Human Reproduction vol.14 no.4 pp.

970975, 1999

The effect of a levonorgestrel-releasing intrauterine

device on human endometrial oestrogen and
progesterone receptors after one year of use

Zhu Pengdi, Liu Xiaoqun, Luo Hongzhi, Gu Zhao,

Cheng Jie, Xu Ruhua, Lian Shizhu, Wu Shangchun,
Wang Jiedong1
National Research Institute for Family Planning, Beijing, 100081,
China
1To

whom correspondence should be addressed

Introduction
A levonorgestrel-releasing intrauterine device (LNG-IUD)
developed in Finland has been in wide clinical use for more
than 15 years. Although its clinical performance, including
long duration of action, low pregnancy rate, reduced menstrual
blood loss and relief of symptoms of dysmenorrhoea is
encouraging, some side effects such as intermenstrual spotting
and amenorrhoea still cause problems for the users. Many
studies have suggested that the mode of action and side effects
of this IUD were, at least in part, based on the changes in
endometrial function and morphology that are produced by
the steroid liberated from the IUD (Nilsson et al., 1984;
970

Materials and methods

Subject selection
Thirty-four healthy women, aged between 23 and 35 years, with
proven fertility and regular menstrual periods were recruited for the
study. The women had no known systematic or gynaecological
disorder, had not received any hormonal therapy, steroid contraceptives
or intrauterine device for at least three cycles, and had no history of
abortion or pregnancy during the last 6 months before the start of
the study. After having obtained written informed consent from each
woman, a levonorgestrel-releasing intrauterine device (LNG-IUD)
was inserted into each subject for a period of 1215 months. The
study was approved by the Ethical Committee of the National
Research Institute for Family Planning, Beijing.
Intrauterine devices
The levonorgestrel-releasing IUD were provided by Leiras Company
(Turku, Finland). Each device contains 46 mg levonorgestrel, and
releases approximately 20 g/day. The effective lifetime of the IUD
is expected to be 7 years.
Sample collection
An endometrial biopsy was taken from the anterior or posterior wall
of the mid uterus on cycle days 1012, just before insertion of the
IUD, and served as the control representing the proliferative phase
of the normal menstrual cycle. One year later, immediately after
removal of the IUD on cycle days 1012, a second biopsy was taken.
In women with amenorrhoea who bore the IUD, the device was
European Society of Human Reproduction and Embryology

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Thirty-four women bearing a levonorgestrel-releasing

intrauterine device, 20 g/day (LNG-IUD-20), for 1215
months were recruited. Endometrial biopsies were collected
during the late proliferative phase of the cycle (on cycle
days 1012) before (control) and after the use of the IUD
for 12 months, and assayed for oestrogen receptors (ER)
and progesterone receptors (PR). An immunohistochemical
technique with the peroxidaseantiperoxidase detection
system (PAP method) was employed. D75 and JZB39 were
the primary antibodies for ER and PR respectively. The
immunostaining semiquantitative analysis was performed
with a computerized microscope image processor, and
expressed as grey value. Both endometrial ER and PR
populations were significantly lower after insertion of the
IUD (P < 0.01) than in control biopsies. The intensity of
nuclear staining and the percentage of positively stained
cells for ER and PR in women with LNG-IUD were
each about 50% of those in control biopsies. The results
suggested that LNG released locally from the IUD has a
depressive action on the ER and PR, which may contribute
to the contraceptive effectiveness of this type of IUD and
also to the possible causes of LNG-IUD-induced irregular
bleeding and amenorrhoea.
Key words: endometrium/immunohistochemistry/levonorgestrel-releasing intrauterine device/oestrogen receptor/progesterone receptor

