Differences in Expression of Activin Receptor 2A at Cytotrophoblast Cells in

Preeclampsia and Normotensive Pregnancy

Muhammad Nooryanto
Fetomaternal Obstetrics and Gynecology Division, Departement of Obstetrics and Gynecology
Faculty of Medicine, Brawijaya University/ dr. Saiful Anwar Hospital, Malang, East Java – Indonesia

Objective : Preeclampsia is a placenta disease of unknown cause. Maternal circulating

concentration of protein markers are altered (mainly increased) in preeclampsia in
comparation with controls. Receptor activin 2A were found to be more elevated in pregnancy
with preeclampsia –eclampsia than in normal.
Methods: Analytic observational design by cross sectional. This research speciments were
placental cytotrophoblast cells from two groups, preeclampsia-eclampsia dan normotensive
pregnancy, collected from February – March 2014. The placenta sample size was 2x2 cm,
washed with normal saline than stored in 200 C , then cutted with rotary microtome and
deparafined, the next step was measure the Activin Receptor 2A using
immunohistochemical technique. There after we use data normality test using Shapiro-Wilk
and the data then analized with Independent Sampel t Test.
Result : Expression Receptor 2A of cytotrophoblast cell in preeclampsia with sgnificant
21.88±7.24%.compared with the controls 12.04±4.19% with p- value =0.000<0.05.
Conclusion : The expression Receptor 2A of cytotrophoblast cell of preeclampsia-
eclampsia pregnancies are significantly different compared with the controls.
Keywords ACVR2A, Cytotrophoblast cell, Preeclampsia

BACKGROUND
Preeclampsia occurs in 5-10% of all pregnancies, and is one of the deadliest trias
along with bleeding and infection. Preeclampsia contributes greatly to maternal morbidity
and mortality (Cuningham, 2010; Varughese, 2010). In preeclampsia there is increased
oxidative stress and maternal systemic inflammation. Oxidative stress is thought to stimulate
the production and secretion of Activin A from the placenta and endothelial cells of
preeclamptic pregnancy. In the last decade, several studies have investigated the possibility
of a biochemical marker in preeclampsia, one of which is Activin A (Muttukrishna, 1997). A
study conducted by Mitswenik et al in 2010 stated that in preeclampsia there was an
increase in the levels of activin A, a protein family of TGF-β which has a role in the invasion
of trophoblasts into the endometrium during the placentation phase. Cells that signal the
activin A protein are a dimer group of transformed growth factor β (TGF-β). The role of TGF-
β has been previously described in a wide variety of cell processes for reproductive function
including cell proliferation, differentiation and apoptosis. Physiologically, activin is needed in
the process of placenta formation because it facilitates trophoblast invasion. However, when
excessive production of this protein occurs, a significant increase in proinflammatory
cytokines induced by cytokines (IL-6, IL-1, TNF-a) will actually result in trophoblast defects,
leading to placental ischemia and preeclampsia. Activin A plays a role in endothelial
regulation and maternal endovascular inflammatory response (Silver, 1999).
 Preeclampsia is associated with an increase in serum activin A in mothers in several
studies allegedly due to genetic factors. The genetic risk of preeclampsia is very complex
and many facts cause preeclampsia in mothers. Linkage's research, in Australia, New
Zealand and Iceland in offspring with preeclamptic mothers has a locus on chromosome
2q22-23. ACVR2A is a receptor classified as TGF-β that binds to activin A and functions to
activate growth genes and angiogenic and non-angigenic factors that stimulate tropoblast
invasion into the endometrial epithelium and decidual stroma (Laivuori et al., 2009).
ACVR2A has been described or encoded by the ACVR2A gene activin receptor on
chromosome 2q22 as a candidate gene (Petraglia et al., 2005). It has been suggested that
the expression of ACVR2A might affect the concentration of activin A and interfere with the
process of decidualization, tropoblast invasion and remodeling of the spiral arteries.
Dysregulation of receptor expression, for example downregulation of ACVR2A expression,
might lead to an increase in serum activin A.
Mose et al. found an association between preeclampsia and Single Nucleotide
Polymorphism (SNP) (rs1424954), located 1800bp at the start of the transcription process in
the ACVR2A gene. To replicate the initial findings, we analyzed the A - G polymorphisms in
the ACVR2A gene associated with preeclampsia in a Finnish study population. The results
obtained were that patients who had Single Nucleotide Polymorphism (SNP) (rs1424954)
were preeclamptic patients. Meanwhile, 53.7% of PE patients gave birth before 37 weeks'
gestation, when compared with the mean of control group with preterm birth of 0.7% (p
<0.001). SGA infants were more frequent in PE patients (28.5%) than in the control group
(0.4%) (p <0.001). Of all PE patients, 62% had severe PE (Roten et al., 2009).
The type I and type II activin receptors are called ACVR1 and ACVR2 are a group of
transmembrane receptor proteins. Activin A binds to ACVR2 associated with ACVR1
phosphorylation. The binding of activin A with its receptor, ACVR2A (Activin receptor A type
2) is alleged to activate intracellular transduction signals that trigger transcription of pro-
inflammatory cytokine genes, activin receptors expressed in the endometrium, placental
tissue, endothelial cells of blood vessels and trophoblasts in early pregnancy (Mitswenik et
al., 2010). Because of the increased expression of the receptor for activin A, namely
ACVR2A in preeclamptic patients, it is likely that ACVR2A is involved in the
pathomechanism of preeclampsia so that it is expected to be one of the biochemical markers
of preeclampsia in the future. Identification of a marker that might predict the presence of an
early pathological process before signs and symptoms manifest can be a major advance in
obstetric science in improving the quality of health of pregnant women. For this reason, it is
necessary to research whether there is also an increase in the receptor levels for activin A,
namely ACVR2A in preeclampsia patients, as well as an increase in activin A levels.

