Accepted Manuscript

Title: Altered expression of progesterone receptor isoforms A

and B in human eutopic endometrium in endometriosis
patients

Author: Monika Martina Wölfler Mareike Küppers Werner

Rath Volker Uwe Buck Ivo Meinhold-Heerlein Irmgard
Classen-Linke

PII: S0940-9602(16)30036-X
DOI: http://dx.doi.org/doi:10.1016/j.aanat.2016.03.004
Reference: AANAT 51027

To appear in:

Received date: 18-1-2016

Revised date: 10-3-2016
Accepted date: 11-3-2016

Please cite this article as: Wddotolfler, M.M., Kddotuppers, M., Rath, W., Buck, V.U.,
Meinhold-Heerlein, I., Classen-Linke, I.,Altered expression of progesterone receptor
isoforms A and B in human eutopic endometrium in endometriosis patients, Annals of
Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.03.004

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Altered expression of progesterone receptor isoforms A and B in human

eutopic endometrium in endometriosis patients

Monika Martina Wölfler a, Mareike Küppers b, Werner Rath c, Volker Uwe Buck b, Ivo

Meinhold-Heerlein c, Irmgard Classen-Linke b

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a
Department of Obstetrics and Gynecology, Medical University of Graz, Auenbrugger Platz

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14, 8036 Graz, Austria.
b
Institute of Molecular and Cellular Anatomy, Medical Faculty of the RWTH Aachen

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University, Wendlingweg 2, 52074 Aachen, Germany.
c
Department of Obstetrics and Gynecology, Medical Faculty of the RWTH Aachen

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University, Pauwelsstrasse 30, 52074 Aachen, Germany.
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Corresponding author:
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Prof. Dr. rer nat. Irmgard Classen-Linke

Institute of Molecular and Cellular Anatomy

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Medical Faculty of the RWTH Aachen University

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Wendlingweg 2

52074 Aachen
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Germany

Fax: +49 241 8082508

Phone: +49 241 8088924

Email: iclassen-linke@ukaachen.de

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Abstract

Recent data implicate an altered expression of progesterone receptor isoform A (PR-A) and

B (PR-B) in the endometrium of endometriosis patients. This prospective exploratory study

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aimed to precisely determine the PR-A and PR-B expression using immunohistochemical

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techniques in eutopic endometrium of women with endometriosis compared with disease-

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free women throughout the menstrual cycle.

All symptomatic patients underwent laparoscopy for the diagnosis of endometriosis and

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histological confirmation of the disease (EO) whereas controls were proven disease-free

(CO). In CO samples (n=10) an increased expression of PR-A and PR-B during the

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proliferative to early secretory phase and a decreased expression of both receptor isoforms

during the mid to late secretory phase was ascertained in accordance with previous studies.
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In patients with endometriosis (n=16) no cycle dependent pattern of PR-A and PR-B

expression was identified in contrast to patients without endometriosis. Moreover, in EO

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samples a huge variety of inter- and intra-individual differences in PR-A and PR-B

expression were detected.

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These data provide further evidence that dysregulation of the PR-A and PR-B expression
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might contribute to the pathophysiology of endometriosis.

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Key words:

endometriosis / eutopic endometrium / PR-A / PR-B / immunohistochemistry / menstrual

cycle

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1 Introduction

Endometriosis is one of the most common benign gynecologic disorders and is characterized

by the presence of endometrial glands and stroma outside of the uterine cavity (de Ziegler et

al., 2010; Meuleman et al., 2009). Millions of women of reproductive age are affected, but the

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pathophysiology still is enigmatic. Regurgitation of viable endometrial cells via retrograde

menstruation is believed to be crucial for the development of endometriosis. However, the

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mechanisms whereby these retrogradely shed endometrial fragments circumvent peritoneal

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immunosurveillance and eradication and implant unimpeded on extrauterine sites to form

endometriotic lesions, remain unclear (Witz et al., 2002). The dislocation of basal

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endometrial layers (Leyendecker et al., 1998) and the model of tissue injury and repair within

the uterus involving local production of estrogen via aberrant aromatase expression (Bulun et

al., 2002; Wolfler et al., 2005) in an estrogen sensitive environment appear to be the basic
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mechanisms for the development of endometriosis and adenomyosis of the uterus

(Leyendecker et al., 2009). Moreover, the eutopic endometrium contains epigenetically

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abnormal cells which inappropriately express pro-survival genes such as steroidogenic factor
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or estrogen receptor ß. Endometriotic lesions, originating from endometrial cells, overexpress

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these factors and activate the cascade of local estrogen and prostaglandin biosynthesis in

endometriotic tissue giving rise to inflammation, resistance to apoptosis and persistence of

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pelvic endometriosis (Bulun, 2009).

