyl posi-

tion (Qian and Song, 1999).

The 17b-HSDs types 4 and 8 are constitutively expressed in

normal human endometrium (Husen et al., 2001; Punyadeera

et al., 2003). No information is available with regard to the

expression levels of these two genes in endometriosis. EST is

also expressed in normal endometrium (Utsunomiya et al.,

2004). It is expressed in the secretory phase only, suggesting

that it is regulated by progestins. In the study of Smuc et al.

(2007) no differences were observed in the expression of

EST in normal endometrium and ovarian endometriosis.

The type 2 17b-HSD is believed to be one of the most import-

ant E2-inactivating enzymes in the endometrium (Tseng and

Gurpide, 1974,1975). There is clear evidence that transcription

of the 17HSD2 gene in normal endometrium is regulated by

progesterone (Tseng and Gurpide, 1975; Kauppila, 1984;

Casey et al., 1994; Mustonen et al., 1998; Zhang et al., 2006),

even though some inconsistent findings have been reported

(Kitawaki et al., 2000; Matsuzaki et al., 2006).

Such contrasting reports were also published with regard to

the expression of 17b-HSD type 2 in eutopic and ectopic endo-

metrium of endometriosis patients. The general consensus is

that the expression of type 2 17b-HSD is reduced or absent

in eutopic and ectopic endometrium of endometriosis patients

(Husen et al., 2001; Bulun et al., 2002a), and that 17b-HSD

type 2 gene expression is insensitive to progestins (Vierikko

et al., 1985; Zeitoun et al., 1998), indicating that endometriotic

tissue may have reduced capacity to inactivate E2. Kitawaki

et al. (2000), however, described exactly the opposite: in endo-

metrium of patients 17b-HSD type 2 expression was increased

in secretory endometrium of endometriosis patients. Also

Smuc et al. (2007) did not observe differences in type 2

17b-HSD expression levels between normal endometrium

and endometriosis. Similar discrepancies were reported for

aromatase. Kitawaki et al. (1997,1999) and Matsuzaki et al.

(2006) found no aromatase expression in endometrium of

disease-free women, whereas Tseng and coworkers

(1982,1984b) clearly showed aromatase activity in normal

cycling endometrium.

The inconsistent findings with regard to the expression and

activity of steroidogenic enzymes in endometrium of controls

and endometriosis patients and endometriotic lesions, can be

attributed to a variety of factors including the use of different

technologies, the quality of the samples (i.e. is the endometrio-

tic tissue contaminated with normal tissue from the local

environment) and the lesion types studied. More efforts are

needed to address these issues.

Most studies have focussed on adenomyosis, leiomyomas

and ovarian and peritoneal endometriosis, but little information

is available about steroidogenic enzymes in the severe

deep-infiltrative type of endometriosis. In the current study,

we evaluated the expression of aromatase, STS, EST and the

types 1, 2 and 4 17b-HSD in eutopic endometrium and endo-

metriotic tissues of patients with deep-infiltrative endometrio-

sis. In addition, expression was assessed in endometria from

disease-free women. Hormonal regulation of the expression

of these enzymes was also investigated in explant cultures of

normal human endometrium.

Materials and Methods

Tissues

Endometrial tissue was collected from 48 women of 26–52 years of

age with regular menstrual cycles, who underwent surgery for

benign indications other than endometriosis. The tissue was collected

from hysterectomy specimens or by pipelle biopsies during laparo-

scopy (Pipelle catheter, Unimar Inc., Prodimed, Neuilly-Enthelle,

France). The women were documented not to be on any kind of

steroid medication. All women signed an informed consent, as

required by the protocol approved by the Medical Ethical Committee

of the University Hospital Maastricht.

Forty-eight biopsies of normal human endometrium were collected.

Twelve were collected in the menstrual phase (M phase), 18 were col-

lected in the proliferative phase: early proliferative phase, n ¼ 9; late

proliferative phase (LP phase), n ¼ 9. Another 18 were collected in the

secretory phase: early secretory phase (ES phase), n ¼ 8; mid-

secretory phase (MS phase), n ¼ 8; late secretory phase, n ¼ 2.

After macroscopic inspection of the uteri by a pathologist, endome-

trium tissue was collected. The endometrium was dated according to

clinical information with respect to the start of the last menstrual

period, which was reconfirmed by histological examination of the

tissue (Noyes et al., 1975).

Endometrium and endometriotic lesions were collected in 14

women of 22–39 years of age who underwent laparoscopic surgery

to remove rectovaginal endometriosis. The endometrium was col-

lected by pipelle biopsies during the operation (Pipelle catheter,

Unimar Inc.). The collected tissues were frozen in Tripure Isolation

Estrogen metabolism in endometrium and endometriosis

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Reagent TM (Roche, Basel, Switzerland) at 2808C until RNA

isolation.

