Human Reproduction vol.14 no.2 pp.536–541, 1999
Characterization of human placental explants:
morphological, biochemical and physiological studies
using first and third trimester placenta
Suren R.Sooranna1, Eugene Oteng-Ntim2,
Rafia Meah1, Timothy A.Ryder2 and
Rekha Bajoria3,4
1Department
of Obstetrics and Gynaecology, Imperial College
School of Medicine, Chelsea and Westminster, 2Queen Charlotte’s
and Chelsea Hospital for Women, London and 3Department of
Obstetrics and Gynaecology, University of Manchester,
St Mary’s Hospital for Women and Children, Whitworth Park,
Manchester M13 OJH, UK
4To
whom correspondence should be addressed
The primary objective of this study was to characterize an
in-vitro model of the human placenta using morphological,
biochemical and physiological parameters. Placental villi
were obtained from normal first trimester and term
pregnancies. The villi were incubated with Dulbecco’s
modified Eagle’s medium: Ham’s F12 nutrient mixture in
a shaking water bath at 37°C for up to 310 min. The
viability was determined by the production of β human
chorionic gonadotrophin (HCG) and lactic dehydrogenase
(LDH) and the incorporation of [3H]thymidine, [3H]Lleucine and L-[U14C]arginine, while ultrastructure was
assessed by transmission electron microscopy. In the first
and third trimester group, the release into the medium
of the intracellular enzyme LDH remained unaltered
throughout the experiment. By contrast, β-HCG concentrations increased linearly and concentrations were higher in
the first trimester than term villi (354.5 K 37.8 versus
107 K 8.1 IU/g villi protein; P < 0.001). Electron microscopy
confirmed preservation of tissue viability for up
to 4 h of incubation. The incorporation of thymidine
(12.2 K 2.9 versus 5.2 K 0.5 nmol/g villi protein; P < 0.05),
leucine (9.4 K 2.1 versus 1.9 K 0.4 nmol/g villi protein; P
< 0.02) and arginine (17 K 4.4 versus 4.2 K 0.5 nmol/g
villi protein; P < 0.05) were markedly higher in early than
in term placenta. Furthermore, placental uptake of Lleucine by the first (9.4 K 2.1 versus 17 F 4.4 mol/g villi
protein; P < 0.001) and third trimester placental villi
(1.9 K 0.4 versus 4.2 F 0.5 mol/g villi protein; P < 0.001)
was less than that of L-arginine. This study describes a
simple technique using placental explants to determine
relative rates of uptake of substrate amino acids throughout
gestation.
Key words: amino acids/electron microscopy/organ culture/
placenta/villi
Introduction
With the recent awareness that an adverse intrauterine environment may predispose the fetus to development of disease in
536
adult life, an understanding of the placental function in relation
to fetal growth and development is crucial.
Although advances in imaging techniques have made the
human fetal circulation accessible, in-vivo studies can only
provide data on direct placental transport of a substance at
almost undisturbed steady state conditions at various stages of
gestation. The main disadvantage of clinical studies, however,
is their inability to provide integral information on placental
uptake and metabolism, as well as the mechanisms and
factors influencing transport of nutrients to the fetus. Ethical
considerations as well as fetal safety further restrict invasive
research in human pregnancy.
Because of its easy accessibility, isolated human placental
tissue has been used widely to overcome some of the restrictions
placed on clinical research. Several techniques have been
described, ranging from membrane vesicles (Eaton and Oakey,
1997), syncytiotrophoblast villous preparations (Palmer et al.,
1997), first trimester placental villi co-cultured with decidual
explants (Babawale et al., 1996) and placental slices (Miller
and Berndt, 1974), to dual perfusion of either the whole organ
(Panigel, 1972) or a single cotyledon (Schneider, 1991).
Although experiments performed with isolated placental tissue
create an artificial situation, such models can provide answers
for those questions which are difficult to obtain in the complex
in-vivo set-up.
