Relationships between TGFbeta proteins and oxygen concentrations inside the first trimester human gestational sac.
ABSTRACT In early pregnancy, the O(2) gradient between the maternal circulation and the gestational sac tissues modulates trophoblast biological functions. The aim was to evaluate if placental partial pressure of oxygen (PaO(2)) modulates in vivo synthesis of specific placental proteins inside the first trimester gestational sac. Matched samples of peripheral venous blood, blood from the placental bed (PB), coelomic fluid (CF) and placental tissue were obtained in 37 normal pregnancies at 6-12 weeks gestation. PaO(2) was measured in PB and CF using an IRMA blood gas monitor. Inhibin A, activin A, sEng, PlGF, sFlt-1 and free VEGF concentrations were measured in all samples. HSP 70 was measured in placental extracts. ANOVA showed approximately 60% increase in PB PaO(2) (P = 0.02) between after 10 weeks gestation. Unpaired Student's T-test between two groups (6-9 weeks vs 9-12 weeks) shows a significant increase in MS Activin A (P = 0.001), CF activin A (P<0.001), MS P1GF (P = 0.001), CF PlGF (P<0.001), MS sFLT-1 (P = 0.03), CF sFLT-1 (P = 0.01), HSP 70 in placental extracts (P = 0.04) and a significant decrease in PB inhibin A levels (P<0.001) and PB sFLT-1 (P = 0.02) . Multiple correlation analysis showed a significant negative correlation between PB inhibin A levels and gestation (r = -0.45, P<0.05) and PB PaO(2) (r = -0.5, P = 0.008) and also between sFLT-1 and PB PaO(2) (P = 0.03). There was a positive correlation (P<0.01) between PlGF, sEng and VEGF levels in the placental extracts. Our results indicate a direct relationship in the early intrauterine PaO(2) in vivo and inhibin A and sFLT-1 concentrations confirming our hypothesis that specific placental proteins are regulated by intrauterine O(2) tension.
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ABSTRACT: Oxygen (O2) free radicals are a potential teratologic threat to the foetal tissues and are known to be involved in the pathophysiology of common human pregnancy disorders such as miscarriage and pre-eclampsia. During the first two months of human gestation, the placenta surrounds the whole gestational sac, the villi contain only a few capillaries located mainly within the centre of the mesenchymal core, the trophoblastic layer is twice the thickness it will be in the second trimester, the foetal red cells are nucleated and the exocoelomic cavity (ECC) occupies most of the space inside the gestational sac. The ECC contains no oxygen transport system, but anti-oxidant molecules that may provide additional protection to the embryo from oxidative damage are present. Ultrasound and anatomical studies have also demonstrated that the intervillous circulation starts in the periphery of the placenta at around 9 weeks of gestation, and that it becomes continuous and diffuse in the entire placenta only after 12 weeks. Overall, these anatomical features provide indirect evidence that the architecture of the human first trimester gestational sac limits foetal exposure to O2 to what is strictly necessary for its development. These results are in agreement with the concept that the placenta and foetus develop in a physiologically low O2 environment and that its metabolism must be essentially anaerobic. Because of these anatomical arrangements, different nutritional pathways to those operating during most of pregnancy must serve the first-trimester foetus. Up to 9 weeks of gestation, foetal nutrition appears to depend on uterine glandular secretions that are delivered into the intervillous space, supplemented by maternal plasma proteins and other molecules that may percolate through the trophoblastic shell. These molecules diffuse through, or are transported by, the trophoblast of the villi and the chorionic plate into the ECC. From here they are absorbed by the secondary yolk sac (SYS), in which the extraembryonic circulation is probably first established. At the end of the first trimester, the SYS and two-thirds of the placental mass degenerate, and the ECC is progressively obliterated by the enlarging amniotic cavity. The trophoblastic plugs occluding the utero-placental arteries are gradually dislocated, allowing maternal blood to flow into the intervillous space, and the uterine glands involute. These major anatomical transformations modify considerably the spatial relationships between the maternal tissues and the developing embryo, and, consequently, the materno-embryonic exchange pathways. Overall the comparison of morphological features with physiological findings reveals that the architecture of the human first trimester gestational sac is designed to limit foetal exposure to oxygen to that which is strictly necessary for its development, and that during early pregnancy alternative nutritional pathways are in use.Placenta 05/2003; 24 Suppl A:S86-93. · 3.12 Impact Factor