From each biopsy, part of the tissue was fixed in 10% buffered for-

malin for histology and immunohistochemistry and another part of the

tissue was snap frozen in lysis buffer (Tripure Isolation ReagentTM,

Roche) and stored at 2808C for RNA isolation.

Explant culture

Endometrium tissues from M phase (n ¼ 8) and LP phase (n ¼ 8) biop-

sies were collected and transported to the laboratory in Dulbecco’s

modified Eagle’s medium (DMEM)/Ham’s F12 medium on ice. The

tissue was minced into pieces of 2–3 mm3. Twenty-four explants

were placed in Millicell-CM culture inserts (pore size of 0.4 mm,

30 mm diameter, Millipore, Billerica, MA, USA) which were placed

in 6-well plates containing phenol red-free DMEM/Ham’s F12

medium (1.5 ml) (Life Technologies, Grand Island, NY). The

medium was supplemented with L-glutamine (1%), penicillin and

streptomycin (1%). The tissue was cultured for 24 h. At the end of

the experiment, part of the explant was collected in formalin and

embedded in paraffin, the rest was collected in lysis buffer (SV Total

RNA Isolation Kit, Promega, Madison, WI, USA) and stored at

2808C until RNA-isolation.

Previous experiments have shown that collagenase activity remains

very low in proliferative endometria during the first 24 h of culture

(Cornet et al., 2002), and that the tissue viability is not affected

after 24 h of culture (Marbaix et al., 1992). Treatments included:

control (0.1% ethanol); E2 (1 nM); progesterone (1 nM) and E2 and

progesterone (1 nM each). The steroid hormones were gifts from

Organon pharmaceuticals (Oss, The Netherlands).

RNA isolation from normal endometrium

Total cellular RNA from explants and uncultured endometrium was

extracted using the SV total RNA isolation kit (Promega) according

to the manufacturer’s protocol, with slight modifications. The concen-

tration of DNase-1 during DNase treatment of the RNA samples was

doubled and the incubation time was extended by 15 min in order to

completely remove genomic DNA. Total RNA was eluted from the

column in 50 ml RNase-free water and stored at 2808C until further

analysis. The quality of the RNA samples was determined with a spec-

trophotometer and agarose gel electrophoresis. All the samples ana-

lysed gave RNA to DNA ratios higher than 1.5. Although primer/probes were designed to overlap exon boundaries, a PCR for a house-

keeping gene glyceraldehyde-3-phosfate dehydrogenase (GAPDH)

was performed on the RNA samples to verify that the samples were

free of genomic DNA.

RNA isolation from endometriotic tissue

Prior to RNA isolation, the tissue was thawed at 48C, cut into small

pieces and homogenized twice for 30 s with an Ultra-Turrax homo-

genizer (Rose Scientific, Edmonton, Alberta, Canada). The samples

were centrifuged 10 min at 12 000 g. To the supernatant 200 ml

chloroform was added. The samples were vortexed 15 s and incubated

at room temperature for 10 min. The samples were centrifuged 15 min

at 12 000 g. The aqueous upper phase was collected and 500 ml iso-

propanol was added to precipitate the RNA. The samples were vor-

texed 10 s and incubated 10 min at room temperature. The samples

were centrifuged 10 min at 12 000 g. The pellets were washed with

1 ml 75% ethanol. After air-drying, the pellets were collected in

RNase-free water and incubated for 10 min at 608C.

cDNA synthesis

For complementary DNA (cDNA) synthesis, total RNA (1 mg) was

incubated with random hexamers (1 mg/ml, Promega) at 708C for

10 min. The samples were chilled on ice for 5 min. To this mixture,

a reverse transcriptase (RT)-mix consisting of 5x RT-buffer (4 ml),

5 mM dNTP mix (2 ml) (Pharmacia, Uppsala, Sweden), 0.1 M dithio-

threitol (2 ml) (Invitrogen, Breda, The Netherlands) and superscript II

reverse transcriptase (200 U) (Invitrogen) was added and the samples

were incubated at 428C for 1.5 h, after which the reverse transcriptase

was inactivated by heating the samples at 958C for 5 min. The cDNA

was stored at 2208C until further use. In each real-time PCR reaction

50 ng of cDNA template was used.

