FOLIA HISTOCHEMICA ORIGINAL STUDY

ET CYTOBIOLOGICA
Vol. 51, No. 2, 2013
pp. 135–140

Expression of estrogen and progesterone receptors

and Ki-67 antigen in Graves’ disease and nodular goiter
Pawel Domoslawski1, Bartosz Pula2, Tadeusz Lukienczuk1,
Marzenna Podhorska-Okolow2, Piotr Dziegiel2, 3
1
Department of General, Gastroenterological and Endocrinological Surgery,
Medical University of Wroclaw
2
Department of Histology and Embryology, Medical University of Wroclaw
3
Department of Physiotherapy, Wroclaw University School of Physical Education, Wroclaw, Poland

Abstract: Graves’ Disease (GD) is an autoimmune disease with higher prevalence in women than in men. The
aim of the study was to correlate the expression of estrogen (ER) and progesterone receptors (PR) as well as Ki-
67 cell proliferation index in thyroid sections of GD and nodular goiter (ND) patients. The study was performed
on archival paraffin blocks of 77 GD and 25 ND patients using immunohistochemistry. Higher expression of
progesterone receptors (p = 0.0276) and Ki-67 index (p < 0.0001) was observed in thyroids of GD as compared
to NG patients. No correlations were found between the particular markers and patients’ clinicopathological
parameters, sex or age. A higher incidence of GD in women was not associated with different thyroid expression
of ER and PR in comparison to men. No correlation was found between the expression of ER and PR and pro-
liferation marker of thyrocytes of GD and ND patients. Significantly higher expression of the Ki-67 antigen in
GD lesions was observed as compared to the NG. (Folia Histochemica et Cytobiologica 2013, Vol. 51, No. 2, 135–140)

Key words: thyrocytes, Graves’ disease, nodular goiter, estrogen receptors, progesterone receptors, Ki-67

Introduction roiditis (HT) affect the thyroid gland and are called
autoimmune thyroid diseases (AITD). They are com-
Graves’ Disease (GD) is a common organ-specific au- plex diseases caused by an interaction of susceptibility
toimmune disorder of recurrent nature and complex genes and other, non-genetic factors like infection [1,
pathogenesis involving genetic and environmental 3]. The background of GD appears to have a strong
factors resulting in majority of the cases in hyperthyro- genetic nature associated with the polymorphism in
idism [1]. Autoimmune diseases (AID) appear when the HLA, CTLA-4 (cytotoxic T-lymphocyte associa-
the host immune system turns against its own antigens ted antigen-4) and protein tyrosine phosphatase type
leading to dysfunction or destruction of tissues and 22 (PTPN22) genes but the pathogenesis is still not
organs. AID may develop in mechanisms involving clear [1]. In GD thyroid-reactive T cells infiltrate the
immune deregulation, genetic predisposition and due thyroid gland and activate B cells to produce thyro-
to influence of environmental factors [2]. Thyroid tropin receptor (TSHR) stimulating autoantibodies,
autoimmune diseases like GD and Hashimoto thy- leading to stimulation of thyroid follicular cells [3].
As a result, the negative control feedback loop of
the pituitary system is superseded by the stimulating
Correspondence address: P. Domoslawski, antibodies leading to constant thyrocyte activation via
Department of General, Gastroenterological
the TSHR and the production of thyroid hormones,
and Endocrinological Surgery,
Medical University of Wroclaw;
thyroxine (T4) and triiodothyronine (T3) leading to
Sklodowskiej-Curie St. 66, 50–369 Wroclaw Poland; hyperthyroidism [3].
tel.: +48 71 784 2162; fax: +48 71 7959619; Like other autoimmune diseases, GD appears
e-mail: [email protected] more frequently in women than in men at a ratio of

©Polish Society for Histochemistry and Cytochemistry

Folia Histochem Cytobiol. 2013 www.fhc.viamedica.pl
10.5603/FHC.2013.0021
136 P. Domoslawski et al.

