Differential Expression of EGFR-1, MMP-3, and MMP-9 in Spontaneous Abortions, Induced Abortions, and Tubal Pregnancies
Mahi BALCI , Gülhan ÖZDEMIR
Department of Pathology, Kırıkkale University Faculty of Medicine , KIRIKKALE, TURKEY
Keywords: Spontaneous abortion, Induced abortion, Epidermal growth factor receptor, Metalloproteinases, Tubal pregnancy
The purpose of our study was to assess trophoblastic and uterine sufficiency in miscarriage pathogenesis with immunohistochemical
methods and to determine if they could be used as a screening tool for the risk of miscarriage in the future.
Material and Method: Placental tissue specimens that were comprised of 20 spontaneous abortions, 23 voluntarily terminated (induced)
abortions, and 12 tubal pregnancies were included in this study. Trophoblastic cells and implantation area were evaluated for staining with
EGFR-1, MMP-3, and MMP-9 by immunohistochemistry.
Results: EGFR-1 expression was more intense and diffuse in decidual cells in the placental bed of spontaneous abortion specimens; this difference
was statistically significant (P=0.004). MMP-3 expression was markedly increased in villous and extravillous trophoblastic cells in induced
abortions; the difference between the groups was found to be statistically significant (P values ranged from < 0.01 to 0.005). MMP-9 expression
tended to be higher in spontaneous abortion and tubal pregnancy specimens, and the results were statistically significant as P values were lower
Conclusion: Higher EGFR-1 expression in the decidual tissue of spontaneous abortion specimens suggests that EGFR-1 triggers the migration
of extravillous trophoblasts, leading to their destructive invasion. Similarly, MMP-9 immunopositivity might be indicative of aggressive invasion
contributing to spontaneous abortion pathogenesis. Relatively high levels of MMP-3 expression in induced abortion specimens used as a control
group might be a predictor of successful implantation, whereas its decreased expression might be indicative of risk for pregnancy loss.
Spontaneous abortion is defined as the rejection of an
embryo or fetus weighing less than 500 g and conceptus
material consisting of placenta and its supplements from
the uterus before 20-22 weeks of pregnancy 1,2
incidence of abortion, the most common complication of
pregnancy, is approximately 15% in clinically diagnosed
pregnancies. Although chromosomal abnormalities are
present in 50% of all spontaneous abortion materials, the
percentage is as high as 70% in miscarriage specimens in the
6th week of gestation 1
. Endocrine disorders, anatomical
abnormalities, multiple gestations, antiphospholipid
antibodies, and medications are among the other etiological
factors of early pregnancy loss. However, the mechanism
underlying abortion has not been precisely elucidated. In
particular, an explanatory cause of recurrent pregnancy
losses cannot be found in 60%-70% cases 1,3-5
Considerable effort has focused on the implantation step,
a very complex process involving interaction between
blastocyst and endometrium. In particular, matrix
metalloproteinases (MMPs) and tissue inhibitors of
metalloproteases (TIMPs) have been extensively studied
for their roles. MMPs are a family of zinc-dependent
proteolytic enzymes with 28 identified isoforms. Several
studies showed that MMP-2 and MMP-9 were expressed
at early stages of placentation and that they degrade
collagen type IV and other basal membrane components
6-8. Specifically, MMP-2 is produced and secreted
predominantly by extravillous trophoblasts during early
stages of the first trimester with a peak between the 6th
and 8th weeks. Similarly, the expression of MMP-9 was
shown in villous cytotrophoblasts and, to a lesser extent,
in extravillous trophoblasts at both the mRNA and protein
levels 9-12. MMP-3 degrades fibronectin, laminins, and
various types of collagen and proteoglycan core proteins. Previous studies have demonstrated that MMP-3 is
weakly expressed in the proximal proliferating layer and is
downregulated during the early invasive period, which is
followed by upregulation in the late invasive period 10,13.