Real-time PCR

Primers and probes for 17b-HSD-1 (Hs00166219-g1), 17b-HSD-2

(Hs00157993-m1), 17b-HSD-4 (Hs00264973-m1), STS

(Hs00165853-m1), EST (Hs00193690-m1) and aromatase

(Hs00240671-m1) were purchased from Perkin-Elmer Applied Bio-

systems as pre-developed assays. Human cyclophylin A

(Hs99999904-m1) was selected as an endogenous RNA control in

order to normalize for the differences in the amount of total RNA

added to each reaction. A pool of total RNA obtained from uncultured

human endometrium tissues was included as positive control. All PCR

reactions were performed using an ABI Prism 7700 sequence detec-

tion system (Perkin-Elmer Applied Biosystems). The thermal

cycling conditions comprised an initial decontamination step at

508C for 2 min, a denaturing step at 958C for 10 min and 40 cycles

of 15 s at 958C followed by 1 min at 608C. Experiments were per-

formed for each sample in duplicate. Quantitative values were

obtained from the threshold cycle number (Ct) at which the increase

in signal associated with the exponential increase of the number of

PCR products was first detected with the ABI Prism 7700 sequence

detector software (Perkin-Elmer, Foster city, CA). The fold-change

in expression was calculated using the DD Ct method, with cyclo-

phylin A mRNA as an internal control (Livak and Schmittgen,

2001). For detailed description of the procedure please refer to the

ABI user manual. (http://www.uk1.unifreiburg.de/core/facility/tagman/user_bulletin_2.pdf)

Immunohistochemistry and western blotting

Novel polyclonal antibodies (pAb) were generated against 17b-HSD

type 1, type 2 and STS (prepared at Pineda Antikorper-Service,

Berlin, Germany, on behalf of Solvay Pharmaceuticals). The pAb

against type 1 17b-HSD (protein accession number NP_000404)

was generated by injecting rabbits with a recombinant 17b-HSD

type 1 peptide (h17HSD) which lacked 42 amino acids at the

C-terminus (generously provided by Christina Fischer, University of

Frankfurt, Germany). The pAb against type 2 17b-HSD (protein

accession number NP_002144) was generated by injecting a cocktail

of the three synthetic peptides, NENGPGAEELRRTC (peptide A),

CNIAGTSDKWEKLEKD (peptide B) and CAKRHFGQDKPM-

PRALR (peptide C). The pAb against STS (protein accession

number NP_000342) was generated by injecting a cocktail of the

three synthetic peptides, CLWEAESHAASRPNII (peptide A), CAH-

VEEVSSKGEIHGGS (peptide B) and CSSKGEIHGGSNGIYK

(peptide C). The peptides were conjugated to a protein carrier

(KLH). Three rabbits were injected with the peptide-conjugate cock-

tails for each antigen, and from each rabbit preimmune serum was col-

lected. The generated antibodies were affinity purified using the

immunizing peptides.

Specificity of the pAbs was demonstrated by western blotting and

immunohistochemistry.

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To confirm specificity of the 17b-HSD type 1 pAb, gels were loaded

with 10 ml (1 mg/ml) HSD1-containing tumor cytosol (Fig. 1, lane 1),

20 ml homogenate (0.55 mg/ml) from a HSD1-positive-cell line

(Fig. 1, lane 2) and 10 ml homogenate (1 mg/ml) from a

HSD1-negative-cell line (Fig. 1, lane 3; for general characteristics

of cells and tumors see Husen et al. 2006), and subsequently probed

with the preimmune serum, the pAb or the pAb after pre-incubation

with the immunizing peptides. Antibody binding was visualized

with an alkaline phosfatase-conjugated anti-rabbit immunoglobulin

G (1:10 000, Sigma-Aldrich, St. Louis, MO, USA) and incubation

with NBT/BCIP-substrate solution (Sigma-Aldrich). Specific

binding of the pAb against type 2 17b-HSD was shown by loading

the individual immunizing peptides, as well as lysates prepared

from placenta. The pAb and preimmune serum were applied at a

1:5000 dilution. Antibody binding was visualized with anti-rabbit

horse-radish peroxidase (1:10 000; Dako, Glostrup, Denmark) and

incubation in a chemiluminescent substrate (SuperSignal West Pico

Chemiluminescent Substrate, Pierce, Rockford, USA). Specific

binding of the anti-STS pAb was demonstrated by loading lysates pre-

pared from endometrium and placenta and purified arylsulfatase C

(Sigma-Aldrich) on the gels. The affinity purified antibody was

applied at a dilution of 1:1000. Antibody binding was visualized in

the same way as for the type 2 17b-HSD.

For immunostaining, paraffin sections of 5 mm were cut from uncul-

tured and cultured explants and from the deep-invasive endometriotic

lesions and uterus from endometriosis patients. In addition, sections

were prepared from placenta and breast cancer tissue to serve as posi-

tive tissue controls. The sections were deparaffinized 2�5 min in

xylene and 2�5 min in 100% ethanol. The endogenous peroxidases

were blocked by incubating 30 min in 0.3% H2O2 in methanol. For

antigen retrieval, the sections were boiled in Tris–EDTA buffer (pH

9.0) in a microwave oven for 20 min. In the preliminary stainings, a

1:1000 dilution was used for the anti-17b-HSD type 1 pAb and

preimmune serum. The Dako-Envision protocol (Dako Diagnostika,

Hamburg, Germany) was used to visualize antibody binding. The pre-

immune serum and pAbs against 17b-HSD type 2 and STS were

diluted 1:2000 and 1:1000, respectively. Antibody binding was visu-

alized with the ChemateTM Envision kit (Dako Diagnostika).