7:1, which might suggest the role of hormonal fac- Histopathological examination and immunohistochemical
tors in its pathogenesis [4]. Recent data showed that reactions (IHC). Tissue samples were fixed in 10% buffered
women are up to 7–10 times more likely susceptible formalin, dehydrated and embedded in paraffin blocks.
to have GD, systemic lupus erythematous or Sjogren’s For histopathological examination, the 6-µm sections were
disease [5]. On the other hand, some other autoimmune stained with hematoxylin and eosin (H&E) and reviewed by
diseases, e.g. rheumatoid arthritis or multiple sclerosis a pathologist to verify the diagnosis. Immunohistochemical
develop only 2-3 times more often in women than in men reactions were performed on 4-µm-thick sections mounted
[5]. When unanticipated dimorphic findings appear be- on Superfrost Plus slides (Menzel Gläser, Braunschweig,
tween sexes, the first consideration is given to sex steroid Germany), deparaffinized in xylene and gradually rehy-
hormones, which are known as potent mediators for sex drated. Then, the sections were boiled in Target Retrieval
differences. Sex steroid hormones include estrogens, Solution buffer (pH 6, 20 min; DakoCytomation, Glostrup,
progesterone and androgens and their action is depen- Denmark), subsequently cooled in distillate water and
dent upon their circulating blood levels and expression 1×PBS. Sections were then incubated with 3% hydrogen
of congruent hormone receptors [5]. peroxide for 5 min to block the endogenous peroxidase.
The protein, Ki-67 antigen is widely used as a Next, the sections were incubated with primary monoclonal
marker of cell proliferation. Its precise function in antibodies directed against ER (clone 1D5, ready-to-use,
cell proliferation is still unknown [6–10]. It was found specific for detecting only the type), PR (clone PgR 636, re-
that Ki-67 may be involved in perinuclear chromatin ady-to-use) and Ki-67 (clone MIB1, ready-to-use) for 45 min
organization and its highest expression is noted in late in room temperature (DakoCytomation). The antigens were
G1, S and G2 phase of the cell cycle [6, 11]. In addition visualized using biotinylated antibodies and streptavidin
to its expression in proliferating cells, Ki-67 expression conjugated with horseradish peroxidase (EnVision™ Mouse
was also noted in processes associated with inhibited HRP, DakoCytomation). Diaminobenzidine (DAB, Dako-
DNA synthesis or in the course of apoptosis [6, 11]. Cytomation) was used as the substrate. All the reactions
Despite some incoherent information concerning the were conducted using negative controls and all the slides
biological significance of Ki-67 in cell physiology, this were counterstained with hematoxylin (DakoCytomation).
antigen is being currently routinely used as marker
of cell proliferation in clinicopathological diagnostic Evaluation of IHC reactions. The IHC sections were eva-
methods, therefore it may also serve as a potent pro- luated using BX-41 light microscope (Olympus, Tokyo,
liferation marker of thyroid follicular cells [11–13]. Japan) by two independent pathologists who were blinded
The purpose of this retrospective study was to to patients’ clinical data. In doubtful cases a re-evaluation
identify the association between the expression of es- was performed using a double-headed microscope and
trogen receptors (ER), progesterone receptors (PR) the staining was discussed until a consensus was achieved.
in patients with GD to determine if these factors have ER, PR and Ki-67 antigen expression in follicular cells was
influence on GD and its higher incidence in women. assessed in whole tissue sections using a semiquantitative fi-
Moreover, the study aimed at examining the effects ve-point scoring system-based cell positivity. It was encoded
of ER and PR on proliferation rate of thyrocytes of as follows: 0 (0% cells stained), 1 (1–5% cells stained), 2
GD patients, by assessing the expression of the Ki-67 (6–10% cells stained), 3 (11–20% cells stained), 4 (21–100%
antigen. cells stained).

Material and methods Statistical analysis. The obtained results were subjected
to statistical analysis using Prism 5.0 (GraphPad, La Jolla,
Patients and tissue specimens. The study was performed US). The Shaphiro-Wilk normality test was used to assess
on archival paraffin blocks of 77 patients with diagnosed the normality of the data distribution. Correlation between
GD and 25 patients with nodular goiter (ND), who were expression intensities of selected markers was examined
operated on at the Department of General, Gastroentero- using Spearman correlation test. The Mann-Whitney U
logical and Endocrinological Surgery of Wroclaw Medical test was applied to compare the differences in expression of
University in the years 2003–2007. The mean age of patients the studied markers between the groups. The results were
with Graves’ disease was 38.89 ± 12.68 (range 16–68) years, considered significant at p < 0.05.
whereas nodular goiter patients were aged 40.28 ± 12.1
(range 21–61) years. In case of GD, 15 (19.5%) patients Results
were male, whereas 62 (80.5%) were of female sex. In the
NG group 6 (24.0%) patients were male and 19 (76.0%) Nuclear expression of ER, PR and Ki-67 was observed
were female. The study was approved by the Bioethical in thyrocytes of patients with GD and compared to
Commission of the Wroclaw Medical University. their expression in patients with NG (Figure 1). The