Human placental development is an example of
organogenesis with a very rapid growth capacity, similar
to that of malignant tumors. The placenta synthesizes and
secretes hormones, growth factors and their receptors
as well as proto-oncogenes needed in both the growth
and differentiation stages. One of the highly detected
growth hormones in the human placenta is the epidermal
growth factor (EGF) and its receptor, EGFR-1. EGF is a
potent epithelial mitogen responsible for trophoblastic
proliferation and has a stimulating effect on trophoblastic
invasion. EGFR-1 is produced by cells in the proximal
portion of the trophoblastic cell column in the early
period. In addition, its expression was reported in other
endometrial tissues and glandular secretions 14-16.
We hypothesized that, in addition to embryonic genetic
defects, implantation failure also plays an important role
in the etiology of early pregnancy loss. The purpose of this
study was to elucidate the decidua-trophoblast relationship
in the early implantation stage and to assess the role of
trophoblasts and decidual cells in miscarriage pathogenesis
with immunohistochemical approaches.
Placental tissue specimens from 55 archival cases that
were comprised of 20 spontaneous abortions, 23 induced
abortions, and 12 tubal pregnancies were included in this
study. Paraffin blocks of formalin fixed tissue specimens were
collected from the archives of the Pathology Department at
the Faculty of Medicine, Kirikkale University. The age of
patients ranged from 20 to 26, and none of the patients had
evidence of psychological disorders. Suction curettage was
performed before 13 weeks of pregnancy for both induced
and spontaneous abortions. The time of surgical removal of
ectopic growth materials varied between 14 and 16 weeks
For immunohistochemistry, 6-μm-thick sections that were
mounted on glass slides coated with organosilane adhesive
were stained using the streptavidin-biotin method. Briefly,
histological sections were deparaffinized in xylene and
rehydrated through a graded series of alcohol washes.
After antigen retrieval, endogenous peroxidase activity was
blocked with 3% hydrogen peroxide incubation for 5 min.
After washes with distilled water, the slides were arranged
in a Sequenza automatic slide stainer (Ventana, Benchmark XT), and phosphate-buffered saline (PBS) was used as the
washing solution. After blocking in Ultra-V-Block protein
blocking Reagent (Labvision, Fremont, CA, USA) for 5
min, slides were incubated with primary antibodies against
EGFR-1 (ab16671, diluted in 1/200), MMP-3 (ab77962,
diluted in 1/100), and MMP-9 (ab51203, diluted in 1/200)
at room temperature in a humidifier overnight. The slides
were then washed with PBS, incubated with biotinylated
secondary goat antibodies for 45 min, and stained with
diaminobenzidine (Thermo Fisher Scientific Anatomical
Pathology, UK) as a chromogen for 20 min. The slides
were counterstained with Mayers hematoxylin for 4 min.
Finally, the sections were dehydrated through a graded
series of alcohol washes, cleared in xylene, and mounted
in Entellan® (Merck, Kenilworth, NJ, USA). Placental tissue
was used as a positive control, and PBS not containing
primary antibodies was used as negative control.
Assessment of Immunohistochemical Staining
Villous and extravillous trophoblasts and placental bed in
serial sections of the specimens were evaluated for staining
with EGFR-1, MMP-3, and MMP-9. We used a semiquantitative
scoring system to evaluate staining intensity
and extent. Every slide was examined by two pathologists.
Specifically, each evaluated area was given a score according
to the intensity of nucleic or cytoplasmic staining (no
staining, 0; weak staining, 1; moderate staining, 2; strong
staining, 3) and the extent of stained cells (0%, 0; < 33%, 1;
34%-66%, 2; > 66%, 3). The final immunoreactive score was
determined by multiplying the intensity score with the score
for extent of stained cells, to achieve a minimum score of
0 and a maximum score of 9 secretions 17. Accordingly,
sum scores were categorized into three levels for statistical
analysis: level 1, 1-3; level 2, 4-6; and level 3, 7-9.
All statistical analyses were performed using the SPSS
15.0® statistical software (SPSS Chicago, IL, USA). The
Chi - square (χ2) test was performed for comparison of
the immunohistochemical expression levels between
two groups. P values < 0.05 were considered statistically
significant with a confidence interval of 95%.
