Effect of Erythropoietin On Feto-Placental Development

N. Yazhan et al.
Original article
The New Journal of Medicine 2007;24: 213-216
Effect of Erythropoietin On Feto-Placental
Development: The Role of Placental Nitric Oxide
Level In Rat
Nuray YAZIHAN 1, Gülnur GÜLER 2, Fethullah KENAR 3, Bülent SALMAN 4, Nilüfer ERKASAP 5,
Kubilay UZUNER 5
1
Ankara University Medical Faculty, Molecular Biology Research and Development Unite, ANKARA
2
Hacettepe University Medical Faculty Department of Pathology, ANKARA
3
Afyon Kocatepe University, Medical Faculty, Ear Nose Throat and Head & Neck Department, AFYON
4
Gazi University Medical Faculty, Department of General Surgery, ANKARA
5
Osmangazi University Medical Faculty, Department of Physiology, ESKøùEHøR
ABSTRACT
Erythropoietin (EPO) is the major hormone that controls
the erythropoiesis and tissue oxygenation. EPO has been
getting interest to the treatment of anemias also during
pregnancy. Erythropoietin con not pass through the
placenta; however its receptors are present in placenta
especially on trophoblasts. Endogen EPO level can
increase under various pathological conditions of
pregnancy, in the present study we aimed to test the
effects of EPO treatment on fetal-placental development
during rat pregnancy period.
We used 16 SD rats that have never been pregnant
before. Rats were divided into control (vehicle treated)
and EPO (100 IU/kg) treated groups (n=8/group).
Vaginal smear method was used for diagnosis of
pregnancy. During the pregnancy; 3 days/week 100 i.u.
/kg EPO was injected s.c, pregnancy was completed with
cessarian section at the 21th day, fetuses, placentas and
blood samples were taken for measurements.
Both fetus length and weights decreased in EPO group.
In pathological examination there was marked congestion
with enlarged placental labrynties and decreased chorion
villus number in EPO group compared to the control.
Placental tissue nitrite level decreased in EPO group.
EPO treatment throughout rat pregnancy period caused
to IUGR and placental abnormalities. Erythropoietin is a
powerful promising agent to treat anemias and decreases
transfusion requirements but it seems its usage during
prengnacy needs more studies to determine EPO
involved mechanisms.
Key Words: Eritropoietin, pregnancy, placenta, nitric oxide
(NO), fetus, intra uterine growth retardation (IUGR)
INTRODUCTION
Erythropoietin (EPO) is a 30 kD glycoprotein;
responsible for the stimulation of red blood cell
production. Decreased oxygen delivery, most
often due to anemia or hypoxemia, is the primary
stimulus to EPO release. It has been shown to be
a deficient in plasma of anemic patients with endstage kidney disease1. Recombinant human EPO
(rHuEPO) is identical biologically to the native
hormone and used in anemic patients especially
with chronic renal failure. The EPO gene is highly
conserved during the species and 80% homology
is found between the human and rodent genes.
EPO is produced in many parts of human body
such as spleen, lung, brain, bone marrow
macrophages, early colony forming cells and
umbilical cord macrophages, but mainly in kidney
and liver2,3. Expression of EPO and its receptor in
placental trophoblasts has also been reported2,4.
However lack of human placental permeability for
EPO has been shown in a dual in vitro perfusion
system of cotyledon isolated from freshly
delivered term placenta5. Chronic renal failure
related anemia is aggravated by pregnancy and
when pregnancy is complicated by hemorrhage, it
may lead to deep anemia. In the treatment of
anemia in pregnancy, the risks of transfusions are
well-known, but our knowledge about the safety
of the use of rHuEPO during pregnancy is still
unclear6.
Adequate utero-placental and feto-placental blood
flows are essential for normal intrauterine growth.
Nitric oxide (NO) production by the feto-placental
unit plays an important role in the regulation of
placental vascular bed resistance and by this
mechanism might be take part in development of
intra uterine growth retardation (IUGR). An
important function of the invading trophoblasts is
to destroy the muscular walls of the uterine spiral
arteries converting them in to large flaccid vessels
that are no longer capable of high conductance.
These physiological changes are required for a
successful pregnancy and failure of spiral artery
213
213
N. Yazıhan et al.
transformation has been reported in preeclampsia,
IUGR
without
maternal
hypertension,
and
miscarriage. The development of placenta is a
highly regulated process with involvement of
hormones, metalloproteins, cytokines, growth
factors, extracellular matrix (ECM) glycoproteins,
vascular endothelial growth factor (VEGF) etc.7,8.
Nitric oxide is thought to be involved in many
aspects of this regulation including trophoblast
apoptosis, motility and also for fetoplasental
perfusion pressure maintenance9. NOS activity is
increased in normal pregnancy and it is important
for physiological vasodilatation of pregnancy.