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ABSTRACT: Onset of the maternal-placental circulation was studied by Doppler ultrasonography in 65 pairs of age-matched normal and abnormal pregnancies. In normal pregnancies intervillous blood flow increased with gestational age, being detected in 9 of 25 cases at 8 to 9 weeks but in 18 of 20 at 12 to 13 weeks (P = 0.001). By contrast, in abnormal pregnancies flow was detected in nearly all cases (22 of 25) at 8 to 9 weeks (P < 0.001). In addition, regional differences were observed between the groups. Early flow was restricted to the peripheral regions of most normal placentas (P < 0.001), whereas in missed miscarriages it was most common in central regions or throughout the placenta (P < 0.05 and P < 0.001, respectively). Immunoreactivity for heat shock protein 70 and nitrotyrosine residues was greater in samples from peripheral than from central regions of normal placentas (P = 0.028 and P = 0.019, respectively), and from missed miscarriages compared to controls (P = 0.005 and P = 0.001, respectively). Our results indicate that oxidative damage to the trophoblast, induced by premature and widespread onset of the maternal placental circulation secondary to shallow trophoblast invasion, is a key factor in early pregnancy loss. High oxygen concentrations in the periphery of normal early placentas may similarly induce local regression of the villi, leading to formation of the chorion laeve.American Journal Of Pathology 01/2003; 162(1):115-25. · 4.52 Impact Factor
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ABSTRACT: Miscarriage and pre-eclampsia are the most common disorders of human pregnancy. Both are placental-related and exceptional in other mammalian species. Ultrasound imaging has enabled events during early pregnancy to be visualized in vivo for the first time. As a result, a new understanding of the early materno-fetal relationship has emerged and, with it, new insight into the pathogenesis of these disorders. Unifying the two is the concept of placental oxidative stress, with associated necrosis and apoptosis of the trophoblastic epithelium of the placental villous tree. In normal pregnancies, the earliest stages of development take place in a low oxygen (O2) environment. This physiological hypoxia of the early gestational sac protects the developing fetus against the deleterious and teratogenic effects of O2 free radicals (OFRs). In miscarriage, development of the placento-decidual interface is severely impaired leading to early and widespread onset of maternal blood flow and major oxidative degeneration. This mechanism is common to all miscarriages, with the time at which it occurs in the first trimester depending on the aetiology. In contrast, in pre-eclampsia the trophoblastic invasion is sufficient to allow early pregnancy phases of placentation but too shallow for complete transformation of the arterial utero-placental circulation, predisposing to a repetitive ischaemia-reperfusion (I/R) phenomenon. We suggest that pre-eclampsia is a three-stage disorder with the primary pathology being an excessive or atypical maternal immune response. This would impair the placentation process leading to chronic oxidative stress in the placenta and finally to diffuse maternal endothelial cell dysfunction.Human Reproduction Update 12(6):747-55. · 8.85 Impact Factor
Relationships between TGFb Proteins and Oxygen
Concentrations Inside the First Trimester Human
Shanthi Muttukrishna1*, Sangeeta Suri1, Nigel Groome2, Eric Jauniaux1
1UCL EGA Institute for Women’s Health, University College London, London, United Kingdom, 2School of Biological and Molecular Sciences, Oxford Brookes University,
Oxford, United Kingdom
In early pregnancy, the O2gradient between the maternal circulation and the gestational sac tissues modulates trophoblast
biological functions. The aim was to evaluate if placental partial pressure of oxygen (PaO2) modulates in vivo synthesis of
specific placental proteins inside the first trimester gestational sac. Matched samples of peripheral venous blood, blood
from the placental bed (PB), coelomic fluid (CF) and placental tissue were obtained in 37 normal pregnancies at 6–12 weeks
gestation. PaO2was measured in PB and CF using an IRMA blood gas monitor. Inhibin A, activin A, sEng, PlGF, sFlt-1 and free
VEGF concentrations were measured in all samples. HSP 70 was measured in placental extracts. ANOVA showed ,60%
increase in PB PaO2(P=0.02) between after 10 weeks gestation. Unpaired Student’s T-test between two groups (6–9 weeks
vs 9–12 weeks) shows a significant increase in MS Activin A (P=0.001), CF activin A (P,0.001), MS P1GF (P=0.001), CF PlGF
(P,0.001), MS sFLT-1 (P=0.03), CF sFLT-1 (P=0.01), HSP 70 in placental extracts (P=0.04) and a significant decrease in PB
inhibin A levels (P,0.001) and PB sFLT-1 (P=0.02) . Multiple correlation analysis showed a significant negative correlation
between PB inhibin A levels and gestation (r=20.45, P,0.05) and PB PaO2(r=20.5, P=0.008) and also between sFLT-1 and
PB PaO2(P=0.03). There was a positive correlation (P,0.01) between PlGF, sEng and VEGF levels in the placental extracts.