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Estrogen and progesterone are essential to control endometrial function throughout the

menstrual cycle. Besides local hyperestrogenism progesterone resistance features hormonal

dysregulation in eutopic endometrium and ectopic lesions, also nourishing the setting for the

establishment and progress of endometriotic lesions (Brosens et al., 2012).

There are two predominant isoforms of the progesterone receptor, PR-A and PR-B, which

are transcribed from the same gene by two different promoters (Kastner et al., 1990). PR-B,

having 164 amino acids more than PR-A, is larger and both isoforms have distinct

transcriptional activities. The PR-A isoform acts as a dominant repressor of PR-B (Vegeto et

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al., 1993). The inhibiting domain in the first 140 amino acids, identical for both isoforms

(Giangrande et al., 1997), can be set aside by the additional activation function AF-3 of the

PR-B isoform (Hovland et al., 1998).

PR-A and PR-B are expressed in the endometrial stromal and epithelial cells and this is

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essential for the endometrium to proliferate, remodel and shed in response to estrogen and

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progesterone (Kim et al., 2013). Among other things, differential expression of progesterone

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receptors in the eutopic and ectopic endometrium was attributed to progesterone resistance

in endometriosis (Bulun et al., 2006; Eaton et al., 2013). Wang et al. reported that the levels

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of both PR isoforms in human endometrium progressively increase during the proliferative

phase, peak immediately prior to ovulation and diminish thereafter, suggesting that estradiol

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stimulates PR levels (Wang et al., 1998). In ectopic endometriotic tissue PR-B was

undetectable and PR-A was markedly lower than in eutopic endometrium (Attia et al., 2000).
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The mechanism responsible for decreased PR expression is still under investigation.

In this pilot study we evaluated the expression of PR-A and PR-B in snap-frozen eutopic
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endometrium of patients suffering from endometriosis compared to disease-free women in

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order to look for an altered expression of PR-A and PR-B throughout the menstrual cycle.
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2 Material and Methods

2.1 Patients, tissue sampling and processing

A total of 62 symptomatic patients aged 18 to 48 years presenting to the Department of

Obstetrics and Gynecology of the RWTH Aachen University for diagnosis and/or treatment of

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dysmenorrhea, dyspareunia, chronic pelvic pain and/or subfertility were recruited within the

prospective-explorative setting of the study, project EK 137/04, which was approved by the

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Ethics Committee of the Medical Faculty of the RWTH Aachen University.

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Prior to enrolment, all participating patients signed an informed consent and estrogen

dependent diseases other than endometriosis or inflammatory disease were excluded by

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gynecologic examination and transvaginal ultrasound. None of the patients had been

diagnosed with endometriosis prior to inclusion. All patients included in the present study had

a regular and natural menstrual cycle and had not taken any hormonal medication for at least
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3 months prior to enrolment.

All patients completed a detailed questionnaire on their symptoms and medical history and
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blood samples for the assessment of the hormonal situation were drawn. Endometriosis was
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diagnosed or excluded (CO) by laparoscopic visualization for the first time followed by
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histopathologic assessment of putative lesions in these patients (EO group). The staging of

the disease was performed according to the revised American Society for Reproductive
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Medicine (rASRM) classification standards (Reproductive, 1997), and for deep infiltrating

endometriosis. In addition, the revised ENZIAN classification was applied (Tuttlies et al.,
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2005). Endometrial samples were collected using Pipelle de Cornier® (KB Biosystems, Ulm,

Germany) for minimal invasive endometrial biopsy during the operative procedure. The

endometrial tissue samples were snap-frozen in liquid nitrogen immediately after surgery and

stored at -40°C.

