ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 49
OBJECTIVE: Placenta previa is associated with the
development of pregnancy complications such as
pregnancy-induced hypertension, preeclampsia, intra­
uterine growth restriction, placental prolapse, and
perinatal mortality. The aim of this study was to eval­
uate morphometric and immunohistochemical changes
in the umbilical cord of placenta previa and normoten-
sive patients.
STUDY DESIGN: Pregnant patients with placenta
previa and healthy pregnant patients between 35 and
38 weeks’ gestation were included in the study. Collected
samples were fixed in 10% buffered formaldehyde. After
routine histological follow-up, 4–6-µm-thick sections
were cut, and histopathological and immunohistochem­
ical examinations were made.
RESULTS: Morphometric examination and endothelial,
fibroblast, smooth muscle cell length, and vessel wall
thickness were significantly different between the
groups. In the placenta previa group there was an
increase in ADAMTS-9 expression in fibroblast and
some inflammatory cells in the subendothelial layer. In
addition, negative expression of E-cadherin was evident
with the separation of the areas of adhesion between
endothelial cells and muscle cells in umbilical cord
sections. Immunohistochemical analysis showed that
endothelial cells, fibroblast cells, and smooth muscle in
the placenta previa group expressed a positive level of
VEGF-A. Increased hypertension and hypoxia occur­
ring during pregnancy is one of the important causes
of placenta previa. Its effect on extracellular matrix
dynamics causes intercellular adhesion. The change
in the production of VEGF-A was thought to play an
important role in fetus development.
CONCLUSION: ADAMTS-9 has been shown to be an
important protein in cell-cell, cell-matrix binding and
promoting vascular development. E-cadherin, which is
the determinant of the cell binding complex, was thought
to be able to increase VEGF-A expression and regulate
angiogenesis by weakening cell adhesion. (Anal Quant
Cytopathol Histpathol 2020;42:49–53)
Keywords: ADAMTS-9; cadherins; E-cadherin;
immunohistochemistry; placenta; placental place­
ment; placenta previa; pre-eclampsia; pregnancy;
pregnancy, high-risk; pregnancy outcome.
Abnormal placental placement, as in placenta
previa, causes misplacement in pregnancies and
increases the risk of placental insufficiency. Pla-
centa previa is also associated with the develop­
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4202-0049/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Morphometric and Immunohistochemical
Examination of Changes in Umbilical Cord
Structure of Placenta Previa and
Normotensive Patients
Uǧur Değer, M.D., and Yunus Çavuş, M.D.
From the Department of Obstetrics and Gynecology, Memorial Hospital, Diyarbakır, Turkey.
Uǧur Değer is Physician.
Yunus Çavuş is Physician.
Address correspondence to: Yunus Çavuş, M.D., Department of Obstetrics and Gynecology, Memorial Hospital, Peyas, Fırat Blv. No.
12, Diyarbakır 21070, Turkey (ycavus1212@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. ment of pregnancy complications associated with
pla­
cental insufficiency, such as pregnancy-induced
hypertension, preeclampsia, intrauterine growth
restriction, placental prolapse, and perinatal mor-
tality.1,2 The umblical cord is covered by an epi­
thelium derived from the enveloping amnion. The
umblical cord is extremely important for correct
fetal development. The network of glycoprotein
microfibrils and collagen fibrils in Wharton’s
jelly has been previously studied.3 The interlaced
collagen fibers and small, woven bundles are
arranged to form a continuous soft skeleton that
encases the umbilical vessels.4 The extracellular
matrix comprises networks of collagenous and
noncollagenous proteins.5
The ADAMTS-9 gene is located on chromosome
3p14.1 and encodes a member of a disintegrin
and metalloproteinase with thrombospondin mo-
tifs (ADAMTS) protein family that has been im-
plicated in the cleavage of proteoglycans, the
control of organ shape during development, and
the inhibition of angiogenesis VEGF. The VEGF-R
system stimulates endothelial cell migration and
proliferation and also plays a key role as a media-
tor of vascular permeability and vasodilation.6
Embryonal development and blastocyst im-
plantation of E-cadherin is a Ca 2+–dependent
cell-cell adhesion molecule expressed in epithelial
cells. The form of E-cadherin is an integral mem­
brane glycoprotein of 120–130 kDa bound to the
cell skeleton via intracellular ligands called cath­
ines α, β, and γ.7 Wharton’s jelly is a vast gelati-
nous matrix that covers the umbilical blood ves­
sels. The mesenchymal cells in the structure secrete
unique molecules for the repair of leakage defects
in the umbilical veins to prevent harm to the
health of the fetus during pregnancy. Therefore,
it is an important area with building blocks for
tissue repair. The aim of this study was to eval-
uate morphometric and immunohistochemical
changes in the umbilical cord of placenta previa–
affected patients and normotensive patients.
