Stereological analysis of terminal villi of the placentas of
pregnant woman with sideropenic anemia
Melisa Lelić1*, Suada Ramić1, Zlata Žigić1, Gordana Bogdanović2, Sergije Marković1
1
Department of Histology and Embryology, Faculty of Medicine, University of Tuzla, Univerzitetska 1, 75 000 Tuzla, Bosnia and Herzegovina.
Clinic for Gynaecology and Obstetrics, University Clinical Center Tuzla, Trnovac bb, 75 000 Tuzla, Bosnia and Herzegovina
2
Abstract
Iron deficiency, causing maternal sideropenic anemia, is one of the most frequent nutritive disorder that develops during the pregnancy. We
collected 30 placentas from anemic mothers and 30 placentas from mothers belonging to the control group. Terminal villi (magnification 10x)
and terminal villi capillaries (magnification 40x) were stereologically analyzed and numerically determined. In the placentas from anemic
mothers we noted the values a) terminal villi: volume density 0,43 mm0, surface density 24.13 mm-1, total volume 185.57 cm3 and total surface
10.27 m2; b) capillaries of terminal villi: volume density 0.53 mm0 and total volume 224.18 cm3. In the placentas from mothers belonging to the
control group we observed the following values a) terminal villi: volume density 0.44 mm0, surface density 22.27 mm-1, total volume 200.17 cm3
and total surface 10.15 m2; b) capillaries of terminal villi: volume density 0.42 mm0 and total volume 197.00 cm3. Compared with the control
group anemic mothers’ placentas have a significant higher values of surface density of terminal villi (p<0.05), volume density (p <0.01) and absolute volume (p<0.0001) of terminal villi capillaries, and significant lower values of absolute volume of terminal villi (p<0.05). In anemic mothers’
placentas, the total volume of terminal villi changes disproportionately to the total surface of terminal villi with statistically significant increase
of terminal villi capillaries compared with control group.
key words: placenta, terminal villi, pregnancy anemia, stereology
Bosn J Basic Med Sci. 2014;14(3):139-143. © 2014 ABMSFBIH
Introduction
The placenta is a highly specialized organ during pregnancy that allows regular fetal growth and development. The
labyrinth layer of the placenta is the only site for nutrients,
gases and wastes exchange between the maternal and fetal
circulations from middle of gestation to term [1]. Placental
morphology and cellular architecture have all the necessary
potential factors for oxygen delivery from the mother to the
fetus [2].
Pregnancy hypoxia develops when expecting mothers are
anemic or living at high altitude [3]. Pregnancy anemia usually develops because of the lack of iron and still is a major
health problem with serious consequences for mother and
developing fetus [4]. For quick and rough estimate of hematological status of pregnant woman, it is sufficient to control
* Corresponding author: Melisa Lelić,
Department of Histology and Embryology, Faculty of Medicine,
University of Tuzla, Univerzitetska 1, 75 000 Tuzla, Bosnia and Herzegovina,
Phone: ++387 35 320 645
Fax: ++387 35 320 601
E-mail: [email protected]
Submitted: 02 December 2013 / Accepted: 15 July 2014
hemoglobin (Hb) and hematocrit (Htc) values, adjusted based
on increase of blood level during the pregnancy [5].
Morpho-functionally, most of the terminal villi are specialized, with sinusoid-type capillaries in close contact with
anuclear regions of syncytiotrophoblast, and significant
increasing the proportion of terminal villi’s endothelial cells
compared with the stromal cells [6]. The syncytiotrophoblast
is a specialized multinuclear epithelial tissue that functions
as a transporting epithelium and endocrine/paracrine organ,
delivering nutrients to the fetus and producing hormones that
sustain pregnancy [7]. Hypoxia in the placenta is associated
with vascular remodeling, metabolic changes, and cell undergoes mitochondrial dysfunction, oxidative and endoplasmic
reticular stress [8].
The placenta regulates fetomaternal exchange. During the
pregnancy it adapts to support increasing fetus’ needs. The
placenta that grows in a hypoxic condition develops morphological changes that influence placenta’s exchange and hemodynamic processes [9]. Pregnancy anemia may result with placental hypertrophy [10] or, if it is combined with malnutrition,
can result with restriction of placental growth and development of small, hypotrophic placenta [11].
