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.
2
Clinic for Gynaecology and Obstetrics, University Clinical Center Tuzla, Trnovac bb, 75 000 Tuzla, Bosnia and Herzegovina

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 abso-
lute 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 hemoglobin (Hb) and hematocrit (Htc) values, adjusted based

on increase of blood level during the pregnancy [5].
The placenta is a highly specialized organ during preg- Morpho-functionally, most of the terminal villi are spe-
nancy that allows regular fetal growth and development. The cialized, with sinusoid-type capillaries in close contact with
labyrinth layer of the placenta is the only site for nutrients, anuclear regions of syncytiotrophoblast, and significant
gases and wastes exchange between the maternal and fetal increasing the proportion of terminal villi’s endothelial cells
circulations from middle of gestation to term [1]. Placental compared with the stromal cells [6]. The syncytiotrophoblast
morphology and cellular architecture have all the necessary is a specialized multinuclear epithelial tissue that functions
potential factors for oxygen delivery from the mother to the as a transporting epithelium and endocrine/paracrine organ,
fetus [2]. delivering nutrients to the fetus and producing hormones that
Pregnancy hypoxia develops when expecting mothers are sustain pregnancy [7]. Hypoxia in the placenta is associated
anemic or living at high altitude [3]. Pregnancy anemia usu- with vascular remodeling, metabolic changes, and cell under-
ally develops because of the lack of iron and still is a major goes mitochondrial dysfunction, oxidative and endoplasmic
health problem with serious consequences for mother and reticular stress [8].
developing fetus [4]. For quick and rough estimate of hema- The placenta regulates fetomaternal exchange. During the
tological status of pregnant woman, it is sufficient to control pregnancy it adapts to support increasing fetus’ needs. The
placenta that grows in a hypoxic condition develops morpho-
* Corresponding author: Melisa Lelić,
Department of Histology and Embryology, Faculty of Medicine, logical changes that influence placenta’s exchange and hemo-
University of Tuzla, Univerzitetska 1, 75 000 Tuzla, Bosnia and Herzegovina, dynamic processes [9]. Pregnancy anemia may result with pla-
Phone: ++387 35 320 645
Fax: ++387 35 320 601 cental hypertrophy [10] or, if it is combined with malnutrition,
E-mail: [email protected] can result with restriction of placental growth and develop-
Submitted: 02 December 2013 / Accepted: 15 July 2014 ment 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 Results

parameters (volume density, total volume, surface density
and total surface of terminal villi, as well as volume density This research presents the results of stereological anal-
and absolute volume of terminal villi’s capillaries) of anemic ysis of 60 placentas that have been divided into two groups:
mother’s placentas and compare them to parameters of pla- 30 placentas of anemic mothers and 30 placentas of the con-
centas of pregnant woman with the regular pregnancy and trol group. Tissue sections, embeded in parafinum, were cut
delivery. on slides of 8μm thikness and stained with hemaotxylin and
eosin. Using multipurpouse test system M 42, terminal villi
Materials and Methods were analyzed on magnification 10x and terminal villis blood
vessels on magnification 40x, in the placentas of anemic preg-
Samples nant woman (Figure 1, A and B) and placentas from healthy
A total of 60 placentas from term delivery pregnan- pregnant woman (Figure 1, C and D).
cies were examined. The placentas were divided into two Anemic mother’s placentas have 8% lower values of total
groups: the anemic group, 30 placentas from pregnancies volume of terminal villi but 8% higher values of terminal villis’
complicated with sideropenic anemia, but without any surface in 1 cm3 of placental tissue. Anemic mother’s placen-
other risk factor or disease that could affect the course and tas have 20% higher values of terminal villi’s capillaries volume
outcome of pregnancy, and the control group, 30 placentas density and 12% higher values of terminal villi’s capillaries total
from pregnancies without signs of anemia and any other volume compared to the control group.
risk factor for pregnancy disorder. Research criteria: single Stereological analyses of terminal villi showed a) anemic
baby pregnancies, spontaneous (vaginal) and term delivery mother’s placentas had significantly lower terminal villi’s sur-
(from 37 to 42 gestational weeks), maternal age from 20 to face density and significantly higher terminal villi’s total volume
35 years, hemoglobin lower than 110 g/l in the first and the compared to the control group; b) volume density as well as
third trimester and lower than 105 g/l in the second trimes- total surface of terminal villi did not differ significantly (Table 1).
ter of pregnancy; hematocrit lower than 0.33 in the first and Terminal villi’s blood capillaries of anemic mother’s pla-
the third trimester and lower than 0.32 in the second tri- centas showed significant increase in 1cm3 of placental tissue
mester of pregnancy. as well as in the total volume of placentas compared to the
control group (Table 2).
Procedures
Discussion
The amnion and umbilical cords were removed from pla-
centas, and then weighed and volume determination based on It is a widely held belief that hypoxia is responsible for the
the amount of displaced liquid. Samples were taken through change in anemic mothers’ placentas and the placentas usu-
the thickness of the entire organ, fixed in 10% formalin solu- ally respond to hypoxia by increased capillary branching [13],
tion, embedded in paraffin, cut into 8μm thick slices and increased trophoblast proliferation, but decreased fusion into
stained with hematoxylin and eosin. The tissues were analyzed syncytiotrophoblast [14]. Hypoxic injury disrupts the syncytial
stereologically in the central region of placenta, between cho- architecture and results with the increased density of syncytial
rionic and basal plate, using multipurpose test system M-42
on magnification 10x and 40x using Nikon microscope, model Table 1. The average values and standard deviations of volume
SE. The sample size was determined according to procedure density (Vd tv), total volume (V tv), surface density (Sd tv) and
total surface (S tv) of terminal villi of the researched placentas
introduced by De Hoff [12].
Anemia mean (SD) Control mean (SD)
We analyzed: volume (Vd tv) and surface (Sd tv) density,
Vv tv (mmo) 0.43 (0.08) 0.44 (0.07) p>0.05
absolute volume (V tv) and absolute surface (S tv) of terminal V tv (cm3) 185.57 (44.56) 200.17 (52.1) p<0.05
villi; volume density (Vd tvc) and absolute volume (V tvc) of Sd tv (mm−1) 24.13 (4.18) 22.27 (4.31) p<0.05
terminal villi capillaries. S tv (m2) 10.27 (2.93) 10.15 (2.72) p>0.05