Barbosa et al., 1995; Gu et al., 1995; Luukkainen and Toivonen,

1995; Xiao et al., 1995).
The endometrium is the target of oestrogen and progesterone.
The steroid hormone may not be directly responsible for the
endometrial functional and morphological changes, as the
hormonal actions not only coincide with the plasma hormone
concentrations but also relate closely to the numbers of
endometrial oestrogen receptors (ER) and progesterone receptors (PR). Some studies have demonstrated the effect of
progestin-releasing intrauterine devices on steroid receptor
expression in the endometrium (Janne and Ylostalo, 1980; Lu,
1991). These studies used radiochemical techniques to identify
total ER and PR populations in whole endometrium, and did
not permit identification of the receptors in particular cell
types. In the present study we used immunohistochemistry to
describe the cellular localization of ER and PR in the endometrium before and after insertion of a LNG-IUD. A semiquantitative analysis was also employed with a computerized
microscope image processor, which was intended to render
results more objective, the main aim of the study being to
help in our understanding of the mechanism of the action and
side effects of the LNG-IUD.

Effect of levonorgestrel IUD on steroid receptors

removed on any day after 12 months of use, and a biopsy taken at

the time of removal. No biopsy could be obtained in 16 cases because
of endometrial atrophy.
Specimen preparation
A small portion of the endometrial specimen was fixed in Bouins
solution and paraffin sections were stained with haematoxylin and
eosin (H&E) for morphology and dating. The major portion of the
specimen was rinsed immediately in cold normal saline, frozen in
liquid nitrogen, and stored at 70C until analysed.

Analysis of endometrial immunoreactivity

In order to assess the effect of LNG-IUD (in terms of ER and PR),
two types of scoring system were used.
Subjective score: the immunostaining intensity of all tissue sections
was scored on a five-point scale, where 0 5 no staining, 1 5 mild,
2 5 moderate, 3 5 high and 4 5 intensive staining. The recorded
immunostaining score was based on the intensity of staining expressed
by the majority of cells in each section. In brief, 310 areas were
chosen randomly in each section according to the size of the sample.
In each area (at 3200 magnification), the percentage of stained cells
of each scale was estimated by eye. The score of each area was

obtained as (IIP), where I is the intensity of staining from 04 and

P is the corresponding percentage. The mean value of all areas gives
the score of the section. All samples were evaluated by the same
observer to rule out interobserver variability.
Objective score: the immunostaining semiquantitative analysis was
performed with a computerized microscope image processor. The
method was described previously by Zhu et al. (1995). Sections were
analysed in blind fashion (Olympus BH light microscope, 3200
magnification). In each case, about 30 positive and negative nuclei
at the level of stroma and glandular epithelium respectively, were
evaluated and expressed as a grey value. The only selection criterion
was the absence of superimposition of the nuclei to obtain the real
optical density. The background was measured at the same time for
each cell in the area immediately outside the cell, in order to eliminate
batch or area differences in staining intensity. The score of each cell
was expressed as background minus nuclear values.
Data management and statistical analyses
The grey values of the endometrial cells (stromal and glandular cells)
in each subject were measured with minicomputer, VAXII, SAS-