Effect of EPO on nitrite level differs from tissue to
tissue10. Effect of EPO on placental tissue nitrite
level is unknown since we know that endogen EPO
levels increase under various stress conditions in
pregnancy, this experiment has been arranged to
test the effect of EPO in normal fetoplacental
development. Our results may also provide some
information to understand the mechanisms of EPO
involvement in IUGR and placental formation
during pregnancies under stress conditions with
increased levels of endogen EPO.
MATERIAL AND METHOD
Design of experiment
We used 16 female Sprague Dawley (SD) rats that
have
never
been
pregnant
before.
All
experimental procedures were approved by the
Osmangazi University Instituonal Animal Care and
Use Committe. Rats were divided in to two groups,
a control and an EPO group (n=8/group). After
coupling night, vaginal smear method was used
for diagnosis of pregnancy and estimation of the
day of pregnancy as well. The first day that sperm
was seen is accepted as the first day of pregnancy.
During the pregnancy; three days in every week
(Monday- Wednesday- Friday mornings), 100
i.u./kg rHuEPO (Eprex, SantaFarma) in EPO group
and physiological saline to the control group were
injected via subcutaneous route; pregnancy was
completed with cesarean section at the 21th day
under anesthesia (ketamine 50 mg/kg and xylazine
hydrochloride-Rompun, 10 mg/kg intraperitoneally),
fetuses and placentas and blood samples were
taken for further measurements. Rats were
housed at an average ambient temperature of
24°C under a 12 h:12 h, light: dark cycle and
were fed with a standard rat chow and allowed to
freely drink tap water during the experimental
procedures.
Pathological Examinations:
For pathological examinations, placental tissues
were removed and fixed in 10% formaldehyte.
Five millimeter sections were cut and stained with
214 214
N. Yazhan et al.
hematoxylin and eosin and used for the
assessment of placental structure and congestion.
Random 25 placental fields were observed using 1
X 10 objectives, and then number of chorion villus
was counted in each area for each placenta10.
Measurement of Hemoglobin and Nitrite levels
Hemoglobin levels of pregnant rats were
measured by an automatic analyzer (Roche).
Placental nitrite level was measured by a method
depends on griess reaction. This method depends
on N-nitrosating of nitrite with an aromatic amine
(sulfanamide) and formation of colorful azo
derivative with N-(1-Naftil) etilendiamine (NEDD).
Then it was measured on spectrophotometrically
at 545-555 nm wave length. Tissue samples were
homogenized in 0.1 M Phosphate Buffer Saline
(PBS) (pH=7,4) and centrifuged. Supernatants
were diluted and used for nitrite measurement11.
For pathological examination, after macroscopic
evaluation, one section was taken from all
placentas for histological examination.
Statistical analysis
Student t paired test is used for comparisons of
nitrite levels of groups. Mann Whitney U used for
pathological examinations. Data are given as
mean–standard error and p<0.05 is accepted as
statistically important.
RESULTS
Hemoglobin Concentrations and Placental
Weights
There was no statistically significant difference
between the placental weights (control:0,71–0,07
g.(n=64);EPO: 0,77– 0,06 g.(n=66) of the two groups
statistically. Hemoglobin concentrations (control:
15,57–0,64; EPO:17,55–0,73 g/dl) increased in EPO
treated rats (p<0.05).
Chorion Villus Numbers
In pathological examination there was marked
congestion in placental labrynties in EPO group.
Placental labyrinth enlarged and chorion villi
decreased in the same group compared to normal.
When chorion villi were counted in 10X power
fields, the mean values for EPO group was 3 and
control group was 8. Higher power field
examination was established in EPO treated group.
(Zeiss Axioscop microscope, ·40 objective) (Figure 1
a-b, 2).
Placental Nitrite Levels
Placental nitrite level was decreased in EPO group.
(p<0.01, Figure 3).
Fetal Weights and Lengths
One fetus was intra uterine ex in control group.
There was no abnormality in external examination
of all the other fetuses. However, both weights
and lengths of fetuses decreased in EPO groups
(Figure 4 a and b respectively).
EPO
2
Fetal
FIGURES:
0
CONTROL
EPO
N. Yazhan
Yazıhan et al.
Fig 4 A
Fetal height (cm)
Fig. 1A
Fig. 1A
Fig 1B
Figure 1 b. 10X view of a placenta from control group.
Congestion is also present but less prominent than EPO
group. Normal chorionic villus are seen
Fig 1B
Fig 1B
Figure 2. High power view (40X) of placenta from EPO
group. There is increased number of inflammatory cells
in EPO treated group
Placental
Placental
NitriteNitrite
LevelsLevels
(MicroM)
(MicroM)
360
360
320
320
280
*
EPO
*
control
EPO
control
280
240
Fig 2.Fig. 3
240
EPO
control
EPO
control
Figue 3. Placental nitrite levels of EPO treated and
*
nontreated
Fig. 3 rats fetuses. (Datas are given as mean±SE,
p<0.01)
Fetal weight
Fetal weight
(g) (g)
Fig 2.
6
*
46
CONTROL
*
24
0
EPO
CONTROL
EPO
02
Fig 4 A
CONTROL
CONTROL
EPO
EPO
A Weights of EPO treated (n:66) and nontreated
FigureFig
4 4a.