Our results indicate a direct relationship in the early intrauterine PaO2in vivo and inhibin A and sFLT-1 concentrations
confirming our hypothesis that specific placental proteins are regulated by intrauterine O2tension.
Citation: Muttukrishna S, Suri S, Groome N, Jauniaux E (2008) Relationships between TGFb Proteins and Oxygen Concentrations Inside the First Trimester Human
Gestational Sac. PLoS ONE 3(6): e2302. doi:10.1371/journal.pone.0002302
Editor: Daniel Tome ´, AgroParisTech, France
Received February 12, 2008; Accepted April 2, 2008; Published June 4, 2008
Copyright: ? 2008 Muttukrishna et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: UCLH charities for early pregnancy
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
THE PLACENTA in mammals is the essential interface
between the maternal circulation carrying oxygen (O2)-rich blood
and nutrients and the fetal circulation. In the past it has been
assumed that the principal function of the organ is to supply the
fetus with as much oxygen as possible, and to a large extent that is
true in the latter half of pregnancy when fetal weight gain is
greatest. Our combined in vivo in vitro investigations have resulted
in a new understanding of the materno-fetal relationship during
the first trimester of pregnancy and led to the hypothesis that the
placenta limits, rather than facilitates, oxygen supply to the fetus
during the period of organogenesis [1,2]. The earliest stages of
development therefore take place in a low oxygen environment,
reflecting to some extent the evolutionary path .
The physiological hypoxia of the early gestational sac protects
the developing fetus against the deleterious and teratogenic effects
of oxygen free radicals but it is also necessary to maintain stem
cells in a fully pluripotent state  for at physiological levels free
radicals regulate a wide variety of molecules, in particular
transcription factors . In addition, it is now well-established
that the first-trimester intra-uterine O2 gradient influences
trophoblast proliferation and differentiation along the invasive
pathway  and villous vasculogenesis .
There is increasing evidence that oxidative stress or an
imbalance in the oxidant/antioxidant activity in utero-placental
tissues plays a pivotal role in the development of placental-related
diseases. Pre-eclampsia stems from a defect in early trophoblast
invasion which may be sufficient to anchor the conceptus but is
insufficient to fully convert the spiral arteries into low-resistance
channels [8–10]. Incomplete conversion of the spiral arteries
results in retention of smooth muscle cells within their walls which
lead not only to diminished perfusion of the intervillous space, but
more importantly to intermittent perfusion . Since the
placenta and fetus continually extract oxygen it is expected that
transient hypoxia will result and that consequently the placenta
suffers a chronic low grade ischaemia-reperfusion type injury
Only a few in vitro studies have investigated the role of vascular
endothelial growth factor (VEGF) , nitric oxide, adhesion
molecules [14,15] and activin A [16,17] on the regulation of
trophoblast invasion. We have previously shown that inhibin A
and activin A levels are higher maternal serum from 15 weeks
gestation in pregnant women who subsequently developed pre-
eclampsia . A recent study has also shown that levels of sFLT-
1 and sEng are elevated around 20 weeks gestation in patients who
subsequently developed pre-eclampsia . Inhibin, activin,
VEGF, PlGF, sFLT-1 and Endoglin are members of the TGF b
PLoS ONE | www.plosone.org1June 2008 | Volume 3 | Issue 6 | e2302
family. Soluble Endoglin is a placenta derived soluble TGF-beta
co-receptor. VEGF is one of the most important factors regulating
angiogenesis . A recent study carried out in bovine aortic
endothelial cells (BAEC) suggests that activin A induces capillary
formation in these cells . Placental growth factor potentiates
the effect of VEGF and sFLT-1 antagonises the effect of VEGF.