Results
Histologically, the cellular components of the endometrium
could be clearly subdivided into glands and surrounding stroma
cells comprising the vasculature. Samples taken before IUD
insertion showed the morphology of the proliferative phase
(Figure 1a) according to the criteria of Noyes et al. (1950)
and Johannisson et al. (1987). Samples from the IUD users
displayed a pattern of uniform suppression (Figure 2a), the
glands being scarce in number and very small. The stroma
showed pseudodecidual features.
Immunoreactivity of endometrial ER and PR in control
samples
As shown in Figure 1b and c, both ER and PR were localized
in the nuclei of epithelial and stromal cells. Luminal and
glandular epithelial cells showed strong staining in nearly all
of their nuclei, and stromal cells were strongly stained in most
nuclei. The glandular cells were slightly more heavily stained,
the percentage of stained cells being greater than that in
stromal cells. The intensity and percentage of cell nuclei
staining for PR were greater than those for ER (Figure 1c;
Table I). Many of the nuclei contained a small focal area,
interpreted as a nucleolus, which was unstained. In contrast to
the densely stained nuclei, the cytoplasm and intercellular
spaces were unstained. Endometrial vascular smooth muscle
cell nuclei and endothelial cell nuclei were consistently
unstained. Results obtained from the two scoring systems were
parallel (Table I). The percentages of stained cells for ER and
PR were not shown.
Immunoreactivity of endometrial ER and PR in LNG-IUD
users
By using a series of adjacent sections, it was shown that
ER and PR immunostaining remained in the nuclei of the
endometrial cells, as before. There was a substantial reduction
(P ,0.01) in both the intensity of nuclear staining and the
proportion of cells stained for ER and PR (Figure 2bd) in
comparison with the control (Figure 1bd). The immunostaining in most cases was almost eliminated, with only
occasional isolated cells showing a variable amount of staining.
However, these occasional isolated cells were quite strongly
stained and contrasted sharply with the minimal staining in
adjacent cells. The nuclear staining intensity and percentage
of stained cells in the gland and stroma were similar (P .0.05),
but the stromal cells were slightly more intensely stained and
the percentage of stained cells was higher than that of the
glandular cells only for ER. During this post-insertion period,
the intensity and percentage of cell nuclear staining for PR
remained slightly greater than that for ER as in the preinsertion period, though no significant difference was found
(Table I). The endometrial vascular cells remained unstained,
as in the controls.
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Immunohistochemistry
Monoclonal antibodies D75 to human ER and JZB39 to human PR
were the generous gifts of Dr G.L.Greene, University of Chicago. A
peroxidaseantiperoxidase method was employed (Wang et al., 1992),
the frozen serial sections of 5 m thickness being fixed in picric
acidparaformaldehyde for 15 min, then immersed in 0.5% hydrogen
peroxide solution for 15 min to block endogenous peroxidase activity.
After washing the sections in phosphate-buffered saline (PBS),
primary antibodies (rat monoclonal antibody to human ER, rat
monoclonal antibody to human PR respectively), secondary (bridge)
antibody (rabbit anti-rat IgG), and rat peroxidaseantiperoxidase
complex were applied to sections sequentially. Between each step the
section was rinsed in PBS. The formation of an immunoprecipitate
in the section was demonstrated with 3,3-diaminobenzidine (Serva,
Heidelberg, Germany). One of the serial sections in each case was
treated with normal rat IgG instead of the primary antibody to
serve as a negative control. A section from receptor-positive tissue,
processed using the same procedure, served as a positive control in
each assay. No sections were counterstained, as this procedure is
thought to reduce the sensitivity of the image analysis system in
terms of recording the integrated optical density of the nuclei.

language Program. Statistical analysis was performed, results were

evaluated with a t-test, and significant differences between the preand post-insertion data were calculated.

Z.Pengdi et al.

Discussion
A number of studies have defined the location and distribution
of ER and PR in the endometrium (Lessy et al., 1988; Snijders
et al., 1992; Bergqvist et al., 1994). The expression of ER and
PR in the endometrium changes cyclically, although some
differences exist among the reports. In general, ER and PR
are located in the nuclei and the positive rate and degree of
expression peak during the mid cycle and then decrease.
Progestins downregulate this expression (Natrajan et al., 1982).
In this study, using an immunohistochemical method, we
showed that the staining for endometrial ER and PR in LNGIUD users was rather similar to the pattern of the mid to
late secretory phase of the normal menstrual cycle reported
previously (Lessy et al., 1988; Snijders et al., 1992; Bergqvist
et al., 1993). There was a substantial reduction in both the
intensity of the nuclear staining and the proportion of the cells
stained for ER and PR in comparison with controls. The
released LNG caused the downregulation of both ER and PR,
which is in agreement with other studies as the populations
of both endometrial oestrogen and progesterone cytoplasmic
receptors were reduced significantly after insertion of a LNGIUD (Janne and Ylostalo, 1980; Lu, 1991).
However, the LNG which is released locally from the IUD
972