After assessing specificity, the immunohistochemistry procedure

was further optimized. The conditions used to stain the clinical speci-

mens were the following. The anti-17b-HSD type 1 pAb and pre-

immune serum were used at 1:4000 and sections were incubated for

1 hr at room temperature. The anti-17b-HSD type 2 pAb and pre-

immune serum were incubated for 2 h at room temperature at a

dilution of 1:2000. The anti-STS polyclonal antibody and preimmune

serum were diluted 1:1000 and sections were incubated overnight at

48C. Antibody binding was visualized by incubating 30 min with

ChemateTM Envision (Dako Dako Diagnostika) and staining with dia-

minobenzidine solution. The reaction was stopped in water. The sec-

tions were briefly counter-stained in haematoxylin, dehydrated and

sealed in Entellan (Merck, Whitehouse Station, NJ, USA).

Staining intensitiy was scored in epithelial and stromal cells sepa-

rately on a 5-point scale: negative (0); weak (1); weak-moderate (2);

moderate (3); moderate-strong (4) and strong (5). In addition, the percen-

tage of postively stained cells within each compartment was assessed.

Statistical tests

Statistical tests were carried out using the Statistical Package for the

Social Sciences 11 (SPSS Inc., Chicago, IL) statistical analysis

package. To evaluate whether expression levels varied significantly

throughout the menstrual cycle, the non-parametric unpaired Mann–

Whitney U-test was used to test for differences between these

expression levels versus the expression level in the M phase. This

was done for both the mRNA and protein data. This test was also

used to test for differences in the expression of the estrogenic

enzymes in normal endometrium versus eutopic and ectopic endo-

metrium of endometriosis patients. The non-parametric Wilcoxon

signed-rank test was used to test for differences between steroid-

treated explants and controls at a confidence level of 95%.

Results

Validation of the antibodies against 17b-HSDtypes 1 and 2 and STS

With immunoblotting, a specific band at the expected molecu-

lar weight of 37 kDa was visible in the cytosol of the tumor cell

line and cells known to express 17b-HSD type 1, which was

not visible with the preimmune serum, and which disappeared

after pre-incubation with the 17b-HSD type 1 fusion protein

(Fig. 1A). Immunostaining in paraffin sections of breast

cancer tissue also disappeared after pre-incubation of the

pAb with excess h17HSD fusion protein (Fig. 1B) indicating

the specificity of the generated pAb.

Only one of the three pAbs generated against type 2 17b-HSD

showed cross reactivity with one of the three peptides (NENGP-

GAEELRRTC, peptide A; Fig. 2A). A specific band was also

present in the placenta lysate at the expected molecular weight

of 43 kDa. The other pAbs only gave a non-specific band in pla-

cental lysates around 75 kDa, which was also seen with the pre-

immune serum (results not shown). These antibodies also

showed strong staining in the syncytiotrophoblast, which could

not be displaced by any of the three peptides (Fig. 2B, polyclonal

2). The candidate antibody, however, showed immunostaining

Figure 1: Western blot analysis (A) and immunohistochemistry(B) with the pAb against 17b-HSD type 1The pAb shows a specific band at the expected molecular weight (A)and specific staining in subcutaneous tumors in immunodeficient micegenerated from 17b-HSD type 1-transfected MCF-7 human breastcancer cells (B). Pre-incubation with the h17HSD1 fusion protein pre-vented antibody binding in both the western blot (A) and immunos-taining (B). Lane 1, 10 ml (1 mg/ml) HSD1-containing tumorcytosol; lane 2, 20 ml homogenate (0.55 mg/ml) from aHSD1-positive-cell line (lane 2); lane 3, 10 ml homogenate (1 mg/ml) from a HSD1-negative-cell line


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mostly in the vessels, which was completely abolished by pre-

incubation with the immunizing peptides (Fig. 2B, polyclonal 1).

To check specificity of the STS pAb, western blot analysis was

performed on purified microsomal steroid sulfatase (arylsulfatase

C; Fig. 3A). Besides the expected bands at 63 kDa, other bands

(�40 and 50 kDa) were visible as well. A similar pattern was

observed when incubated with the preimmune serum (Fig. 3A)

and lower dilutions of the pAb (results not shown), and these

bands could not be blocked with the immunizing peptides. In

lysates of endometrium and placenta tissues also the 63 kDa

band is visible (Fig. 3B), however, the 40 and 50 kDa bands

were more abundant. Based on the known size of the active

enzyme and the post-translational modifications that could

occur, it is not possible that the 40 and 50 kDa bands represent

STS. In contrast, immunohistochemical staining was completely

prevented after pre-incubation of the pAb with the immunizing

peptide A, but not peptides B and C (Fig. 3C). This antibody

appears therefore to be suitable for immunohistochemistry and

not for western blotting.