Of the various systems available, perfusion of the placenta
mimics in-vivo conditions closely as it retains the complexity
of the intact organ and allows control of experimental variables
(Schneider, 1991; Bajoria et al., 1996). Despite numerous
advantages of the placental perfusion model, this experimental
system cannot be used to study uptake and transfer function
during first trimester pregnancies. Furthermore, it is also not
technically feasible to perfuse placentae from pregnancies
complicated with systemic lupus, severe pre-eclampsia and
fetal growth restriction.
Alternatively, isolated cytotrophoblast cells which can be
grown in primary cultures to form multinucleated cells may
be used, but they are difficult to purify and have a limited
lifespan (Sooranna, 1996). Furthermore, they are more removed
from the in-vivo situation because of limited cell–cell interactions, and cells isolated from placentae obtained from
abnormal pregnancies may revert to ‘normal’ functions when
in culture. These preparations do, however, have advantages
over perfusion systems in that they allow comparisons of
several parameters within one experiment and only small
quantities of experimental substances (e.g. radiolabelled substrates) are needed (Bajoria et al., 1997). Another possibility
is the use of choriocarcinoma cell lines, of which the BeWo
line has been the most extensively characterized (Pattillo and
© European Society of Human Reproduction and Embryology
Characterization of human placental explants
Figure 1. (A) Release of lactic dehydrogenase (LDH) by placental villi (n 5 12). Data from first and third trimester placental villi were
pooled together as there was no difference between two groups. (B) Release of β human chorionic gonadotrophin (HCG) by the first
trimester villi (–d–) and term villi (–s–). The data in each group are shown as mean 6 SEM of six experiments.
Gey, 1968; van der Ende et al., 1987). Although these will
grow very easily in culture, experiments with these should be
treated with caution because they are transformed cells.
In order to obviate some of the limitations of the perfusion
as well as the cell culture systems, we developed a simple invitro model of the human placenta using placental explants.
In this paper we characterize the biochemical, morphological
and physiological viability of placental villi obtained from
early and term placenta.
Materials and methods
Methyl [3H]thymidine, [3,4,5–3H(N)]-L-leucine and tissue culture
reagents were obtained from Sigma Chemical Co. Ltd, Poole,
Dorset, UK and L-[U14C]arginine monochloride from Amersham Life
Sciences, Slough, UK. All other laboratory reagents were of the
purest grade available from BDH Chemicals Ltd., Poole, Dorset, UK.
Villous preparation
First trimester human placentae between 7–12 weeks were obtained
from women undergoing legal abortion. Placentae were also obtained
between 37 and 41 weeks gestation following Caesarean section or
vaginal deliveries from women with normal pregnancies. Gestational
age was determined in all cases by ultrasound scan. From term
placentae, 10–15 1 cm3 pieces of tissue were removed at random and
placed in cold phosphate buffered saline (PBS) containing 100 units/
ml penicillin, 100 mg/ml streptomycin and 100 units/ml nystatin.
From termination of pregnancy the whole placenta was placed in
PBS. The tissue was washed several times with PBS to remove as
much blood as possible. The placental tissue was teased into small
pieces, carefully removing any obvious blood vessels or clots. This
tissue was re-washed several times with PBS. Approximately 15 ml
of settled tissue was made up to 30 ml with Dulbecco’s modified
Eagle’s medium: Ham’s F12 nutrient mixture (1:1) containing 1%
bovine serum albumin, 50 units/ml penicillin and 50 µg/ml
streptomycin. This suspension was swirled to provide a homogeneous
preparation prior to sampling for addition to incubation tubes. The
incubation was carried out in sterile centrifuge tubes in DMEM:
Ham’s F12 medium.
Experiment protocol
Forty two experiments were undertaken using either placentae from
first trimester (n 5 23), or term (n 5 19) pregnancies. In the first
trimester group, we did six experiments each to study the production
of β human chorionic gonadotrophin (HCG) and lactic dehydrogenase
(LDH), or the uptake of L-arginine and L-leucine, while five experiments were performed with thymidine. In the term group, six
experiments were done to estimate β-HCG and LDH, while five
incubations were performed with thymidine only. The uptake of
L-arginine and L-leucine was studied using six first trimester and five
third trimester placentae.