does not mimic the effect of physiological progesterone on the

endometrium. The endometrium of LNG-IUD users displayed a
pattern of uniform depression. In the IUD users, the endometrial
steroid receptor population was uniformly decreased independent of the cell types, while in the normal cycle it was cellspecific, there being a major difference between epithelial and
stromal cells. Interestingly, Critchley et al. (1993) reported
that the immunoreactive PR population remained high in the
endometrium of Norplant users, whereas the population of ER
was relatively low. These findings are significantly different
from those of the present study, and may suggest that the
effect of LNG released locally on the endometrium is different
from that of circulating LNG. Moreover, Lau et al. (1996)
showed an increase in immunoreactive PR population, but a
reduction in PR mRNA levels in Norplant endometrium, while
in the normal menstrual cycle the protein is consistent with the
mRNA. It would be interesting to investigate the endometrial
expression of isoforms of ER and PR in both LNG-IUD users
and Norplant users.
In this study, the endometrial blood vessels were not stained
in either controls or after insertion of the LNG-IUD. However,
conflicting data have been obtained regarding this issue. Lessy
et al. (1988) demonstrated no immunostaining at all, whereas

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Figure 1. Light micrographs of endometrial sections for the immunoreactivity of oestrogen receptors (ER) and progesterone receptors (PR)
in IUD users. Before insertion, at cycle day 12. (a) A haematoxylin and eosin-stained section shows a normal late proliferative phase
endometrial pattern. Epithelial cells and the stromal cells in the upper functionalis exhibit strong immunoreactivity for ER (b) and PR (c)
with the specific nuclear staining using D75 and B39 as primary antibodies (PAP method). No specific cytoplasmic staining was observed.
No immunoreactivity for PR or ER was detected in the vascular endothelial cell nuclei and smooth muscle cell nuclei. (d) Background for
PR (ER not shown) in the control section. Scale bars 5 100 m.

Effect of levonorgestrel IUD on steroid receptors

others reported the presence of both ER and PR in vascular

smooth muscle cells of endometrial blood vessels (PerrotApplanat et al., 1988, 1994). A earlier study in this laboratory
reported immunostaining for PR in the endothelial cells from
human decidua (Wang et al., 1992), while Perrot-Applanat
et al. (1988, 1994) took tissue at the end of the luteal phase
or during pregnancy. It is possible that endometrial blood
vessels express the receptors when endometrium becomes
decidualized. The endometrium of LNG-IUD users also showed
a pseudodecidual pattern, but this was uniformly and heavily
depressed, including the vessels which were small with a thin
wall that was only single-layered in most cases. In addition,
combining results of the present study with those reported
previously (Lessy et al., 1988; Snijders et al., 1992; Critchley
et al., 1993; Bergqvist et al., 1994; Lau et al., 1996), it is
likely that locally released LNG affects the endometrium in a
different manner from other circulating progestins. However,
Rogers et al. (1996) demonstrated highly varied ER and PR
immunoreactivity in the vascular smooth muscle cells of
endometrial blood vessels, ranging from 0% to 85%, with a
tendency towards greater immunoreactivity in the late secretory
phase. We could not find an adequate explanation for the
difference between our results and those of Rogers et al.

(1996), who identified receptors as being present in blood

vessels throughout the menstrual cycle, albeit with high variability. However, the fact that the antibodies used in these
studies were different might provide an explanation.
Many studies suggested that the changes in endometrial
function and morphology are caused mainly by the local
effect of progestin on the endometrium (Nilsson et al., 1984;
Luukkainen and Toivonen, 1995). Previous studies of ovarian
function in LNG-IUD users have suggested that the majority
of women studied had ovulatory cycles (Nilsson et al., 1984;
Barbosa et al., 1995; Xiao et al., 1995). The intrauterine release
of levonorgestrel results in high local tissue concentration in
the endometrium (Nilsson et al., 1982). Oral administration of
a dose 10-fold higher than the daily dose administered with
LNG-IUD resulted in endometrial concentrations of levonorgestrel which were similar to those in adjacent tissues, and
the effects on the endometrium were correspondingly much
weaker than with locally released LNG (Nilsson et al., 1982).
Many LNG-IUD usersthough none of the LNG intracervical
device usersdeveloped amenorrhoea, which confirms the
local effect of LNG-IUD (Kurumaki et al., 1984).
Several studies have indicated that endometrial morphology
in women bearing a LNG-releasing IUD shows profound and
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Figure 2. Light micrographs of endometrial sections stained for the immunoreactivity of ER and PR in IUD users. Post-insertion; cycle day
12. (a) A haematoxylin and eosin-stained section showing typical uniform suppression of endometrium. A substantial reduction in both the
intensity of nuclear staining and the proportion of cells stained for ER (b) and PR (c) in comparison with the control. Specific cytoplasmic
staining was not observed. The endometrial endothelial cells and vascular myometrial cells remained unstained for ER and PR, as in the
control. (d) Background for PR (ER not shown) was shown in the control section by using normal non-immunized rat immunoglobulin in
place of monoclonal anti-receptor antibody, B39.