Expression of E2 synthesizing and metabolizing enzymesthroughout the menstrual cycle

The expression of type 2 17b-HSD mRNA increased in normal

endometrium during the secretory phase of the menstrual cycle

compared with the proliferative phase of the cycle (Fig. 4). Tran-

script levels increased in the ES phase and remained high

throughout the secretory phase. At the protein level, however,

17b-HSD type 2 expression was observed mostly in the glandular

epithelium (Fig. 5). Levels peaked during the MS phase, the

implantation window (Fig. 6). Little staining was also observed

in the stroma, but expression in the stroma did not change

throughout the menstrual cycle.

Aromatase gene transcript levels in normal endometrium were

near the detection limit, and no cyclical variations were observed.

Figure 4: Gene transcript levels of 17b-HSD type 2 and EST in thenormal human endometrium throughout the menstrual cycleData are presented as means + SEM. EP, early proliferative phase;ES, early secretory phase; LP, late proliferative phase; LS, latesecretory phase; MS, mid-secretory phase. *versus M phase (P ,0.05)

Figure 2: Western blot analysis (A) and immunohistochemistry (B)with the polyclonal antibody against 17b-HSD type 2Immunostaining of pAb 1 can be prevented by pre-incubation with theimmunizing peptides (B); this is not the case for pAb 2. The anti-type2 17b-HSD pAb 1, cross reacted with immunizing peptide A (A)

Figure 3: Western blot analysis (A and B) and immunohistochemis-try in placenta (C) with the pAb against STSThe immunostaining pattern is similar to that of the type 2 17b-HSDpAb, and can also be completely blocked with the immunizing pep-tides (C). (A) Western blotting showed specific bands around 63, 50and 40 kDa in the blot loaded with arylsulfatase C. (B) The samebands were also present in homogenates of endometrial tissues andplacenta (P)

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No expression of 17b-HSD type 1 mRNA was observed

(results not shown). Surprisingly, however, prominent

expression was observed at the protein level (Figs 5 and 6).

These levels did not vary between stages of the cycle (Figs 5

and 6).

Transcript levels of 17b-HSD type 4 and STS were similar

to that of 17b-HSD type 2, but the levels did not increase in

the secretory phase (results not shown). Expression of STS

protein did also not change during the menstrual cycle accept

for the LS phase were it was significantly downregulated

compared to the menstrual phase (Figs 5 and 6). STS protein

was expressed in both epithelial and stromal cells.

EST transcript levels were lower (P , 0.05) than that of

17b-HSD types 2 and 4, but expression levels increased signifi-

cantly in the secretory phase (Fig. 4). Despite these differences

in expression levels, a highly significant correlation was found

between the cyclic variations in mRNA expression of

17b-HSD type 2 and EST (correlation 0.79, P , 0.0001).

Steroid regulation of E2 synthesizing and metabolizingenzymes

The production of aromatase, the 17b-HSD types 1 and 4 and

STS mRNA in cultured endometrium explants prepared from

M-phase and LP-phase explants was not affected by E2 or pro-

gesterone (data not shown).

The levels of EST and 17b-HSD type 2 gene transcripts were

significantly induced by progesterone in both, M-phase and

LP-phase explants versus control (Fig. 7). Treatment with E2

alone had no effect on the 17b-HSD type 2 and EST mRNA

expression in M-phase and LP-phase explants. Tissues col-

lected from the LP phase were significantly (P , 0.05) more

responsive then M-phase tissue to the action of progesterone

for both EST and 17b-HSD type 2.

Steroid regulation at the protein level in these short-term

cultures was not confirmed with the semi-quantitative

immunohistochemistry.

Figure 5: Representative photographs of 17b-HSD type 1, type 2 andSTS protein expression in normal endometrium collected on menstrualcycle days (CD) 11, 17 and 21

Figure 6: Expression of 17b-HSD type 1, type 2 and STS protein throughout the menstrual cycleThe staining intensity and the percentage of cells stained are presented and analysed separately. Data are presented as means + SEM. *P , 0.05versus M phase


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E2 synthesizing and metabolizing enzymesin endometriotic tissues

Compared with normal endometrium, transcript levels of

17b-HSD type 1 and aromatase were significantly higher in

the endometrium (P , 0.01) and endometriotic lesions (P ,

0.01) of endometriosis patients compared with normal endome-

trium (Fig. 8). Aromatase mRNA levels were found also to be

significantly higher (P , 0.05) in the endometriotic lesions

compared with the endometrium of endometriosis patients

(Fig. 8). The 17b-HSD type 1 protein is expressed in all

cells, but staining intensity is significantly lower in epithelial

(P , 0.01) and stromal cells (P , 0.01) of the eutopic and

ectopic endometrium of the endometriosis patients compared

with the normal endometrium (Figs 9a and 10).