Incubations were performed in a shaking Mickle® water bath
(Fission Ltd, Dorset, UK) (70 oscillations/min) at 37°C. When
radioactive substances were present the final concentrations of
thymidine, leucine and arginine were 1.5 µM (175 000 d.p.m./µmol),
0.42 mM (43106 d.p.m./µmol) and 0.7 mM (700 000 d.p.m./nmol)
respectively. Incubations were carried out in duplicate for up to
310 min. At the end of the incubations, villi from each incubation
tube were centrifuged for 5 min at 1420 g. The supernatant was
stored for estimation of β-HCG and LDH concentrations, and the
pellet washed three times with cold PBS. After the final wash the
pellet was solubilized with 2 ml of 1 M sodium hydroxide overnight
at 37°C. The solubilized pellets were then diluted to 10 ml with
distilled water and vortex mixed for 1 min; 2.0 ml of scintillant
(Packard Ultima gold XR®; Meriden, CT, USA) was added to 231
ml aliquots of solubilized pellet and counted on a scintillation counter.
Aliquots of the solubilized pellets were also kept for determination
of protein concentration by the method of Lowry et al. (1951).
Transmission electron microscopy (TEM)
Samples of placental villi at 0, 4 and 8 h post-incubation were fixed
for 1–2 h in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2,
washed in fresh buffer and post-fixed in 1% osmium tetroxide.
Samples were dehydrated through a graded series of alcohol and
embedded in araldite epoxy resin. Ultrathin sections were cut
perpendicular to the plane of the membrane using a diamond knife.
Sections were stained with uranyl acetate and lead citrate before
examination in an Hitachi HU 12A transmission electron microscope
operated at 50 kV.
Analytical methods
The concentration of β-HCG was measured by a radioimmunoassay
kit purchased from ICN Biochemicals Ltd, Thame, Oxfordshire, UK.
The assay had a coefficient of variation of 10–14%. The lower limit
of detection was 0.1 IU/ml.
Lactic dehydrogenase activity was measured by a commercially
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S.R.Sooranna et al.
Results
Concentration of β-HCG and LDH
The biochemical viability of the placental villi during
incubation as assessed by estimation of LDH and β-HCG
concentrations in the extracellular medium are shown in Figure
1. As the concentration of LDH in early and term placenta
was comparable, data in both groups were pooled together
and remained constant during incubation of up to 3.5 h of
(6 6 1.4 at 0 min to 9.3 6 1.9 at 210 min; n 5 12) % total
LDH activity. By contrast, β-HCG concentrations in first
trimester placental villi increased markedly from 18.4 6 5.11
IU/g of villi at 0 min to 354.5 6 37.8 at 210 min (n 5 6).
This was markedly greater than those produced by third
trimester villi (8.4 6 2.32 U/g of villi at 0 min to 82.1 6 10.7
at 210 min; n 5 6).
Ultrastructure of the tissue
Samples of placental villi were examined at 0, 4 and 8 h of
incubation (Figure 2). The surface structure of the trophoblast
epithelial cells, i.e. microvilli, were not adversely affected by
the experimental procedure up until 4 h. The plasma membranes
and cytomembrane system of the cells remained intact, and
the mitochondria were also unaffected. However, under similar
experimental conditions, formation of intracellular vacuoles
was noted at 8 h incubation (Figure 2C).
Uptake of essential amino acids
First trimester
When placental villi from first trimester were incubated with
a known concentration of thymidine, the uptake increased
from 2.8 6 1.0 nmol/g of villi at 0 min to 12.2 6 2.9 at
120 min (P , 0.001) (Figure 3B). L-leucine incorporation into
the placental villi increased with time from 0.6 6 0.2 nmol/g
of villi at 0 min to 9.4 6 2.1 at 180 min (Figure 3C). A
similar increase in placental incorporation of L-arginine was
also found with time (0.9 6 0.2 nmol/g of villi at 0 min to
17.0 6 4.4 at 180 min; Figure 3A). At each time point, the
uptake of L-arginine was greater than L-leucine (P , 0.001).