2.2 Sample selection

Frozen tissue samples were embedded in Tissue Tek® (Miles 4583, Elkhart, USA), cut into 8

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µm sections and transferred to APES coated glass slides. For internal quality, control

sections of each specimen were stained with Toluidine Blue and after visualization of intact

endometrial tissue, serial sections were prepared for the immunohistochemical reactions.

Recent studies on endometrial biopsies repeatedly showed contamination with connective

tissue cells, blood and cellular debris forming a heterogeneous mixture of cells which is not

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representative of endometrium and would not allow precise immunohistologic interpretation

of receptor distribution (Bergqvist et al., 1997; Bukulmez et al., 2008). Therefore, we applied

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rigorous criteria and only admitted samples with continuous endometrial tissue containing

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endometrial epithelial and stromal cells warranting significant and representative

immunohistochemical staining and data. Samples containing only cellular debris and blood

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were excluded from further processing and analysis.
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2.3 Immunohistochemistry

After internal quality control, 26 tissue samples were processed as follows: the cryostat

sections were air dried and incubated at room temperature for 10 minutes in 3.7% PBS
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buffered formalin, then fixed at -20°C in methanol for 4 minutes and in acetone for 2 minutes.
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For rehydration, the sections were washed in PBS three times for five minutes. To avoid non-
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specific background staining the sections were blocked, subjected to a 3% H2O2/methanol

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mixture, incubated for 10 minutes in the dark and washed repeatedly. Potentially loaded

proteins like collagens of the extracellular matrix were blocked by the serum blocking solution
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of Histostain-SP® kit for monoclonal antibodies (Zymed Laboratories Inc., San Francisco,

USA) for 10 minutes. Established and validated primary antibodies to progesterone receptor

isoforms: Ab-7 (Clone hPRa7) mainly for PR isoform A and Ab-6 (Clone hPRa6) for PR

isoform B (both: Lab Vision, Fremont, USA) were used (Clarke et al., 1987; Mote et al., 1999;

Mote et al., 2001). The incubation was performed in a humid chamber at room temperature

for 60 minutes (dilution 1:20).

After washing with PBS for 3 x 10 minutes the tissue sections were incubated with the

biotinylated secondary antibody of the Histostain-SP® kit for 10 minutes, then washed for 3 x

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10 minutes and incubated with streptavidin-peroxidase-conjugate of the Histostain-SP® kit for

10 minutes. Specific binding of the antibody was visualized by incubation with the chromogen

aminoethylcarbazole (AEC). Finally, slides were washed in distilled water and mounted in

glycerol gelatin.

Single tissue sections were additionally counterstained with Hematoxylin for better distinction

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of the endometrial glands and stroma. However, to avoid interference with the antigen-

antibody complex and potential misinterpretation this was not applied to all tissue sections

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subjected to the final analysis.

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As a negative control, the primary antibody was omitted or replaced with nonimmune mouse

immunoglobulin G at the same concentration. None of the negative controls revealed a

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positive staining.
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2.4 Cycle dating

The menstrual cycle phase was determined using a combination of reported cycle day,

assessment of serum hormone profiles (in particular progesterone and 17-β-estradiol) and
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endometrial histologic dating, which was performed as described by Hendrickson and

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Kempson (Hendrickson, 1997). When cycle dating, hormonal status and reported cycle day
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diverged and the menstrual cycle phase could not be dated exactly or the patient turned out
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to be on hormonal medication, the samples were excluded from further analyses. The

histologic dating and allocation to the menstrual phase was performed by two researchers
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(MK, ICL).

2.5 Interpretation of data

The PR-A and PR-B staining was semi quantitatively analyzed by two researchers (MK, ICL)

using the Allred-Score (Allred et al., 1998; Mohsin et al., 2004). Four representative details

with a magnification of x400 were evaluated for each sample using an Axiophot microscope

(Zeiss/Jena, Germany) with a Nikon Digital DXM 1200F camera. For the semi quantitative

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data analysis positive staining throughout the whole tissue section was incorporated and the

mean score was taken.

3 Results

A total of 62 patients were recruited for participation in this study. However, for the final

immunohistochemical analysis merely 26 out of the 62 endometrial samples met the rigorous

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criteria and provided sufficient epithelial glands and endometrial stromal cells which were not

contaminated with blood or cell debris to guarantee significant and representative results.