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Melisa Lelić et al.: Stereological analysis of terminal villi of the placentas of pregnant woman with sideropenic anemia
The aim of our research was to determine quantitative
parameters (volume density, total volume, surface density
and total surface of terminal villi, as well as volume density
and absolute volume of terminal villi’s capillaries) of anemic
mother’s placentas and compare them to parameters of placentas of pregnant woman with the regular pregnancy and
delivery.
Materials and Methods
Samples
A total of 60 placentas from term delivery pregnancies were examined. The placentas were divided into two
groups: the anemic group, 30 placentas from pregnancies
complicated with sideropenic anemia, but without any
other risk factor or disease that could affect the course and
outcome of pregnancy, and the control group, 30 placentas
from pregnancies without signs of anemia and any other
risk factor for pregnancy disorder. Research criteria: single
baby pregnancies, spontaneous (vaginal) and term delivery
(from 37 to 42 gestational weeks), maternal age from 20 to
35 years, hemoglobin lower than 110 g/l in the first and the
third trimester and lower than 105 g/l in the second trimester of pregnancy; hematocrit lower than 0.33 in the first and
the third trimester and lower than 0.32 in the second trimester of pregnancy.
Procedures
The amnion and umbilical cords were removed from placentas, and then weighed and volume determination based on
the amount of displaced liquid. Samples were taken through
the thickness of the entire organ, fixed in 10% formalin solution, embedded in paraffin, cut into 8μm thick slices and
stained with hematoxylin and eosin. The tissues were analyzed
stereologically in the central region of placenta, between chorionic and basal plate, using multipurpose test system M-42
on magnification 10x and 40x using Nikon microscope, model
SE. The sample size was determined according to procedure
introduced by De Hoff [12].
We analyzed: volume (Vd tv) and surface (Sd tv) density,
absolute volume (V tv) and absolute surface (S tv) of terminal
villi; volume density (Vd tvc) and absolute volume (V tvc) of
terminal villi capillaries.
Statistical Analysis
Student t-test was used to determine the statistical significance of the results. Hypotheses were tested at level of signification α = 0.05 i.e., differences between samples was considered significant when p<0.05.
Results
This research presents the results of stereological analysis of 60 placentas that have been divided into two groups:
30 placentas of anemic mothers and 30 placentas of the control group. Tissue sections, embeded in parafinum, were cut
on slides of 8μm thikness and stained with hemaotxylin and
eosin. Using multipurpouse test system M 42, terminal villi
were analyzed on magnification 10x and terminal villis blood
vessels on magnification 40x, in the placentas of anemic pregnant woman (Figure 1, A and B) and placentas from healthy
pregnant woman (Figure 1, C and D).
Anemic mother’s placentas have 8% lower values of total
volume of terminal villi but 8% higher values of terminal villis’
surface in 1 cm3 of placental tissue. Anemic mother’s placentas have 20% higher values of terminal villi’s capillaries volume
density and 12% higher values of terminal villi’s capillaries total
volume compared to the control group.
Stereological analyses of terminal villi showed a) anemic
mother’s placentas had significantly lower terminal villi’s surface density and significantly higher terminal villi’s total volume
compared to the control group; b) volume density as well as
total surface of terminal villi did not differ significantly (Table 1).
Terminal villi’s blood capillaries of anemic mother’s placentas showed significant increase in 1cm3 of placental tissue
as well as in the total volume of placentas compared to the
control group (Table 2).