Materials and Methods
The study protocol was approved by the Medi­
cal Committee of Diyarbakır Memorial Hospital,
and informed consent was obtained from all
subjects involved in the study. Twenty patients
with placenta previa and 20 age-matched, healthy
pregnant women were enrolled in this study (40
pregnant women in total). All umbilical cords
were provided from the Department of Obstetrics
and Gynecology, Diyarbakır Memorial Hospital.
Umbilical cords of infants born at 35–38 weeks of
pregnancy were removed. Placenta previa (n=20)
and normal umbilical cords (n=20) (a total of 40
units) were received.
Immunohistochemistry Staining
The immunohistochemical method was performed
according to the procedure described by Baloğlu
et al.8 Samples of umbilical cord tissue were im-
mersed in 10% buffered formaldehyde. After rou­
tine histological follow-up, 4–6-µm-thick sections
were cut. Sections were brought to distilled water
and washed in phosphate-buffered saline (PBS)
3×5 min (Catalog no. 10010023, Thermo Fisher
Scientific, Fremont, California, USA). Antigen re-
trieval was done in a microwave (Bosch, 700 watt)
for 3 min×90°C. They were subjected to a heating
process in a microwave oven at 700 watts in a
citrate buffer (pH 6) solution for proteolysis. Sec­
tions were washed in PBS 3×5 min and incubat­
ed with hydrogen peroxide (H2O2) (K-40677109,
64271 H2O2, Merck, Germany) (3 mL 30% H2O2+
27 mL methanol) for 20 minutes. Sections were
washed in PBS 3×5 min and blocked with Ultra
V Block (lot #PHL150128, Thermo Fisher) for 8
minutes. After draining, primary antibodies E-
cadherin, mouse monoclonal (1/100) (lot #ab1416,
Abcam, USA), VEGF-A mouse monoclonal (1/100)
(lot #ab1316, Abcam), and ADAM-9 antibody
(1/100) (lot #ab186833, Abcam) were applied. Sec­
tions were incubated and left overnight at 4°C.
Sections were washed in PBS 3×5 min and then
incubated with secondary antibody (Histostain-
Plus Kit, Invitrogen, Carlsbad, California, USA)
applied for 20 minutes. After washing with PBS,
Streptavidin Peroxidase was applied to sections
for 20 minutes. Sections were washed in PBS 3×5
min, and 3,3′-Diaminobenzidine (DAB) was ap-
plied to the sections for up to 10 minutes. Slides
showing reaction were stopped in PBS. Counter­
staining was done with Harris Hematoxylin for
45 seconds, dehydrated through ascending alco-
hol, and cleared in xylene. Slides were mounted
with Entellan (lot #107961, Sigma-Aldrich, St.
Louis, Missouri, USA) and examined under an
Olympus BH-2 lightmicroscope (Olympus).
Evaluation of Morphometric Parameters
Morphometric study was performed in 10 differ-
ent areas. All measurements were done using an
ocular micrometer.
50 Analytical and Quantitative Cytopathology and Histopathology®
Değer and Çavuş

3. Results
Nonparametric Mann-Whitney U test was used
to analyze significance between the groups. Mean
ranks of each group and p values were evaluated.
Statistically, p<0.05 was accepted as significantly
different. The characteristics of patients including
morphometric and immunohistochemical features
are summarized in Table I.
In the control group umbilical cord section,
positive ADAMTS-9 expression was observed in
fibroblast cells in Wharton’s jelly in blood vessel
subendothelial layer, while negative ADAMTS-9
expression was observed in muscle cells in the
media layer (arrow) (Figure 1A). In the placenta
previa group, positive ADAMTS-9 expression
was seen in the fibroblast and some inflammatory
cells in the subendothelial layer, and positive
ADAMTS-9 expression was observed in the cells
with separation of collagen fibers in Wharton’s
jelly layer and muscle cells (Figure 1B).
Positive E-cadherin expression between endo­
thelial cells and E-cadherin positive expression
in adhesion areas between muscle cells were ob-
served in the umbilical cord vessel sections of the
control group (Figure 1C). Negative expression of
E-cadherin was observed in the placenta previa
group of umbilical cord sections, with separations
in the areas of adhesion between endothelial cells
and between muscle cells (Figure 1D).
In the control group, VEGF-A expression was
positive in endothelial cells in the umbilical cord
vein section, while negative VEGF-A expression
was observed in Wharton’s jelly and muscle cells
(Figure 1E). Positive VEGF-A expression was ob-
served in endothelial cells and small inflammatory
cells in the subendothelial layer of umbilical cord
sections of the placenta previa group, while VEGF-A
expression was observed in Wharton’s jelly and
muscle cells (Figure 1F).