©Polish Society for Histochemistry and Cytochemistry

Folia Histochem Cytobiol. 2013 www.fhc.viamedica.pl
10.5603/FHC.2013.0021
ER, PR and Ki-67 in Graves’ disease 137

A B

C D

Figure 1. Low (A, C) and high (B, D) nuclear expression of PR (A, B) and ER (C, D) in thyroids of Graves’ disease pa-
tients. Arrowheads indicate positive nuclear reaction. Magnification × 400

Table 1. Distribution of ER, PR and Ki-67 in thyrocytes of Graves’ disease and nodular goiter patients
IHC marker score Graves’ disease No. cases (%) Nodular goiter No. cases (%) All patients
No. cases (%)
ER
0 (0%) 36 (46.7) 8 (32.0) 44 (43.1)
1 (1–5%) 19 (24.7) 12 (48.0) 31 (30.4)
2 (6–10%) 12 (15.6) 4 (16.0) 16 (15.7)
3 (11–20%) 6 (7.8) 1 (4.0) 7 (6.9)
4 (21–100%) 4 (5.2) 0 (0.0) 4 (3.9)
PR
0 (0%) 32 (41.6) 15 (60.0) 47 (46.1)
1 (1–5%) 16 (20.8) 7 (28.0) 23 (22.5)
2 (6–10%) 13 (16.8) 2 (8.0) 15 (14.7)
3 (11–20%) 11 (14.3) 1 (4.0) 12 (11.8)
4 (21–100%) 5 (6.5) 0 (0.0) 5 (4.9)
Ki-67
0 (0%) 19 (24.6) 19 (76.0) 38 (37.4)
1 (1–5%) 41 (53.3) 5 (20.0) 46 (45.1)
2 (6v10%) 12 (15.6) 0 (0.0) 12 (11.6)
3 (11–20%) 3 (3.9) 1 (4.0) 4 (3.9)
4 (21–100%) 2 (2.6) 0 (0.0) 2 (2.0)

IHC — immunohistochemical, IHC scores were estimated as described in Material and methods

distribution of the studied markers in both studied proliferation rate in comparison to NG as measured by
groups was summarized in Table 1. We noted a si- the expression of the Ki-67 antigen (1.07 ± 0.89 vs. 0.32
gnificantly higher PR expression in GD as compared ± 0.69; p < 0.0001; Figure 2B). No differences were
to ND (1.23 ± 1.31 vs. 0.56 ± 0.82; p = 0.0276; Figure noted between the two groups regarding the ER expres-
2A). Thyrocytes affected in the GD exerted also higher sion (GD: 1.00 ± 1.19 vs. ND: 0.92 ± 0.81; p = 0.7349).

©Polish Society for Histochemistry and Cytochemistry

Folia Histochem Cytobiol. 2013 www.fhc.viamedica.pl
10.5603/FHC.2013.0021
138 P. Domoslawski et al.