Soluble Endoglin inhibits the formation of blood vessels and
induces vascular permeability and hypertension .
Increased oxidative stress in the placenta of women presenting
with pre-eclampsia is well documented . We hypothesise that
changes in intrauterine PaO2 modulates placental protein
secretion and synthesis as early as in the end of the first trimester
of human pregnancy and could reflect a progressive defect in the
normal placentation which is associated with the development of
pre-eclampsia during the second half of pregnancy. To evaluate
this hypothesis we have investigated ex vivo, the relationships
between PaO2and specific placental proteins inhibin A, activin A
and pro and anti angiogenic factors PlGF, sFLT-1, sEng and
VEGF concentration inside the early human gestational sac. Heat
shock protein content (HSP 70) in the placental extracts was
measured to study the effect of oxygen variation between
gestations in early pregnancy.
Materials and Methods
Subjects and Samples
We have investigated a series of coelomic fluid (CF), blood
from the decidua under the placental bed (PB) peripheral venous
blood and villous tissue samples in 37 women undergoing
surgical termination of pregnancy for psychosocial reasons at 6–
12 weeks gestation. The study only included uncomplicated
pregnancies and gestational age was determined from the first
day of the last period and confirmed by ultrasound measure-
ment of the fetal crown-rump length. Written consent was
obtained from each woman after receiving complete information
on the procedure. This study was approved by The University
College London Hospitals Committee on the Ethics of Human
In all cases, maternal peripheral venous blood was obtained
from an antecubital vein prior to the surgical procedure. All
patients received small dose (400–800 mg) of misoprostol 30–
60 min before the procedure to prime the cervix. None of the
women presented with any side effect and our previous studies
have shown this medication does not have an effect on the
samples collected . CF and PB samples were all collected
before the surgical termination of pregnancy, under general
anaesthesia, by means of transvaginal needle aspiration under
ultrasound guidance as previously described . The blood
samples were collected in pre-heparinized plastic gas-tight
syringe. CF and PB O2concentration (PaO2) was immediately
measured on the IRMA blood gas analyser (IRMA True point
blood analysis system, Edison, New Jersey, USA) within the
operating theatre. The IRMA analyser was calibrated using a
disposable calibration cartridge set-up at the temperature value
obtained in vivo. Only six CF samples were collected in group 2
(9–12 weeks) as the Coelomic cavity becomes smaller with
advancing gestation and the amniotic cavity expands. CF was
snap frozen in liquid nitrogen. Maternal and the remainder of
the placental blood samples were centrifuged at 3000 rpm for
10 minutes. Placental villous tissue was collected at the end of the
surgical procedure, rinsed in sterile PBS and snap frozen in liquid
nitrogen. Fluid, serum and tissue samples were all stored at
280uC until assayed.
Fluid and Serum Assays.
were measured in all biological fluids in duplicates using our in
house ELISAs as described elsewhere [24,25]. The intra and inter
assay variation was ,10%. The minimum detection limit for
inhibin A was 2pg/ml and activin A was 50pg/ml.
Commercial ELISAs from R&D system (Abingdon, Oxford,
UK) were used to measure PlGF, sEndoglin, sFLT-1, and ‘‘free’’
VEGF in fluids and serum samples according to the manufactur-
er’s protocol. All samples were assayed in duplicates and the intra
and inter assay variations were ,12% for all assays. The
minimum detection limit for the respective assays was PlGF:
3.9pg/ml, sEng: 62.5pg/ml, sFlt-1 31.3pg/ml and free VEGF
Placental protein extracts.
homogenised and extracts were prepared as described elsewhere
. The placental extract total protein contents was estimated by
a commercial Bradford protein assay from PIERCE (UK) using a
BSA standard. The extracts were assayed for the specific proteins
investigated in this study and normalised against the total protein
content. HSP 70 (R&D systems, Oxfordshire, UK) was also
measured in placental extracts in addition to the above proteins.