Discussion
It is a widely held belief that hypoxia is responsible for the
change in anemic mothers’ placentas and the placentas usually respond to hypoxia by increased capillary branching [13],
increased trophoblast proliferation, but decreased fusion into
syncytiotrophoblast [14]. Hypoxic injury disrupts the syncytial
architecture and results with the increased density of syncytial
Table 1. The average values and standard deviations of volume
density (Vd tv), total volume (V tv), surface density (Sd tv) and
total surface (S tv) of terminal villi of the researched placentas
Vv tv (mmo)
V tv (cm3)
Sd tv (mm−1)
S tv (m2)
Anemia mean (SD)
0.43 (0.08)
185.57 (44.56)
24.13 (4.18)
10.27 (2.93)
Control mean (SD)
0.44 (0.07)
200.17 (52.1)
22.27 (4.31)
10.15 (2.72)
p>0.05
p<0.05
p<0.05
p>0.05
Table 2. The average values and standard deviations of volume
density (Vd tvc) and total volume (V tvc) of terminal villis’
capillaries of the researched placentas
140
Vd tvc (mmo)
V tvc (cm3)
Anemia mean (SD)
0.53 (0.11)
224.18 (66.59)
Control mean (SD)
0.42 (0.12)
197 (65.84)
p<0.01
p<0.0001
Melisa Lelić et al.: Stereological analysis of terminal villi of the placentas of pregnant woman with sideropenic anemia
A
B
C
D
Figure 1. The placenta of anemic pregnant woman (A and D) and healthy pregnant control (C and B).
knots and vasculosyncytial membrane thickness that promotes the release of soluble syncytial factors [2]. Study conducted on animal models, showed that iron-deficiency anemia in pregnant rats increase the expression of hypoxia and
inflammatory markers in the placenta [15]. Despite the fetal
resistance to maternal deficiency, any stress that alters placental development and function may have consequences for the
developing fetus [16]. The placenta does not respond in a single way to hypoxia because of its large reserve capacity, and
hypoxic lesions may not always result in poor fetal condition
or outcome. On the other hand, very acute, in utero, hypoxic
events, followed by prompt delivery, may not be associated
with placental pathology, and many poor perinatal outcomes
can be explained by an etiology other than hypoxia [17].
Sideropenic anemia is the most common type of nutritive anemia in pregnancy [18]. It develops because of iron
deficiency. Other types are rare, and caused by folic acid and
vitamin B12 deficiency, and some types of hemoglobinopathies [19]. Iron deficiency anemia accounts for 75% of all anemia in pregnancy [20]. According to WHO recommendation, there are three types of maternal anemia in pregnancy
mild: (Hb 109-100 g/l), moderate (Hb 99-70 g/l) and severe
(Hb<70 g/l) [21].
Pregnancy anemia is one of the risk factor for abnormal
placental growth, that lead to development of either hypertrophic or hypotrophic placenta [22]. Stereology applied to placental section allows minimal-biased and economical quantification of the three-dimensional structure of the placenta
from molecular to whole-organ level [23].
According to data, volume density of terminal villi counts
from 0.41 mm0 at older pregnant women [24] to and 0.62 mm0
in the regular pregnancy [25]. Placentas from mother with preeclampsia showed significant increase of terminal villi density
but significant decrease of villis’ blood vessels [26,27]. In our
research, volume density of terminal villi of anemic mothers’
placentas count 0.43 mm0, without significant difference, and
our results are similar to values of older pregnant woman [24].
Among the results of different researchers, Reshetnikova
et al. [11] indicate that total volume of chorionic villi (terminal
and others) of the placentas of the mother with sideropenic
anemia counts 157.80 cm3 and is significantly lower compared
to the control group counting 234.5 cm3. Other researchers indicate that the total volume of placental villi seems to
increase significantly compared to the control group [10]. The
placenta is potentially subjected to stressful conditions during
delivery, and sample collection may be delayed by routine
postpartum inspection by clinical staff [28]. The results of
morphometrical analysis of placental tissues are partly contradictory because numerous factors can influence the results
of the stereological analysis such as number of analysed placentas and possible relation of maternal anemia and malnutrition [22] or pregnancy at high altitude [14]. Placental hypertrophy is related to mild or moderate anemia thus the placental
enlargement seems to be a uniform physiological compensatory mechanism [29]. Our research indicate that absolute volumes of terminal villi of the anemic mothers’ placentas count
185.57 cm3, and thus it is significantly lower compared to control group where it count 200.17 cm3. A long-lasting hypoxia,
141
Melisa Lelić et al.: Stereological analysis of terminal villi of the placentas of pregnant woman with sideropenic anemia
related to duration of anemia (during each trimester of pregnancy), seems to disrupt the compensatory mechanisms of
placenta and cause the shrinkage of terminal villi. A hypoxic
environment during long-lasting hypoxia in sideropenic anemia can cause shape deformation of terminal villi, capillaries
branch extensively and form fist-like terminal villi with short
multiple invaginations that enlarge terminal villis’ surface [29].