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3.1 Final results

The remaining 26 patients were aged between 23 and 47 years and had a regular and

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natural menstrual cycle. At laparoscopy, 16 out of 26 (61.5%) patients exhibited

endometriosis (EO) and 10 out of 26 (38.5%) were proven disease-free (CO). The mean age
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of the patients was comparable and not significantly different (EO: 34.5±2.2 years; CO:

35.1±2.9 years; p = n.s.). The stage of disease was determined for every patient and we

found stage rASRM I endometriosis in 3, rASRM II endometriosis in 8, rASRM III

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endometriosis in 4, and rASRM IV endometriosis in 1 patient. Due to deep infiltrating

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endometriosis, 2 patients were additionally classified using the revised ENZIAN classification
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and we found deep infiltrating endometriosis of the pelvic wall (ENZIAN B2) and deep
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infiltrating endometriosis of the rectovaginal septum (ENZIAN A2). Besides deep infiltrating

endometriosis, the patients showed peritoneal and ovarian endometriosis. Details are
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displayed in Table 1.

According to the patients´ record, cycle dating and the hormonal status, 10 patients in the EO

group and 4 patients without endometriosis were in the proliferative phase of the menstrual

cycle while 6 EO patients and 6 CO patients were in the secretory phase, respectively. By

means of immunohistochemistry we found, in subjects without endometriosis, an increased

expression of PR-A and PR-B during the proliferative to early secretory phase of the

menstrual cycle and a decreased expression of both receptor isoforms during the mid to late

secretory phase, especially in the epithelial cells. In endometrial stromal cells PR-A was

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detectable until the late secretory phase and PR-B vanished in the late secretory phase (Fig.

1).

In patients with endometriosis, however, no such cycle dependent pattern was identified.

Moreover, in EO samples a huge variety of inter-individual and intra-individual differences in

PR-A and PR-B expression was detected. PR-A was detectable in the proliferative and

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secretory phase in stromal and epithelial cells, but in the late secretory phase only in stromal

cells (Fig.1, 2). PR-B was not detectable in some EO samples of the proliferative and mid-

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secretory phase, both in epithelial and in stromal cells (Fig. 1; Fig. 2 s,t,w). However, in the

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late secretory phase, we found an unexpectedly strong positive reaction for PR-B in

endometrial epithelium and stroma (Fig. 1 and Fig. 2x).

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Representative immunohistochemical reactions are shown in Fig. 2 to illustrate and compare

PR-A and PR-B detection during the menstrual cycle for patients with and without
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endometriosis. Due to the limited number of samples applicable for the final analysis, data

are displayed and discussed descriptively.

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4 Discussion
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This is the first study to address eutopic endometrium of endometriosis patients throughout
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the menstrual cycle to allow for differentiation of PR-A and PR-B expression in endometrial

stromal and epithelial cells. In this pilot study we found an altered expression of progesterone
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receptor isoform A and B in eutopic endometrium of endometriosis patients compared to

disease-free patients. For disease-free patients we confirmed the findings of earlier studies

(Snijders et al., 1992; Wang et al., 1998) and of former projects performed in our laboratory

(Rebhan, 2005) showing a cyclic pattern of PR-A and PR-B and a decrease in progesterone

receptor density from the mid-secretory phase onwards by immunohistochemical as well as

quantitative Western Blot analyses.

PR-A and PR-B co-expression in target cells of the human uterus mediating distinct

pathways of progesterone action in the glandular epithelium and stroma of the uterus
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throughout the menstrual cycle is well established (Mote et al., 1999). However, for

immunohistochemical techniques it has been shown that the selection of antibodies

recognizing each isoform PR-A and PR-B individually in endometrial tissue, is essential for

the accurate evaluation of PR positivity in clinical specimens (Mote et al., 2001). Therefore,

in the present study, we used such validated primary antibodies. The specificity for clone

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hPRa6 which is PR-B specific by immunoblot analysis (Clarke et al., 1987) has been

demonstrated by staining a cell line expressing only PR-B. Although the hPRa7 antibody

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recognizes both isoforms on immunoblot analysis it failed to detect PR-B on

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immunohistochemistry in this PR-B expressing cell line. This suggests that the additional N-

terminal sequence in PR-B may affect the conformation of the region of the molecule in

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which the hPRa7 epitope is located in a manner reducing its accessibility in

immunohistochemistry in fixed tissues (Mote et al., 1999; Saito et al., 2006).