MATERIAL AND METHODS

This research was carried out in the Obstetrics and Gynecology care unit of
Department of Obstetrics and Gynecology, Saiful Anwar Hospital Malang.
The speciments were placental cytotrophoblast cells from two groups, preeclampsia-
eclampsia dan normotensive pregnancy in third trimester ( > 28 weeks) , collected from
February – March 2014. The placenta sample size was 2x2 cm, washed with normal saline
than stored in 200 C , then cutted with rotary microtome and deparafined, the next step was
measure the Activin Receptor 2A using immunohistochemical technique.
After data is collected, data were analyzed using SPSS version 16 software. Analysis
of normality performed test using Shapiro-Wilk. If data was normally distributed, we used T-
tes to compare the mean of ACVR2A levels in the normotension and preeclamptic
pregnancy groups, namely the independent sample t test. Differences and correlations were
considered significant if P <0.05.

RESULTS
In the study the sample consisted of 2 groups, namely the group of normal pregnant
women who were selected as the control group and the group of pregnant women with
preelampsia as the preeclampsia / PE group (cases). The two groups each consisted of 25
people who had been selected based on inclusion and exclusion criteria.
Sample Characteristics
p-
Control Preeclampsia
Sample characteristics valu
(n=25) (n=25)
e
a. Maternal Age (year) 28.16±3.88 26.56±4.46 0.279
b. Education ( year) 8.64±2.34 8.36±2.34 0.705
c. Gestasional age (weeks) 38.32±2.56 38.48±2.82 0.834
d.Blood pressure
- Systolic 118.0±6.46 155.2±8.23 0.000
- Diastolic 78.0±5.78 97.2±6.14 0.000
e.Parity
9(36%) 18(72%) -
- primigravidy
16(64%) 7(28%) -
- Multigravidy
f. Apsgar score (AS)
- Normal (AS >=7) 18 (72.0%) 17(68.0%) -
- Mild asphyxia (AS=6) 7 (28.0%) 7 (28.0%) -
- Moderate asphyxia (AS 4-5) 0 (0%) 1 (4%) -
g. Length of stay
- 1-3 days 10 (40%) 3 (12%) -
- 3-5 days 11 (44%) 20 80%) -
- > 5 days 4 (16%) 2 (8%) -
h.BMI 29.79±2.28 32.1±3.32 0.006
Negatif(100% 23 Negatif(92%)
i.History of Severe Preeclampsia
) 2(8%)
Negatif(100%
j.History of Severe Preeclampsia in Family Negatif(100%)
)
k.Outcome 0.244
- Normal 21(84%) 20 80%)
- Low Birth Weight 4 (16%) 5 (20%)

Based on maternal age data , selecting research samples based on the control group
and the PE group obtained a homogeneous age of pregnant women, because the
distribution of data on the age of pregnant women was almost the same and not statistically
significant.
Systolic blood pressure data, showed that there was a very significant difference
between the control group and PE. Similarly, the diastolic blood pressure data were
distributed to the control group shows a very significant difference between the control group
and PE.
Apgar score data (AS) to be distributed in the control group with AS more than 7
people, including normal there are 18 people (72%), AS = 6 mild asphyxia there are 7
people (28%), and moderate asphyxia is absent. Meanwhile, AS seemed to be spread in the
PE group with AS more than 7 people, including 17 people (68%), AS = 6 mild asphyxia, 7
people (28%), and 1 person (4%) moderate asphyxia. There is no significant difference data
of Apgar score.
Data on length of stay appeared to be scattered in the control group with a length of
stay of 1-3 days there were 10 people (40%), at 3-5 days there were 11 people (44%), and
more than 5 days there were 4 people (16%). While the length of stay seemed to be spread
in the PE group with a length of stay of 1-3 days there were 3 people (12%), at 3-5 days
there were 20 people (80%), and more than 5 days there were 2 people (8%). There is no
significant difference data on length of stay.
BMI data for pregnant women were distributed to the control group with mean ±
stan.dev. was 29.79 ± 2.28 and in the preeclampsia group the mean ± stan.dev. is 32.1 ±
3.32There was a very significant difference (p = 0.006 <α) in the BMI data between
normotensive and preeclamptic pregnancies.
In the outcome data, it appears that there is no significant difference between the PE
group and control group.
Based on gestational age in the group / control, the largest mean ± standard
deviation was 38.32 ± 2.56 weeks, whereas in the pre-eclampsia group the mean ± standard
deviation was 38.48 ± 2.82 weeks. It appears that the distribution of gestational age data
shows almost the same value. So this means there is no significant difference in gestational
age.
The distribution of data characteristics based on parity in this study showed that in
the preeclampsia group there were 18 primigravidas (72%) and 7 multigravidas (28%).
Whereas in the normotensive group, 9 people (36%) were found primigravida and 16 (64%)
multigravidas had a previous history of preeclampsia. Preeclampsia pregnancies are more
common in primigravidas than in multigravidas.
Pregnant women who have had preeclampsia in the first pregnancy have 7x the risk
of getting preeclampsia in the second pregnancy (Duckitt, 2005). From this study,
Preeclampsia tends to have a previous history of preeclampsia.