Transcript levels of 17b-HSD type 2 were significantly

lower (P , 0.01) in the endometriotic lesions than in the endo-

metrium of patients and controls. No significant differences

Figure 8: Gene transcript levels of steroidogenic enzymes in eutopicand ectopic endometrium from endometriosis patients and normalendometrium. Data are presented as means+SEM. *P , 0.05,**P , 0.01 versus normal endometrium; #P , 0.05; ##P , 0.01versus eutopic endometrium of the same patient

Figure 9: Protein expression of 17b-HSD-1 (A), 17b-HSD-2 (B) andSTS (C) in eutopic and ectopic endometrium from endometriosispatients and normal endometriumStaining intensity and the percentage of stained cells are analysed andpresented separately. Data are presented as means + SEM. EP, epi-thelium; S, stroma. *P , 0.05; **P , 0.01 versus normal endome-trium; #P , 0.05 versus eutopic endometrium of the same patient

Figure 7: Effects of E2 and progesterone (P) on gene transcript levelsof 17b-HSD-2 and EST in M-phase and LP-phase explantsData are presented as means + SEM. *P , 0.05 versus control;**P , 0.05 versus M-phase explants

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were found between the normal endometrium and the eutopic

endometrium of patients (Fig. 8). The 17b-HSD type 2

protein is expressed mostly in epithelial cells. However, the

number of positive stromal cells was slightly increased (P ,

0.05) in the eutopic endometrium of patients, whereas staining

intensity was significantly reduced (P , 0.05) in stromal cells

of the lesions compared with the normal endometrium (Figs 9b

and 10).

The mRNA levels of 17b-HSD type 4 were significantly

(P , 0.01) lower in the lesions and endometrium of endome-

triosis patients when compared with the normal endometrium

(P , 0.01). No difference was found between the lesions and

the endometrium of endometriosis patients (Fig. 8).

No differences were observed between the STS mRNA

levels of endometrium and lesions of endometriosis patients

and endometrium of patients without endometriosis (Fig. 8).

STS protein was expressed both by stroma and epithelium.

STS protein levels were significantly (P , 0.05) lower in epi-

thelial cells of endometrium of endometriosis patients com-

pared with those of normal endometrium (Fig. 9c and 10).

No difference in STS protein levels was observed between epi-

thelial cells of the lesions and the normal endometrium

(Fig. 9C).

EST mRNA levels were significantly (P , 0.05) higher in

endometriotic tissue when compared with normal endometrium

(Fig. 8).

Discussion

In this study, we have investigated the expression of six

enzymes involved in the synthesis and inactivation of E2 in

eutopic and ectopic endometrium of patients with

deep-infiltrative endometriosis. Three pAb were generated to

study types 1 and 2 17b-HSD and STS protein expression in

the tissues. We show that there are parallels between

deep-infiltrative endometriosis and the reports on adenomyo-

sis, ovarian and peritoneal endometriosis, with regard to the

expression of steroidogenic enzymes, but we also report

some novel findings.

With regard to the E2 synthesizing enzymes, we found very

low levels of aromatase and 17b-HSD type 1 mRNA in normal

human endometrium, which is in line with earlier reports

(Bulun et al., 1993; Matsuzaki et al., 2006). Aromatase and

17b-HSD type 1 expression was not responsive to E2 and pro-

gesterone in the explant cultures. This is in contrast with

finding of Tseng (1984a) who showed that aromatase activity

in isolated endometrial stromal cells was induced up to

40-fold by progesterone, which was further enhanced 20–

100-fold by E2. Whether this is due to the fact that in the

tissue context, estrogen metabolism may be different than in

isolated cell compartments, or that prolonged exposure is

needed to induce aromatase expression remains to be

elucidated.

In contrast to the low levels of 17b-HSD type 1 mRNA in the

normal human endometrium, we did however detect 17b-HSD

type 1 protein. The fact that we were unable to detect 17b-HSD

type 1 mRNA transcripts in the endometrium of disease-free

women, suggests that these transcripts either degraded

quickly or are synthesized in low quantities. Reports on the

expression of 17b-HSD type 1 in normal endometrium are con-

flicting. Miettinen et al. (1996) reported low expression of

17b-HSD type 1 mRNA in endometrium, whereas Casey

et al. (1994) found no expression of 17b-HSD type 1 mRNA

in normal human endometrium, and Utsunomiya et al. (2001)

found no 17b-HSD type 1 protein expression in normal endo-

metrium. In this study, we used a new pAb and we clearly

demonstrated expression of type 1 17b-HSD in normal endo-

metrium. Steroidal regulation, however, was not observed in

the explant cultures. The presence of type 1 17b-HSD

protein agrees with the observed E2 conversion into estrone

in normal proliferative endometrium (Delvoux et al., 2007).