(n:64) rat fetuses (Values are given as mean±SE, * p<0.01)
l height
ht
(cm) (cm)
*
40
50
50
40
CONTROL
EPO
30
CONTROL
Figure 1 a. Low power view of a placenta from EPO and
the control groups. Low power view of a placenta from
EPO group showing prominent congestion. Villus number
Fig. 1A
per area decreased
Fig 2.
50
EPO
Fig 4B
Figure 4 b. Heights of EPO treated (n:66) and nontreated
(n:64) rat fetuses (Values are given as mean±SE, p<0,01)
DISCUSSION
In the present study we found that use of rHuEPO
or increased levels of maternal serum EPO
concentration may lead to IUGR and placental
abnormalities. Placental vascular growth begins in
early days of first trimester and from the 12
weeks formation of villous trees occurs by
invasion of capillaries into trophoblasts12. In our
study, we used rHuEPO in all trimesters of
pregnancy beginning from the first day, so it can
be expected that all stages of placental formation
would be influenced. Pathological examinations
showed the decreased number of chorion villi and
increased congestion in placentas of EPO given
group.
Nitric oxide has an important role in the invasion
of cytotrophoblasts. It is a very labile molecule.
Nitrite is one of the metabolites of the nitric oxide.
Decreased levels of placental nitrite in EPO group
may be the cause of pathological abnormalities
seen
in
our
study.
Increased
maternal
erythropoietin may have caused decreased nitrite
levels of placenta and inhibited trophoblast
invasion and motility. It is well known that chorion
villus number is very important for the
nourishment of fetuses. The cause of IUGR in EPO
group might be decreased number of villi.
Nitric oxide is also a potent vasodilator which has
major roles in the physiological control of vascular
tone in many tissues including placenta.
Preeclampsia has been considered as an
endothelial cell disorder and it is associated with
deficiency of antithrombotic and vasodilatator
factors. Nitric oxide has an important role in
pathogenesis of preeclampsia. Nitric oxide synthase
enzyme activity is decreased and erythropoietin
level is increased in maternal serum of preeclamptic
patients without any anemic condition. It may be
a response to hypertension or may be the direct
cause of hypertension. It is reported that the
genetically high blood erythropoietin concentration
may
have
responsibility
in
hypertension
development13. It is reported that rHuEPO exerts a
215
215
*
CONTROL
EPO
N. Yazıhan et al.
N. Yazhan et al.
direct contractile effect on human placental
vessels14. Also Kashiwagi et al15 reported a
pregnancy with renal anemia, initially normal
blood pressure followed by a rapid rise in
hemotocrit and development of hypertension. In
this study nitrite level of placental tissue has been
found fo be decreased in EPO treatment group
compared
to
control.
Increased
maternal
erythropoietin causes decrease in nitrit level of
placenta and inhibit trophoblast invasion and
motility. It may be true for the condition of
excessive endogen maternal blood erythropoietin
concentrations. It may also take part in the
pathogenesis of placental abnormalities of
preeclampsia.
Mayhew found that placental morphology differs in
human placentas from normal and abnormal
pregnancies. Alterations in intervillous space were
isomorphic in diabetic and smoking women but
anisomorphic in maternal anemia, pre-eclampsia
and in mothers who live at high altitude16. It is
known that, erythropoietin levels are decreased in
diabetic and smoking mothers and increased in
the condition of high altitude, preeclampsia and
anaemia17,18. Although a lot of complex mechanisms
are involved in, according to this information, it
might be concluded that intervillous morphology
directly related with mother’s erythropoietin
serum levels.
EPO is a mitotic and proliferative factor for many
structures like red blood cells, vessels etc. It
causes increased synthesis of VEGF19. In the
pathological examination of EPO group there was
increased sinusoidal structures and labyrinths. In
the study of Mayhew, they found that in the
placentas of smoking and diabetic mother’s there
are decreased vascularization but in the preeclampsia and high altitude groups there was
increased vascularization16.
EPO uses tyrosine kinase related receptor and acts
via JAK/STAT way. Activation of JAK/STAT pathway
activates proliferation and it can also cause to
destruction of newly instructed villus structures in
early days of pregnancy with induction of
inflammatory cytokines14. This may be another
reason of decreased villus formation in EPO group.
In conclusion; in the present study, EPO treatment
throughout rat pregnancy period caused to IUGR
and
placental
abnormalities
by
probably
interfering with NO metabolism.
There has been growing evidence suggesting that
erythropoietin is a powerful agent to treat
anemias and it decreases transfusion requirement.
However, according to our results, it seems that
further studies are still needed to determine
physiological and pathophysiological EPO induced
mechanisms during pregnancy and various
diseases.
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Correspondence :
Nuray YAZIHAN MD, PhD
Ankara University, Faculty of Medicine, Molecular Biology , Shhiye-Ankara
e-mail: [email protected]
Acceptance date: 16.11.2007