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In samples from patients with endometriosis we could not detect a cyclic pattern of PR-A and

PR-B expression. This partly stands in contrast to findings of Attia et al. (2000) who
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postulated a cyclic pattern of PR-B in eutopic endometrium, but no expression of PR-B in

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ectopic endometrium in paired samples of endometriosis patients. However, in their study,

homogenates of endometrial samples were assessed by Western blotting not allowing for
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discrimination between connective tissue cells and endometrial glandular structures as well
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as endometrial stromal and epithelial cells. Another study from Igarashi et al. is in line with

our findings (Igarashi et al., 2005). In their study on eutopic endometrium of endometriosis
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patients, an altered PR-A/PR-B ratio due to low PR-B expression was ascertained, but the

endometrial homogenates studied derived only from the late proliferative phase.

Bukulmez et al. (2008) showed a pattern of staining of PR-A and PR-B similar to our results,

in their series of eutopic endometrial samples from endometriosis patients, obtained

exclusively in the secretory phase of the cycle. In particular, they found a more intense

staining of PR-A in the nuclei of the stromal cells compared to the epithelium and a persistent

PR-B staining in epithelial cells. They postulated that the changes might be due to pro-

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inflammatory alterations induced by increased cyclo-oxygenase 2 levels in the eutopic

endometrium. In our series, we repeatedly found an unexpectedly strong positive reaction for

PR-B in endometrial epithelium and stroma in some of the endometriosis samples in the mid

and late secretory phase. The receptor expression, however, varied widely within even one

sample.

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Ultrastructural analyses of eutopic endometrial tissue from women with endometriosis

revealed heterogeneous responses to the disease as well. Biopsies often showed a delay in

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the maturation sequence in combination with characteristics of later phenotypes particularly

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in the mid- and late secretory phase of the menstrual cycle (Jones et al., 2009). In

endometriosis samples, inter-individual and intra-individual changes in the eutopic

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endometrium were detected showing adequate and delayed glandular transformation within

the same sample. These data underline an endometrial dysregulation in endometriosis

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patients which may also explain the heterogeneous and inadequate PR-A and PR-B

expression we found in our series.

Endometriosis, as a chronic inflammatory disorder, disrupts coordinated progesterone

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response and leads to a condition which has been termed “progesterone resistance”
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(Brosens et al., 2012) as described by several authors (Attia et al., 2000; Bulun et al., 2010;
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Burney et al., 2009) and might occur at the level of the PR isoforms, but also at the steroid
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receptor co-activators or downstream target genes. The transcriptome analysis performed by

Burney et al. (2007) revealed molecular dysregulation of the proliferative to secretory

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transition in eutopic endometrium of endometriosis patients and, in accordance with our

study, the progesterone receptor was found to be one of the genes dysregulated in women

with endometriosis.

As discussed above, the heterogeneous appearance of endometrial samples deriving from

endometriosis patients was the most striking finding, suggesting a dysregulation of the

eutopic endometrium in patients affected by endometriosis. The different stages of the

disease might account in part for the heterogeneity, however, there is plenty of evidence that

the stage of disease determined by rASRM staging system does not reflect the activity of the

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endometriotic lesions or the symptoms of the disease (Fauconnier and Chapron, 2005;

Vercellini, 1997; Vercellini et al., 2007). The severity of pain, additional symptoms and the

severity of deep infiltrating endometriosis assessed according to the revised ENZIAN

classification are correlated (Haas et al., 2013a; Haas et al., 2013b). In this study, however,

only two patients were diagnosed with deep infiltrating endometriosis and the alterations of

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PR expression was in line with the findings in patients with superficial peritoneal and ovarian

endometriotic lesions.