Among rare data considering terminal villis’ surface density
in preplacental hypoxic environment we contrive that Huang
et al. [10] claims that the terminal villis’ surface density does
not significantly differ between placentas of anemic pregnant
women compared with the control group.
In our research, stereological analysis showed that surface density of terminal villi in anemic mothers’ placenta is
24.13 mm-1 and in control group 22.27 mm-1, with statistically
significant difference. Such results may be explained by a characteristic shape of terminal villi. Because of increased number
of capillaries, villi become deformed, with multiple invaginations [29], and thus increased share of terminal villis’ surface
in organ volume.
Specific pregnancy condition, such as pregnancy diabetes
or multiple pregnancies increase the placental mass [3]. The
placental surface increase in hypoxic condition in anemic
mother and pregnancies at high altitude. Described placental
enlargement may result in increase syncytial knots’ ripping
off, that undissolved in maternal blood may undergo necrosis
and leed to preeclamptic symptoms [3]. Severe maternal anemia, with hemoglobin lower than 70g/l results in decrease of
placental villis’ surface, and increase of blood vessels’ surface
compared to the control group 30. Total surface of all chorionic
villi in anemic mothers’ placenta is 9.50 m2 [11], and in healthy
pregnant woman it counts from 11.04 m2 [24] to 14.90 m2 [11].
Terminal villis’ surface at term pregnancy counts 10.34 m2 [24].
During our research, terminal villi were analyzed as a
separate variable. The results showed that in the placentas
of anemic mothers terminal villi’s total surface is 10.27 m2
and in control group 10.15 m2, with no significant difference
among researched groups. Our results are partly diverse with
the results of other researchers, especially considering that
we did not represent the values of other villis’ total surface as
cited authors [10,11,30]. On the other hand, pregnant women
were not severely anemic with extremely low hemoglobin
values, lower than 70g/l, that may be an explanation why the
significant shrinkage of terminal villis total volume did not
occur.
We concluded that the anemic mothers’ placentas develop
changes of terminal villis’ total volume, but not the surface,
and our data are consistent with the belief that the anemic
mothers’ placentas show anisomorphic changes i.e., the surface of villi change disproportionately with the volume of the
villi and vice versa [9].
Transplacental oxygen transport is one of the numerous placental villis’ functions, but is also a cause that influences growth and differentiation of villi more than any other
factor [7]. The placenta from maternal anemia show signs
of chronic preplacental hypoxia that causes prevalence of
branching angiogenesis with intense trophoblast knotting
as a result of increased tangential sectioning across irregular
villous surface [29]. According to our research data, there is
statistically significant increase of volume density of terminal villis’ capillaries in placentas of mothers with sideropenic anemia where it counts 0.53 mm0 and compared to the
control group that counts 0.42 mm0. The total volume of
terminal villis’ capillaries in our research is 224.18 cm3 and is
significantly higher compared to the control group (197.00
cm3). Placenta modulates to hypoxia by increased branching
and number of capillaries and thinning the placentas’ barrier
[31] and during sideropenic anemia, the terminal villi capillaries undergoes adaptation that results in volume increase.
Placental villi exposed to low oxygen partial pressure may
show an increase of fetal capillaries, even there are no exact
evidence of capillary proliferation and changes are result of
capillary dilatation [32].
Hypoxia during pregnancy anemia impacts structure and
developing of placental blood vessels, particularly the capillaries of terminal villi, the most important structure required
for fetomaternal exchange. Therefore, terminal villi have been
analyzed as an independent variable, to give the knowledge of
placentas’ compensatory pathways.
Conclusion
Our research indicates that mild and moderate maternal
sideropenic anemia lead to significant morphological and
structural changes in terminal villi. Improved changes have
been signed as anisomorphic, we noted the significant reduction of the total volume of terminal villi but the total surface
did not differ significantly. The capillaries of terminal villi
showed the significant increase both volume density and total
volume in the placentas of mothers with sideropenic anemia
compared with the control group.
Declaration of Interest
The authors declare no conflict of interest.
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