Aromatase and type 1 17b-HSD mRNA levels were higher

in eutopic and ectopic endometrium of endometriosis patients

than in the endometrium of controls, which is in agreement

with the findings of others (Noble et al., 1996; Matsuzaki

et al., 2006). Surprisingly, however, 17b-HSD type 1 protein

levels were lower in epithelial and stromal cells of endo-

metrium and lesions of endometriosis patients compared with

the normal endometrium. For 17b-HSD type 1 mRNA, two iso-

forms have been described, 1.3 and 2.3 kb (Miettinen et al.,

1996). Only the 1.3 kb mRNA is correlated with 17b-HSD

type 1 protein expression and activity (Miettinen et al.,

1996). In normal human endometrium, the 1.3 kb 17b-HSD

type 1 mRNA expression is low in normal human endo-

metrium, but the 2.3 kb mRNA is abundantly present (Mietti-

nen et al., 1996). Expression of the type 1 17b-HSD gene

may therefore not necessarily reflect changes at the functional

protein level. The type 1 17b-HSD is still considered a rate-

limiting enzyme in the production of E2, since the major

product of the aromatase activity in humans is estrone and

not E2 (Simpson et al., 1994), and is therefore still an interest-

ing alternative target for therapy.

Figure 10: Representative photographs of immunostainings for17b-HSD type 1, type 2 and STS in eutopic and ectopic endometriumof endometriosis patients


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At the IXth World Congress on Endometriosis in Maastricht,

The Netherlands (2005), Fusi et al. (2005) showed that both

endometrium and endometriotic tissues have high STS activity,

which suggests that these tissues can generate high levels of

estrone from the serum pool of estrone-3-sulfate. This agrees

with the report of Benedetto et al. (1990), who also showed

STS activity in human endometrial cells, but is not in line

with the finding of Utsunomiya et al. (2004), who showed

immunohistochemically that STS protein is not present in

normal human endometrium using another pAb directed

against STS purified from placenta. In this study, however,

we demonstrated STS gene expression in disease-free endo-

metrium, but in contrast to the finding of Smuc et al. (2007)

in ovarian endometriosis, we did not observe higher STS

mRNA levels in endometriotic lesions. Using our own pAb,

we clearly demonstrated immunostaining in the endometrium.

In contrast to Benedetto et al. (1990) who showed that STS

activity increased in the presence of E2 and medroxyprogester-

one acetate, we did not observe steroid responsiveness in our

short-term explant cultures. This may be due to the fact that

prolonged incubation (8–15 days) was required to evoke this

response, which would not be visible in the 24 h-explant

cultures.

The differences we observed in the mRNA expression levels

of aromatase, type 1 17b-HSD and STS between eutopic and

ectopic endometrium from endometriosis patients and normal

endometrium are significant, but small. In deep-infiltrative endo-

metriosis, it is possible that differences in gene expression may

be masked due to the presence of normal tissue from the local

environment, which does not express steroidogenic enzymes

(Noble et al., 1996). This also explains why Matsuzaki et al.

(2006) found much higher levels of aromatase in the ectopic

than the eutopic endometrium, because the ectopic endometrium

was isolated from its environment with laser capture microdis-

section. Despite the relatively small effects, we still observed

significantly higher levels of aromatase and type 1 17b-HSD

in deep-infiltrative endometriosis.

With regard to the E2-inactivating enzymes we confirmed

that type 2 17b-HSD mRNA levels are significantly lower in

endometriotic lesions compared with levels in normal endo-

metrium (Zeitoun et al., 1998; Matsuzaki et al., 2006). We

also show for the first time that the type 4 17b-HSD is

expressed in endometriotic lesions and that the mRNA levels

are lower than in normal endometrium. EST mRNA levels

were not different between the groups, which agrees with the

findings of Smuc et al. (2007). Using a new antibody,

however, we could not confirm that type 2 17b-HSD protein

is expressed at lower levels in endometriotic tissue, which is

in contrast to the observation of Zeitoun et al. (1998).

We showed in our steroid-responsive explant cultures that of

all enzymes investigated, only the 17b-HSD type 2 and EST

were responsive to progesterone which agrees with the findings

of others (Tseng and Gurpide, 1974; Liu and Tseng, 1979; Pack

et al., 1979; Maentausta et al., 1991; Casey et al., 1994; Husen

et al., 2001). The lower expression of 17b-HSD type 2 in the

ectopic endometrium in patients was postulated to be the

result of reduced sensitivity to progesterone (Bulun et al.,

2000,2002a,b). We observed, however, that in contrast to

type 2 17b-HSD, expression levels of EST were not reduced

in the endometriotic lesions, whereas in normal endometrium

the expression patterns of 17b-HSD type 2 and EST were

strongly correlated. This suggests that the expression of

17b-HSD type 2 is selectively suppressed.