rrently, the data concerning the expression of these

receptors in thyrocytes of GD remain obscure. In
women progesterone plays an important role during
pregnancy. Its elevated levels keep the uterus in com-
plete quiescence, acting through its nuclear PR [14].
Progesterone is also known for its anti-inflammatory
role in human myometrial cells, as it acts antagonisti-
cally to NF-kB activation and induces cyclooxygenase 2
expression, the crucial enzyme in prostaglandin produc-
tion and inflammation [14]. Therefore, progesterone
besides its endocrine effects in pregnancy, acts as an
‘immunosteroid’ with local immunosuppressant functions
[15]. Progesterone acts through PR via the induction of
Progesterone Induced Blocking Factor (PIBF), which in
turn may affect B lymphocytes. In result, the interleukin
expression profile of these cells switches to a more non-
cytotoxic, therefore supporting the immunosuppressive
role of progesterone and the PR [16].
Interestingly, in our study we observed the incre-
ased expression of PR receptors in GD patients as
compared to NG patients. PR expression was also
studied in rheumatoid arthritis (RA), a disease with
higher prevalence in women [17]. Ishizuka et al. [17]
found a significant difference in the relative expres-
sion levels of progesterone receptor type B (PR-B)
in synovial tissue. In light of those results, it seems
that elevated PR in thyrocytes of GD patients may
Figure 2. Differences in the expression of PR (A) and Ki-67 represent a protective, immunosuppressive mecha-
antigen (B) between the Graves’ disease and nodular goiter
nism in this disease, but further research should be
patients. *p < 0.05; ****p < 0.0001 (Mann-Whitney test)
undertaken with inclusion of patients detailed clinical
data to clarify such a hypothesis.
No significant correlations were noted between Two types ER are recognized, the estrogen recep-
the studied markers and patients age in the particular tor a (ERa) encoded by ESR1 gene (locus 6q25.1)
study groups and when both groups were analyzed and estrogen receptor b (ER b), which is encoded by
together (Spearman correlation test). Expression the ESR2 gene (locus 14q23), which mediate actions
intensities of the studied markers did not differ re- of estrogen in multiple tissues [1]. The ESR2 gene
garding patients age and sex in GD and NG lesions. is situated close to the GD locus at 14q31 [3, 18].
Moreover, no differences in patients age and sex were In our study we estimated the expression of ERa,
observed between the two analyzed lesions. however, we failed to note any significant differen-
ces in its expression in GD and NG. The ERa was
Discussion shown to promote proliferation in the tissue of the
uterus, mammary gland and cells of the immune
In this study we have investigated the association system [4, 19], therefore we analyzed its expression
between expression of ER and PR in patients with in GD thyroids. Until now, only one study analyzed
GD to determine whether these factors may have in- the relationship between the ER in thyroid cells of 40 GD 
fluence on the proliferation of thyrocytes and overall patients and compared to its expression noted in
higher incidence of GD in women. We found higher 15 control cases encompassing healthy thyroid tissu-
level of PR expression in thyrocytes of GD patients es [20]. In contrast to the results obtained by us on
in comparison to NG patients. Moreover, we noted a larger patients cohort, Wang et al. found a significant
a significantly higher expression of the Ki-67 antigen correlation between ERa expression in GD thyrocytes
in GD lesions as compared to the NG. as compared to normal thyroid [20]. Although in our
Cellular responses to sex hormones are dependent study we did not noted any significant differences
on the expression of hormone receptors that include in ER expression between GD and NG, we may not
two types of ER, PR and androgen receptors. Cu- directly compare our results with the results obtained

©Polish Society for Histochemistry and Cytochemistry

Folia Histochem Cytobiol. 2013 www.fhc.viamedica.pl
10.5603/FHC.2013.0021
ER, PR and Ki-67 in Graves’ disease 139

by Wang et al. as the ER expression of ER was not Acknowledgement

studied in NG lesions [20]. The results of studies
analyzing ER expression in NG were inconsistent, as This study was supported by the grant of Wroclaw
some studies reported even higher ER expression as Medical University, No. ST-654.
this noted in our studies, whereas some did not report The authors thank Mrs. Aleksandra Piotrowska,
ERa expression in thyrocytes [21–23]. Studies aimed Mrs. Bozena Przygodzka, Mrs. Agnieszka Baranska
at examining the associations between ER and GD and Mrs. Elzbieta Polejko from the Department of
found, that the ESR2 gene was not associated with GD Histology and Embryology, Medical University of
incidence in the Japanese population [24], whereas Wroclaw for their technical support.
in the Polish study of Kisiel et al., the authors found
a significant association between this gene and GD References
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Submitted: 18 December, 2012

Accepted after reviews: 27 May, 2013

©Polish Society for Histochemistry and Cytochemistry

Folia Histochem Cytobiol. 2013 www.fhc.viamedica.pl
10.5603/FHC.2013.0021