The majority of cytotrophoblastic, syncytiotrophoblastic
and extravillous trophoblastic cells showed level 3
EGFR-1 immunostaining in all three clinical groups. A
statistically significant difference was detected between
cytotrophoblasts of induced abortion and tubal pregnancy
cases (p=0.012; Table I
). A level 3 MMP-3 immunostaining
was observed in cytotrophoblastic cells of 21 of a total of 23 induced abortion cases, which was much lower
in spontaneous abortion and tubal pregnancy cases
(p<0.01 or equal to 0.001; (Table I
) (Figure 1
level 3 immunostaining was determined for MMP-3 in
syncytiotrophoblastic cells in most induced abortion
cases when compared with spontaneous abortions and
tubal pregnancy (p< 0.01) (Table II
) (Figure 1
spontaneous abortion and tubal pregnancy specimens had
level 3 syncytiotrophoblastic MMP-9 expression, whereas
the majority of spontaneous abortion cases displayed
level 2 MMP-9 immunostaining (p < 0.01) (Table II
In extravillous trophoblasts, all spontaneous abortions
and tubal pregnancy specimens showed level 3 MMP-9 immunoreactivity while moderate or weak nuclear staining
was observed in induced abortions (p< 0.01) (Table III
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|Table I: The sum value of the EGFR-1, MMP-3, MMP-9, positivity in cytotrophoblasts between the groups (n)
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|Table II: The sum value of the EGFR-1, MMP-3, MMP-9, positivity in syncytiotrophoblasts between the groups (n)
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|Table III: The sum value of the EGFR-1, MMP-3, MMP-9, positivity in extravillous trophoblasts between the groups (n)
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|Figure 1: Intense and diffuse nuclear MMP-3 expression in
villous and some extravillous trophoblasts in induced abortion
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|Figure 2: Diffuse and strong MMP-9 positivity in extravillous
trophoblasts with atypical nuclear features in the vascular lumen
and stroma in spontaneous abortion (IHC; x100).
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|Figure 3: Diffuse and intense immunoreaction with MMP-9 in
extravillous trophoblasts in ectopic pregnancy (IHC; x100).
The expression of EGFR-1, MMP-3, and MMP-9 was
evaluated only in spontaneous and induced abortion
specimens as a decidual reaction is minimally observed in
tubal pregnancy. The results demonstrated that significantly
more spontaneous abortion cases exhibited level 3 EGFR-
1 expression in decidual cells of the implantation area,
compared with induced abortions (p= 0.004) (Table IV)
(Figure 4). There was also a significant difference between
MMP-3 and MMP-9 expressions among corresponding
groups (p=0.003 and p<0.01, respectively) (Table IV).
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|Figure 4: Strong cytoplasmic EGFR-1 positivity in implantation
site decidual cells in spontaneous abortion (IHC; x200).
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|Table IV: The sum value of the EGFR-1, MMP-3, MMP-9, positivity in implantation area desidual cells between the groups (n)
The adherence of competent blastocyst on the receptive
endometrium includes a complex and tightly regulated
process of mutual signaling where maternal-fetal dialogue
is established. Further, successful implantation requires
an optimally maturated decidua, well developed capillary
plexus, and adequate glandular development prior to implantation. Of these, functional decidualization is
essential for pregnancy to regulate maternal immune
tolerance and facilitate placentation 18,19
implantation and placentation, a variety of growth
factors, adhesion molecules, proto-oncogenes, cytokines,
and proteases play active roles in remodeling of the
endometrium, trophoblast invasion, proliferation, and
In this study, we showed intense and diffuse EGFR-1
expression in both villous and extravillous trophoblasts in
all clinical groups. However, the rate of cases displaying
strong and diffuse EGFR-1 expression in decidual cells of
implantation area was higher in spontaneous abortions
than induced abortions. Our findings suggested that the
number of EGFR-1 receptors in the placental bed might
be upregulated to compensate for invasion of stimulated
trophoblasts. Conversely, EGFR-1 in decidual cells might
exhibit an overexpressed mutant type that is the result of
molecular defects affecting DNA in spontaneous abortion
cases, which warrants future molecular analysis. Large et
al. demonstrated that the coordinate actions of EGFR are
critical for the successful progression of early pregnancy.