Statistical Analysis Table 2. The average values and standard deviations of volume
density (Vd tvc) and total volume (V tvc) of terminal villis’
Student t-test was used to determine the statistical signifi- capillaries of the researched placentas
cance of the results. Hypotheses were tested at level of signifi- Anemia mean (SD) Control mean (SD)
cation α = 0.05 i.e., differences between samples was consid- Vd tvc (mmo) 0.53 (0.11) 0.42 (0.12) p<0.01
ered significant when p<0.05. V tvc (cm3) 224.18 (66.59) 197 (65.84) p<0.0001

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 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 pro- According to data, volume density of terminal villi counts
motes the release of soluble syncytial factors [2]. Study con- from 0.41 mm0 at older pregnant women [24] to and 0.62 mm0
ducted on animal models, showed that iron-deficiency ane- in the regular pregnancy [25]. Placentas from mother with pre-
mia in pregnant rats increase the expression of hypoxia and eclampsia showed significant increase of terminal villi density
inflammatory markers in the placenta [15]. Despite the fetal but significant decrease of villis’ blood vessels [26,27]. In our
resistance to maternal deficiency, any stress that alters placen- research, volume density of terminal villi of anemic mothers’
tal development and function may have consequences for the placentas count 0.43 mm0, without significant difference, and
developing fetus [16]. The placenta does not respond in a sin- our results are similar to values of older pregnant woman [24].
gle way to hypoxia because of its large reserve capacity, and Among the results of different researchers, Reshetnikova
hypoxic lesions may not always result in poor fetal condition et al. [11] indicate that total volume of chorionic villi (terminal
or outcome. On the other hand, very acute, in utero, hypoxic and others) of the placentas of the mother with sideropenic
events, followed by prompt delivery, may not be associated anemia counts 157.80 cm3 and is significantly lower compared
with placental pathology, and many poor perinatal outcomes to the control group counting 234.5 cm3. Other research-
can be explained by an etiology other than hypoxia [17]. ers indicate that the total volume of placental villi seems to
Sideropenic anemia is the most common type of nutri- increase significantly compared to the control group [10]. The
tive anemia in pregnancy [18]. It develops because of iron placenta is potentially subjected to stressful conditions during
deficiency. Other types are rare, and caused by folic acid and delivery, and sample collection may be delayed by routine
vitamin B12 deficiency, and some types of hemoglobinopa- postpartum inspection by clinical staff [28]. The results of
thies [19]. Iron deficiency anemia accounts for 75% of all ane- morphometrical analysis of placental tissues are partly con-
mia in pregnancy [20]. According to WHO recommenda- tradictory because numerous factors can influence the results
tion, there are three types of maternal anemia in pregnancy of the stereological analysis such as number of analysed pla-
mild: (Hb  109-100  g/l), moderate (Hb 99-70  g/l) and severe centas and possible relation of maternal anemia and malnutri-
(Hb<70 g/l) [21]. tion [22] or pregnancy at high altitude [14]. Placental hypertro-
Pregnancy anemia is one of the risk factor for abnormal phy is related to mild or moderate anemia thus the placental
placental growth, that lead to development of either hypertro- enlargement seems to be a uniform physiological compensa-
phic or hypotrophic placenta [22]. Stereology applied to pla- tory mechanism [29]. Our research indicate that absolute vol-
cental section allows minimal-biased and economical quan- umes of terminal villi of the anemic mothers’ placentas count
tification of the three-dimensional structure of the placenta 185.57 cm3, and thus it is significantly lower compared to con-
from molecular to whole-organ level [23]. trol group where it count 200.17 cm3. A long-lasting hypoxia,