Z.Pengdi et al.

Acknowledgements
Table I. The immunoreactivity score of endometrial cells for oestrogen and
progesterone receptors (arbitrary units) before and after insertion of
LNG-IUD-20. Values are mean 6 SD
Index

Stroma cells
Scorea

Epithelial cells
Grey valueb

Scorea

Grey valueb

Oestrogen receptor
Before
2.97 6 0.17
(n 5 34)
After
1.69 6 0.48*
(n 5 16)

14.45 6 1.80
(n 5 34)
8.09 6 1.35*
(n 516)

2.94 6 0.24
(n 5 34)
1.43 6 0.79*
(n 5 7)

15.25 6 1.12
(n 5 34)
8.02 6 1.97*
(n 5 7)

Progesterone receptor
Before
3.62 6 0.49
(n 5 34)
After
1.43 6 0.51*
(n 5 14)

17.55 6 0.97
(n 5 34)
8.66 6 1.81*
(n 5 14)

3.74 6 0.45
(n 5 34)
1.80 6 0.79*
(n 5 10)

17.98 6 1.20
(n 5 34)
9.80 6 3.73*
(n 5 10)

,0.01 versus control.

aSubjective quantification.
bObjective grey value.

uniform suppression which is independent of the stage of the

menstrual cycle (Zhu et al., 1989; Gu et al., 1995). Locally
released levonorgestrel causes atrophy of the endometrium,
such that the glands are scarce and the stroma shows pseudodecidual features. This effect on the endometrium is seen as
early as 1 month after insertion, and is maintained as long as
levonorgestrel is released. The mechanism is likely to be a
complex process (Hsueh et al., 1976), and many factors may
be involved. For example, the decidualized cells produce
insulin-like growth factor binding protein-1, which can block
insulin-like growth factor I that is thought to be a mediator of
oestrogen-induced mitotic effects (Pekonen et al., 1992). From
the present data, we might suggest that the continuous high
concentration of locally released LNG inhibits the expression
of ER, which in turn makes the endometrium insensitive to
circulating oestradiol and provokes an anti-proliferative effect
during the use of LNG-IUD. Thus, endometrial proliferative
activity is totally arrested, and this is thought to be the main
contraceptive action of the IUD.
Taken together, the results of the present study indicate that
there is a statistically significant decrease of ER and PR in
the endometrium of LNG-IUD users, when compared with
controls, and confirms that progesterone downregulates its
receptor. However, the decrease of ER and PR populations in
LNG-IUD users was somewhat uniform, and not cell-specific
a situation which is different from that in the normal menstrual
cycle (Lessy et al., 1988; Snijders et al., 1992; Bergqvist et al.,
1994). In addition, the immunoreactivity of ER and PR in the
LNG-IUD endometrium is significantly different from that in
the Norplant endometrium (Critchley et al., 1993; Lau et al.,
1996), though it is unclear why such differences exist. However,
the data acquired in the present study show that 16 out of 34
subjects failed to produce a tissue samplea proportion similar
to that seen in Norplant users (Hadisputra et al., 1996). As,
therefore, data are unavailable from these patients, the samples
that were collected may not be truly representative, and so
care must be taken when interpreting our results.
974

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*P

The authors are highly appreciative of the gifts of LNG-IUD and

anti-receptor monoclonal antibodies from Prof. Luukkainen of Leiras
Company, Turku, Finland, and Dr G.L.Greene of Ban May Institute,
University of Chicago, USA. We are also very grateful to Dr Elisabeth
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manuscript. The present study was supported by WHO Special
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Received on April 27, 1998; accepted on December 23, 1998

975