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5 Conclusions

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In summary, this study on snap-frozen eutopic endometrium throughout the menstrual cycle

provides considerable evidence on alterations of progesterone receptor expression in the

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eutopic endometrium of endometriosis patients compared to controls. The observed

dysregulation of the regular cycle dependent expression of PR-A and PR-B isoforms in the
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eutopic endometrium of the EO group may play a role in disturbed maturation of the

endometrium and vice versa with the clinically observed subfertility of endometriosis patients.
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To date, it is impossible to distinguish whether altered progesterone receptor regulation is a

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cause or a consequence of progesterone resistance in this disease. The small sample size,

however, is a limitation of the study. This is due to the strict criteria for the IHC evaluation
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using exclusively endometrial samples with sufficient glandular structures of eutopic

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endometrium samples. Further investigations are necessary to better understand the role of

altered PR regulation in endometriosis.

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6 Acknowledgements

Thanks are due to Sabina Hennes-Mades and Diana Seelis-Schmidt for skillful technical

assistance and to all patients for the participation in this project.

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7 Conflict of Interest Statement

The authors declare that they have no conflict of interest.

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9 Tables

9.1 Table 1

entity of
ID cycle phase cycle day endometriosis rASRM rENZIAN
endometriosis patients

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139 early proliferative phase 3 PE II n.a.

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137 early proliferative phase 4 PE II n.a.
49 early proliferative phase 6 PE II n.a.

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174 early proliferative phase 6 PE I n.a.
76 early proliferative phase 7 PE I n.a.
153 mid proliferative phase 8 PE, DIE II A2

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80 mid proliferative phase 9 PE II n.a.
160 mid proliferative phase 9 PE I n.a.
135 mid proliferative phase 9 OV III n.a.

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147 late proliferative phase 13 PE II n.a.
13 early secretory phase 18 PE II n.a.
85 early secretory phase 19 PE, OV III n.a.
15 mid secretory phase 20 PE, DIE IV B2
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101 mid secretory phase 22 PE, OV III n.a.
129 late secretory phase 26 OV III n.a.
133 late secretory phase 28 PE II n.a.
d
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Cycle day and stage of disease of endometriosis patients included in the study. The stage of
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disease in endometriosis patients is displayed according to the rASRM classification and the
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revised ENZIAN classification for deep infiltrating endometriosis. Besides deep infiltrating

endometriosis (DIE) the patients revealed peritoneal (PE) and ovarian endometriosis (OV).
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Data of disease-free controls are not displayed since the staging according to rASRM and

ENZIAN classification is not applicable. ID patients identity; rASRM revised American

Society for Reproductive Medicine classification; rENZIAN revised ENZIAN classification (for

deep infiltrating endometriosis only); n.a. not applicable.

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10 Legends to Figures

10.1 Legend to Figure 1

Evaluation of PR-A and PR-B expression in eutopic endometrium of patients with and without

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endometriosis on different days of the menstrual cycle using the Allred Score.

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In samples of patients without endometriosis (control group) we found an increased

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expression of PR-A and PR-B during the proliferative to early secretory phase. A decreased

expression of both receptor isoforms during the mid to late secretory phase, especially in the

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epithelial cells, could be discerned. In endometrial stromal cells PR-A, but not PR-B, was still

detectable until the late secretory phase.

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Samples of patients with endometriosis (endometriosis group) showed no specific cycle

dependent pattern and displayed a variety of interindividual differences in PR-A and PR-B
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expression. In particular, PR-B was not detectable in some samples of the mid-proliferative

and mid-secretory phase in epithelial as well as in stromal cells. In the late secretory phase
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we found an unexpectedly strong positive reaction of PR-B in endometrial epithelium and

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stroma compared to the control group. # = patients’ identification number; day = menstrual

cycle day.
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10.2 Legend to Figure 2

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Immunohistochemical staining of PR-A (a-l) and PR-B (m-x) in representative samples

throughout the menstrual cycle. Comparison of endometrium from patients of the control

group without endometriosis (normal endometrium: a-f; m-r) with eutopic endometrium from

patients with endometriosis (g-l; s-x). Especially PR-B is dysregulated in endometrium of

patients with endometriosis and cannot be detected in samples of the mid-proliferative and

mid-secretory phase in endometrial epithelium and stromal cells (s,t,w).

Scale bars = 20 µm. PR-A without Hematoxylin counterstaining (d,f,i,j,k); PR-B without

Hematoxylin counterstaining (p,u,w).

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Figure 1

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Figure 2

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