Figure 2. Electron micrograph of the placental villi sampled (A) at
0 h, (B) 4 h and (C) 8 h of incubation. The placental villi at time
4 h shows syncytiotrophoblast (syn) with regular cylindrical
microvilli (M). The preservation of the cytoplasmic organelles is
similar to that seen at 0 h. The placental villi show well preserved
densely stained mitochondria and there is no evidence of
intracellular oedema or vacuoles. The villi at 8 h show evidence of
intracellular oedema and formation of vacuoles. Bars 5 2 µm.
available colorimetry assay kit from Sigma, with a coefficient of
variation of 7–12%. The sensitivity of the assay was 1 U/ml.
Data analysis
Values were expressed as mean 6 SEM per g of placental villi
protein unless otherwise indicated. Data between two groups as a
function of time were compared by two-way analysis of variance.
One-way analysis of variance was used to compare blocked variables
between groups. P values , 0.05 were considered significant.
538
Third trimester
Similar to first trimester data, the uptake of thymidine
(1.3 6 0.2 nmol/g of villi to 5.0 6 0.5), L-arginine
(0.7 6 0.08 nmol/g of villi to 4.2 6 0.5), and L-leucine
(0.5 6 0.2 nmol/g of villi to 1.9 6 0.4) by term placental villi
increased with time. At each time point, uptake of L-arginine
was greater than L-leucine (P , 0.001).
The uptake of thymidine (P , 0.05), L-leucine (P , 0.02),
and L-arginine (P , 0.05) at each sample point by term
placenta was significantly less than in the first trimester group
(Figure 3D).
Discussion
We have developed a simple in-vitro model of the human
placenta using short-term placental explants to study the uptake
and transport of nutrients by the placenta. In this article we
characterized this system using biochemical and morphological
parameters. Furthermore, we used this model to study the
Characterization of human placental explants
Figure 3. Incorporation into first trimester (–d–) and third trimester (–s–) placental villi. (A) 3H-thymidine (n 5 5 in each group);
(B) L-leucine (n 5 6 for first and n 5 5 for third trimester; and (C) L-arginine (n 5 6 for first trimester and n 5 5 for third trimester).
(D) compares uptake of leucine (u), arginine (j) at 180 min and the thymidine (e) at 120 min by first trimester with term placentae. The
data are shown as mean 6 SEM. *P , 0.001.
effect of gestational age on the uptake of a neutral and a
cationic amino acid.
Various investigators have used similar systems to study
placental function (Ahmed and Murphy, 1988; Haning et al.,
1988; Barnea et al., 1992; Polliotti et al., 1995). However, no
study has systematically characterized this model to evaluate
its suitability to determine placental transport of nutrients.
Initially, we undertook experiments to optimize the system
using various parameters. Viability of the placental tissue
was maintained for up to 3.5 h as assessed by measurement
of β-HCG in the culture media. Like in-vivo data, our results
also showed an eight-fold increase in β-HCG production in
early pregnancy as compared to term placenta. The documented
decrease in the ratio of cytotrophoblast to syncytiotrophoblasts
(Kaufmann and Castellucci, 1997) may explain the differences
between early and late placental β-HCG production. We also
measured LDH concentration during the experimental period
as an index of cellular leakage during incubation period.
LDH is a cytoplasmic enzyme and minimal alteration in its
concentration in the medium further substantiates that placental
membrane integrity was preserved during the experiment. At
the beginning of the experiment β-HCG and LDH were present,
reflecting the release of these proteins into the medium during
the first wash. Similarly values for incorporation of radioactive
substrates into tissues were also seen at the start of the
experiment.
Cellular function in terms of the incorporation of thymidine, an index of cellular proliferation, was also assessed.
[3H]Thymidine was chosen for this purpose because it is only
incorporated during the S-phase of the cell cycle and therefore
considered to be a reliable proliferative marker (Kaufmann
and Castellucci, 1997). Our data showing an almost linear
increase in cellular incorporation of thymidine over 2 h further
confirms cellular viability. The levelling off in incorporation
seen is not surprising because the DNA synthetic ability of
the trophoblast is limited. The finding of greater incorporation
of thymidine by the first trimester as compared to term placenta
agrees with the evidence that proliferation and differentiation
of placental cells decrease with advancing gestation (Kaufmann
and Castellucci, 1997).