Discussion
Placenta previa is a negative condition that occurs
in the lower segment of the uterus. The implanta­
tion of the placenta into the weakly vascularized
lower uterine segment was reported to cause in-
adequate uteroplacental perfusion, affecting fetal
oxygenation and growth. The placenta vessels can
be separated during the transition between the
amnion and chorion before they reach the pla­
centa. Wharton’s jelly is a soft connective tissue
consisting mainly of fibroblasts and macrophages
embedded in a homogeneous, jelly-like intercel­
lular substance.9 Wharton’s jelly acts as a protec­
tive barrier on the wall structure of the veins and
arteries. In the placenta previa group, the disorder
of collagen fibers in Wharton’s jelly leads to ex-
pansion in space, which might lead to deteriora­
tion in the structure of blood vessels. In a pre-
vious study, it was shown that the wall of the
umbilical cord artery is decreased in preeclamp-
sia patients.10 Junek et al11 reported increased
thickness of the tunica media and intima in the
arteries and an increased rate of duplication of
the internal elastic lamina in preeclamptic cords.
In our study, the placenta previa group demon­
strated decreased thickening of the artery and
vein walls (Table I). Sumeda et al12 showed that
ADAMTS-9 was expressed in the umbilical cord
insertion in the embryo and in the placenta
containing the gel transition regions of Wharton.
It was observed in the adventitia of umblical ves­
sels, Wharton’s jelly mesenchyme, and endothelial
cells of the umblical vein and vascular smooth
muscle cells.
In our study, positive ADAMTS-9 expression
was found in placenta previa cord sections and
fibroblast cells in the subendothelial layer in
some inflammatory cells and positive ADAMTS-9
expression in cells separating collagen fibers in
muscle layer and Wharton’s jelly. It is thought
that it plays an important role in regulating cel-
lular and fiber regulation in the extracellular ma-
trix, inflammation, and muscle activity.
Increased concentration of VEGF-A in fetal cir­
culation and allantoic and amniotic liquids sug-
gests that umbilical vessels, amniotic epithelium,
and allantoic duct epithelium can be, like the pla­
centa, a source of VEGF-A.13 It is thought that one
of the complications that arises as a result of in-
Volume 42, Number 2/April 2020 51
Umbilical Cord Structure in Placenta Previa
Table I Morphometric Parameters in Both Groups
Placenta Control
previa group
(n=20) (n=20) p Value
Arterial wall thickness 85.3 69.1 <0.001
Vein wall thickness 72.4 52.2 0.004
Basement membrane thickness 8.12 4.32 <0.001
Endothelial cell length 12.22 14.01 0.008
Fibroblast cell length 14.44 16.22 0.007
Smooth muscle cell length 13.56 10.22 <0.001
E-cadherin expression 1.24 3.8 <0.001
sFlt-1 expression 3.48 1.86 0.006
ADAMTS-9 expression 1.48 3.98 <0.001

4. creased hypertension, preeclampsia, may cause
an increase in VEGF production. In our study,
the increase of VEGF-A expression in small in-
flammatory cells in the blood vessel endothelial
cells and subendothelial layer of the placenta
previa samples was parallel to the increase in
hypertension.
E-cadherin is a cell-cell adhesion transmem­
brane molecule. It plays important roles in cell
adhesion and morphogenesis.14 Tahaoglu et al15
showed that E-cadherin expression in the umbil­
ical cord vascular endothelial cells showed a
positive reaction in smooth muscle as a result
of gestational diabetes. However, E-cadherin cell
separation and connection may play an important
role in the separation of the complex. A significant
decrease in E-cadherin expression was observed
between the endothelial cells in the blood vessels
of the placenta previa group and the adhesion sites
between muscle cells. However, it is thought that
apoptotic process may begin after placenta previa
may weaken cellular integrity.
Conclusion
ADAMTS-9 has been shown to be an important
component in the binding of extracellular matrix
dynamics and aiding pathway to cellular regula­
tion for umbilical cord vascular development. It
52 Analytical and Quantitative Cytopathology and Histopathology®
Değer and Çavuş
Figure 1
(A) Control group. Endothelial
cells (yellow arrow),
muscle cells (black arrow).
Immunostaining, ADAMTS-9.
(B) Placenta previa group.
Inflammatory cells (red
arrow), muscle cells (blue
arrow). Immunostaining,
ADAMTS-9. (C) Control group.
Endothelial cells (yellow
arrow), muscle cells (blue
arrow). Immunostaining,
E-cadherin. (D) Placenta
previa group. Endothelial cells
(yellow arrow), muscle cells
(blue arrow). Immunostaining,
E-cadherin. (E) Control group.
Endothelial cells (yellow
arrow), muscle cells (blue
arrow). Immunostaining,
VEGF-A. (F) Placenta previa
group. Endothelial cells
(yellow arrow), muscle cells
(blue arrow). Immunostaining,
VEGF-A.

5. is thought that cell adhesion may be weakened
due to decreased expression of E-cadherin and
may increase angiogenesis with increased expres­
sion of VEGF-A.
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