They identified over 3,000 misregulated genes in the absence
of EGFR. Bioinformatics analysis revealed widespread
alterations including genes involved in growth factor and
kinase signaling, the cell cycle, DNA replication, and cell
survival 20. Wright et al. found that the expression of α1
and α6-adhesion molecules which are essential for invasion
or interaction with the extracellular matrix were decreased
in the extravillous trophoblastic cell line by blocking
epidermal growth factor receptor (EGFR/HER1) activation
21. In contrast, EGFR is overexpressed in approximately
50% of endometrial tumors and is significantly associated
with decreased survival in patients with Type II endometrial
Increased level of EGFR-1 expression observed in
decidua of spontaneous abortions in our study could be
interpreted as exaggerated decidualization. We suggest
that this aberrant decidual phenotype would respond
paradoxically to embryonic HCG stimuli. In addition,
EGF might reduce maternal immune tolerance. EGFR-
1 has a number of ligands including EGF, transforming
growth factor (TGF-α), heparin-binding (HB)-EGF,
amphiregulin, betacellulin, and epiregulin; many
inflammatory cells, such as eosinophils, neutrophils,
mast cells, and macrophages can produce these ligands.
EGFR activation leads to cell migration, proliferation,
and upregulation of cytokines modulating innate immune
system 24,25. Successful reproduction relies on limited
inflammation during implantation, anti-inflammation
during the mid-pregnancy, and inflammation again during
parturition. Uterine natural killer cells (NK) and regulatory
T cells (Tregs) are abundant during the early stages of
pregnancy. NK cells and Tregs have supposed to undergo
numerical or functional changes in patients with a history
of spontaneous abortion, implantation failure or other
pregnancy complications. Tregs acquire an inflammatory
phenotype and produce inflammatory cytokines instead of
immunosuppressive IL-10 and TGF-β 26-28.
Numerous findings have suggested that a substantial
portion of the effects attributed to EGFR antagonist
treatment in cancer might not be based on direct influence
on the tumour itself. Instead it may be based on indirect
effects, potentially mediated via the immune system. EGFR
antagonist treatment could be impacting tumor growth by
blocking macrophage and regulatory CD4+ T cell function
Disruption of balance between activating and inhibiting
regulatory factors controlling immunity in the implantation
might lead to the accumulation of potential cytotoxic
decidual macrophages and Tregs that trigger immune
mediated pregnancy loss. However, further research will
be needed to clarify the relationship between EGF and
In this study, we evaluated the invasive capability
of trophoblasts, an important step of implantation,
extracellular matrix degradation, and remodeling of the
matrix by MMPs secreted by these cells. MMP-2 and MMP-
9 play key roles in the destruction of matrix components and
were extensively studied in the context of early implantation.
However, there are a limited number of well controlled
studies on recurrent pregnancies or spontaneous abortions,
with conflicting results. For example, while some studies
determined that the expression of MMPs and their tissue
inhibitors were increased in recurrent miscarriages, others
reported decreased levels of these factors. The distribution
of MMP-3 and MMP-9 expression in our study differed
between different clinical groups. The expression of MMP-
3 tended to be higher in induced abortion cases, whereas
MMP-9 exhibited evident immunopositivity in specimens
from spontaneous abortions and tubal pregnancies.
In an in vivo model of induced abortion, where Kanca et al.
inhibited pregnancy at mid-gestation with a progesterone
receptor antagonist in ten domestic animals, MMP-2 and
MMP-9 activity were evaluated 30. They also included
seven animals with normal pregnancy as the control group
and five spontaneous abortion cases. They determined
that MMP-2 and MMP 9 expression in induced abortion
specimens were lower than those in spontaneous abortion
cases, with lowest expression in the control group. These
findings were consistent with the results of our study.