The viability of the villous tissue during the first 4 h of the
experimental procedure was also evaluated. The ultrastructure
of the syncytiotrophoblast showed no apparent changes over
a 4 h incubation period. The plasma membrane remained intact
with regular cylindrical microvilli. There was no evidence of
intracellular oedema or damage and mitochondria, in particular,
appeared to be well preserved with organized cristae. However,
under similar experimental conditions, formation of intracellular vacuoles was noted at 6 and 8 h incubation. Because
of these changes we studied uptake by placental villi for no
longer than 3 h.
After having established the cellular viability of the system,
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S.R.Sooranna et al.
we then studied the uptake of leucine and arginine by first and
third trimester placental villi obtained from normal pregnancies.
Leucine was chosen because it is a branched neutral amino
acid and its uptake occurs by membrane bound sodiumdependent l transporter (Moe, 1995). Placental uptake of
arginine was also determined because of its important role in
regulation of fetal growth and pre-eclampsia. Furthermore, the
uptake of arginine is mediated by a sodium-independent system
y1 and y1 l (Moe, 1995).
Our data clearly demonstrate that the uptake of both neutral
and cationic amino acids by the human placenta decreases
with advancing gestational age. This observation is at odds
with reports on other placental transport systems such as
system A in the human (Mahendran et al., 1994) and the rat
placenta, and system y1 and y1 l in the rat placenta, all of
which increase with gestation (Malandro et al., 1994; Novak
et al., 1997). Similarly, in relation to glucose transport by the
human placenta, there is an increase in GLUT-1 from late
second trimester (Jansson et al., 1993). However, these data
agree with the observation that the concentration of free amino
acid in the placental homogenate decreases with gestational
age. The decrease in L-arginine uptake with gestational age
parallels the changes we have found recently in nitric oxide
synthase (Sooranna et al., 1995). Moreover, recent evidence
that the concentration of L-arginine required for eNOS and
iNOS activation are one to two orders of magnitude below the
intracellular concentrations of L-arginine (Bogle et al., 1992;
Myatt et al., 1993) suggests that nitric oxide synthesis is
dependent upon the extracellular rather than intracellular concentration of L-arginine. It is possible that greater uptake of
L-arginine by first trimester villi may be responsible for
increased nitric oxide synthesis and hence NOS activity in
early pregnancy. Increased nitric oxide synthesis in early
gestation therefore may be one of the mechanisms responsible
for reduced vascular resistance.
In addition, our data also indicate that for a given gestational
age, the uptake of L-arginine was twice as high as that of
L-leucine. This discordance in placental uptake of two essential
amino acids is not altogether surprising (Rosso, 1975). In
human pregnancies over a gestational age ranging between
20–37 weeks, placental transport of leucine is significantly
more rapid than glycine (Cetin et al., 1995). Less is currently
known about regulation of amino acid transepithelial flux. We
speculate that one of the following mechanisms may be
responsible for difference in uptake between leucine and
arginine by placental villi.
We speculate that the difference in uptake between leucine
and arginine by placental villi could be attributed to the
differences in (i) intracellular concentration of the substrate,
(ii) rate of protein synthesis, catabolism and production of
individual amino acid within the placenta, (iii) placental
permeability or affinity of the membrane-bound protein transporter. However, further studies are required to characterize
the regulatory mechanism of amino acid uptake by the
human placenta.
In summary, our data for the first time suggest that the
uptake of leucine and arginine decreases with increasing
gestational age. The current approach provides an alternative
540
simple technique to characterize relative rates of uptake of
amino acids across the human placenta throughout gestation
as well as the ability to determine differences in metabolism
between placentae from normal and pathophysiological conditions such as pre-eclampsia and growth restriction. This model
has an added advantage that placental uptake functions can be
studied in parallel to intracellular amino acid concentration.
This is likely to improve our understanding of transepithelial
regulatory mechanisms of amino acid flux.
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