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 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 preg- Transplacental oxygen transport is one of the numer-
nancy), seems to disrupt the compensatory mechanisms of ous placental villis’ functions, but is also a cause that influ-
placenta and cause the shrinkage of terminal villi. A hypoxic ences growth and differentiation of villi more than any other
environment during long-lasting hypoxia in sideropenic ane- factor [7]. The placenta from maternal anemia show signs
mia can cause shape deformation of terminal villi, capillaries of chronic preplacental hypoxia that causes prevalence of
branch extensively and form fist-like terminal villi with short branching angiogenesis with intense trophoblast knotting
multiple invaginations that enlarge terminal villis’ surface [29]. as a result of increased tangential sectioning across irregular
Among rare data considering terminal villis’ surface density villous surface [29]. According to our research data, there is
in preplacental hypoxic environment we contrive that Huang statistically significant increase of volume density of termi-
et al. [10] claims that the terminal villis’ surface density does nal villis’ capillaries in placentas of mothers with siderope-
not significantly differ between placentas of anemic pregnant nic anemia where it counts 0.53 mm0 and compared to the
women compared with the control group. control group that counts 0.42 mm0. The total volume of
In our research, stereological analysis showed that sur- terminal villis’ capillaries in our research is 224.18 cm3 and is
face density of terminal villi in anemic mothers’ placenta is significantly higher compared to the control group (197.00
24.13 mm-1 and in control group 22.27 mm-1, with statistically cm3). Placenta modulates to hypoxia by increased branching
significant difference. Such results may be explained by a char- and number of capillaries and thinning the placentas’ barrier
acteristic shape of terminal villi. Because of increased number [31] and during sideropenic anemia, the terminal villi capil-
of capillaries, villi become deformed, with multiple invagina- laries undergoes adaptation that results in volume increase.
tions [29], and thus increased share of terminal villis’ surface Placental villi exposed to low oxygen partial pressure may
in organ volume. show an increase of fetal capillaries, even there are no exact
Specific pregnancy condition, such as pregnancy diabetes evidence of capillary proliferation and changes are result of
or multiple pregnancies increase the placental mass [3]. The capillary dilatation [32].
placental surface increase in hypoxic condition in anemic Hypoxia during pregnancy anemia impacts structure and
mother and pregnancies at high altitude. Described placental developing of placental blood vessels, particularly the capil-
enlargement may result in increase syncytial knots’ ripping laries of terminal villi, the most important structure required
off, that undissolved in maternal blood may undergo necrosis for fetomaternal exchange. Therefore, terminal villi have been
and leed to preeclamptic symptoms [3]. Severe maternal ane- analyzed as an independent variable, to give the knowledge of
mia, with hemoglobin lower than 70g/l results in decrease of placentas’ compensatory pathways.
placental villis’ surface, and increase of blood vessels’ surface
compared to the control group 30. Total surface of all chorionic Conclusion
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]. Our research indicates that mild and moderate maternal
Terminal villis’ surface at term pregnancy counts 10.34 m2 [24]. sideropenic anemia lead to significant morphological and
During our research, terminal villi were analyzed as a structural changes in terminal villi. Improved changes have
separate variable. The results showed that in the placentas been signed as anisomorphic, we noted the significant reduc-
of anemic mothers terminal villi’s total surface is 10.27 m2 tion of the total volume of terminal villi but the total surface
and in control group 10.15 m2, with no significant difference did not differ significantly. The capillaries of terminal villi
among researched groups. Our results are partly diverse with showed the significant increase both volume density and total
the results of other researchers, especially considering that volume in the placentas of mothers with sideropenic anemia
we did not represent the values of other villis’ total surface as compared with the control group.
cited authors [10,11,30]. On the other hand, pregnant women
were not severely anemic with extremely low hemoglobin Declaration of Interest
values, lower than 70g/l, that may be an explanation why the
significant shrinkage of terminal villis total volume did not The authors declare no conflict of interest.
occur.
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