Similarly, an immunohistochemical study of MMP-9 and
TIMP-3 conducted by Liu et al. compared 30 spontaneous
abortion cases with 20 normal pregnancies. In cases
of spontaneous abortion, they detected that MMP-9
expression was higher in the decidua from spontaneous
abortions than that of normal pregnancies. In addition,
they did not detect a difference in TIMP expression
between clinical groups, which was similar to that observed in controls. They concluded that an increased ratio of
MMP-9/TIMP-3 might trigger spontaneous abortion
31. Nissi et al. assessed serum levels of MMP-2, MMP-
9, and their tissue inhibitors in 40 normal pregnancies
and 89 spontaneous abortions. The authors found that
serum MMP-9 and MMP-2/TIMP-2 ratio were elevated
in spontaneous abortions, whereas tissue inhibitors were
at lower levels in normal pregnancy cases 32. Sundrani
et al. reported higher mRNA levels of MMP-1, -2 and -9
in the placentae of those delivering preterm as compared
to term labor. Additionally, MMP-9 gene promoter was
hypomethylated in preterm placentas compared to term
placenta in this study 33.
It has been shown that placental leukocytes from human
term pregnancies are able to secrete large amounts of MMP-
9, and that the production of the enzyme is enhanced by
labor. The authors demonstrated that endogenous MMP-3
plays a major role in the MMP-9 activation process 34.
These findings are in accordance with our suggestion that
inflammation may contribute to creating the collagenolytic
microenvironment that induces miscarriage like term
labor. Some studies have focused on genotype analysis
of matrix metalloproteinases including the MMP-9 gene
polymorphism but found no significant differences
between spontaneous abortion or other placental disorders
and control groups 35,36.
The role of MMPs was previously investigated in tubal
pregnancy materials as well. Since there is no decidualization
in tubal mucosa, Neratzoula and Judith reported that
trophoblasts could exhibit a highly invasive phenotype
in tubal pregnancy 37,38. A study on unruptured
tubal pregnancy specimens by Qui et al. suggested that
the levels of MMPs were significantly increased in the
ectopic implantation site and that the expression of tissue
inhibitors of MMPs were decreased. They concluded that
dysregulation of the balance between MMPs and their
tissue inhibitors could result in widespread and severe
destruction of extracellular matrix in ectopic implantation
39. Both the mRNA level and immunoreactivity of MMP-
9, TIMP-1 and -3 has found to be increased, while those
of TIMP-2 decreased concurrent with the progression of
pregnancy during weeks 3-9 in tubal pregnancy 40.
The data obtained from these studies investigating MMPs
to understand implantation, remodeling, and placentation
showed that MMP-9 played a more important role than
other MMPs and that invasion could not occur without
MMP-9 in vitro.
The findings in our study suggested that increased
MMP-9 expression in spontaneous abortion and tubal
pregnancy might result from an exaggerated, uncontrolled
trophoblastic invasion as part of the miscarriage
pathogenesis. In addition, increased EGFR-1 expression
in decidual cells of spontaneous abortion cases suggested
that EGFR-1 might stimulate the migration of extravillous
trophoblasts, leading to destructive invasion. Overall, these
findings indicated that aggressive invasion, might play a
role in spontaneous abortion pathogenesis.
We detected relatively moderate MMP-3 expression
in spontaneous abortion specimens compared to other
groups, which suggests that there is an inverse correlation
between MMP-3 and MMP-9. The presence of relatively
high MMP-3 expression in induced abortion cases that
comprised the control group in this study might be a
predictor of successful implantation. Consequently,
decreased MMP-3 level might be indicative of increased
risk for pregnancy loss. Future comparative studies are
needed to determine if serum MMP levels could be utilized
to assess pregnancy progression and to prevent unwanted
abortions via screening.
We thank Dr. Mustafa Emre Ercin and Dr. Selçuk Yıldız
for providing assistance in data collection and statistical
CONFLICT of INTEREST
The authors declare no conflict of interest.
Our study was financially supported by Kirikkale University
Research Project Unit. The Project number: 2012-80.
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