It is clear that enzyme activities are different in the epithelial

and stromal compartments, as is the expression of the different

steroidogenic enzymes. Aromatase activity was shown to be

present in both stroma and epithelium, but activity in the

stroma was �3–4 times higher (Tseng, 1984b). For this

reason, most in vitro studies have been performed with

stromal cells isolated from the endometriotic lesions. Aroma-

tase mRNA (Matsuzaki et al., 2006) and protein (Kitawaki

et al., 1997), however, were found to be expressed mostly in

the epithelial cells. The importance of intercellular communi-

cation with regard to the regulation of steroidogenesis was

illustrated by for instance the paracrine regulation of type 2

17b-HSD expression in epithelial cells by factors released by

the stroma (Cheng et al., 2007), and the presence of progester-

one receptor in the stromal cells (Yang et al., 2001). Endo-

metrial cell-specific production of metabolites was

demonstrated for tibolone (Groothuis et al., 2006). Stromal

cells were shown to contain 3a- and b-aldoketoreductases

which converted tibolone into 3a- and 3b-hydroxytibolone,

whereas in the epithelial cells tibolone is directly converted

into D4-tibolone. It is therefore important to consider that the

net production of E2 synthesis is also influenced by cellular

interactions, which has to be accounted for in the experimental

designs, i.e. by using co-cultures or explant cultures.

Finally, it has to be noted that even though much attention

was given to the expression and activities of individual

enzymes, there is no information with regard to the actual

output of E2 by the ectopic endometrium. An important

aspect in this regard is the fact that the intracellular redox

state of cells is an important determinant of the enzymatic con-

versions by HSDs (Sherbet et al., 2007). The nicotinamide

cofactors required for the reduction and oxidization activities

are abundant in cells, reaching millimolar concentrations. In

cells the major reducing cofactor is NADPH (NADPH/NADPþ ratio �100:1) and the major oxidizing cofactor is

NADþ (NADH/ NADþ ratio �1:1000; Krebs, 1973; Reich

and Sel’kov, 1981). In addition, it was shown that low micro-

molar concentrations of NADPH potently inhibits NADþ-

dependent oxidative reactions (Steckelbroeck et al., 2004).

Collectively, this suggests that in vivo the reductive activity

is dominant (Steckelbroeck et al., 2004). This would for

instance mean that the type 1 17b-HSD activity would domi-

nate type 2 activity. In addition, P450 enzymes like aromatase

also need NADPH and molecular oxygen and will irreversibly

oxidize the steroid nucleus (Miller, 2005). These observations

indicate that increases in the expression of E2-generating

enzymes will have a greater impact on the net production of

E2 than a reduction in the expression of E2-inactivating

enzymes.

The endometrium is also a source of drug metabolizing

enzymes usually present in the liver. Reddy et al. (1981)

showed that human endometrial tissue was able to metabolize

E2 into the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol.

Dassen et al.

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It has now been shown that the human endometrium expresses

CYP3A4, known to convert estrone into 16a-hydroxyestrone

and E2 into 2-hydroxyestradiol (Hukkanen et al., 1998), and

CYP1B1, known to convert E2 into 4-hydroxyestradiol (Tsuchiya

et al., 2004). Furthermore, the human endometrium expresses

catechol-O-methyltransferase, which converts 2-hydroxy- and 4-

hydroxyestradiol into 2- and 4-methoxyestradiol (Salih et al.,

2007). Another group of potential E2-inactivating enzymes was

also identified in the endometrium, the uridine diphospho-

glucuronosyltransferases (UGTs) (Lepine et al., 2004). The endo-

metrium appears to be a rich source of UGTs, which are able to

conjugate both estrogens and catecholestrogens on different pos-

itions. However, their relevance with regard to the development

of endometrial pathologies is not known.

Summary

We confirm that aromatase and 17b-HSD type 1 expression is

higher in deep-invasive endometriosis than in eutopic and

normal endometrium. Even though no 17b-HSD type 1 gene

transcripts were detected in normal endometrium, we did

observe a clear expression of the 17b-HSD type 1 protein,

which was not elevated in the ectopic endometrium.

Expression of the STS was prominent at both the mRNA and

protein level, but was not elevated in endometriotic tissues.

Type 2 17b-HSD gene transcript levels were lower in

deep-invasive endometriosis. Type 2 17b-HSD protein

levels, however, were not lower in lesion compared with the

eutopic or normal endometrium. We show for the first time

that the E2-inactivating enzyme 17b-HSD type 4 is expressed

in deep-invasive endometriosis, and that it is also down-

regulated in endometriosis. Together with the continuous pre-

sence of aromatase and the substantial levels of STS, this

may result in an increase in the local production of E2 in the

endometriotic lesions. Inventarization of the net metabolic con-

versions in the whole endometriotic tissue, would aid in the

selection of the appropriate combination of enzyme inhibitors

to optimize medical therapy of endometriosis.

In normal endometrium both the 17b-HSD type 2 and EST

were responsive to progesterone. The fact that in deep-infiltra-

tive endometriosis only the expression of type 2 17b-HSD was

suppressed and not that of EST, suggests local and selective

dysregulation of type 2 17b-HSD expression.

Funding

This study was funded by Solvay Pharmaceuticals Research

Laboratories, Hannover, Germany.

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Submitted on February 26, 2007; resubmitted on July 12, 2007; accepted onJuly 24, 2007

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