All data were log transformed to obtain
a normal distribution. Statistical analysis was carried out using the
log transformed data. PB O2 was analysed in three separate
groups using ANOVA. All collected samples measurements were
analysed as two separate subgroups according to gestational age;
early first trimester: 6–9 weeks (n=16), and late first-trimester 9–
12 weeks (n=21). Unpaired Student’s T-test was carried out to
investigate the difference between the two groups of pregnant
women. Pearson correlation was carried out to study the
relationship between the measured parameters using SPSS
statistical package (SPSS, Chicago Illinois, USA). Results were
considered statistically significant at p,0.05. Graph pad prism
(Graph Pad software inc, San Diego, California, USA) was used to
do the normality test and to plot the graphs.
Inhibin A and ‘‘total’’ activin A
Frozen placental tissue was
The median (range), gestational age (days) maternal age (years)
and BMI in group 1 were; 55 days (42–63), 24 years (17–37) and
26.77 (19–32) respectively and in group 2 were; 72 days (64–96),
24.5 years (20–38) and 24.3 (18–29) respectively. Maternal age
and BMI were not significantly different between the two
TGFb proteins distribution in the different compartments
Significantly (P,0.001) higher concentrations of inhibin A and
activin A in PB and in placental extracts compared to MS (table 1)
confirms a placental origin of these proteins.
High concentrations of sEng were measured in MS, PB and
placental extracts. sEng levels were significantly (P,0.01) lower in
CF than in the other compartments.
SFLT-1 was in significantly (P,0.001) higher concentrations in
CF than in the other compartments whereas PlGF was
significantly lower (P,0.001) in CF than in other compartments.
‘‘Free’’ VEGF levels were below the detection limit of the assay
in MS, CF and PB.
Changes with gestational age
PB PaO2 significantly increased (,60%, P,0.02, figure 1a)
after 10 weeks gestation compared to PaO2 at 6–10 weeks
gestation. Regression analysis showed a significant positive
Oxygen and Placental Proteins
PLoS ONE | www.plosone.org2 June 2008 | Volume 3 | Issue 6 | e2302
association (r=0.42, P=0.009) between PB PaO2and gestational
age (figure 1b).
Inhibin A in the PB significantly decreased (,75%, P,0.001)
from 6–9 weeks to 9–12 weeks (12276282pg/ml, vs 270630pg/
ml). MS, CF and placental inhibin A concentration did not change
with advancing gestation in the first trimester (fig 2a). Activin A
levels in both the MS (,2 fold, P,0.001, 522678pg/ml to
10206152pg/ml) and CF (,4 fold, P,0.001, 4556162pg/ml to
21096281pg/ml) increased significantly with gestation (fig 2b).
However, there was no difference in the levels of sEng between
the two subgroups (figure 3a). P1GF levels in MS increased by
.100% (1562 vs. 3269pg/ml, P=0.001) between the two
groups. P1GF levels did not change in the PB or placental tissue
extract with advancing gestation. CF PlGF concentration only
showed a significant decrease with advancing gestation (P,0.001,
Levels of sFLT-1 decreased significantly with advancing
gestation in PB (1665 vs 6.361.6ng/ml; P=0.02) and an increase
in MS ( P,0.03) and CF (P=0.01, figure 3c). sFLT-1 levels did
not change significantly in placental extracts between early and
late first-trimester periods,
Placental extract contained very low concentrations of free
VEGF and they did not change with advancing gestational age
HSP 70 concentration in the placental extracts were signifi-
cantly higher (P=0.04) at 9–12 weeks (82.55+6ng/mg protein)
compared to 6–9 weeks (51.3+5.7ng/mg protein) placentae.
Multiple regression analysis
Table 2 shows all the significant relationships between the
different molecules in the same compartment. An association
between the same molecules in the different compartments is also
shown in table 2. PB oxygen tension was negatively correlated to
sFLT -1(r=20.44, P,0.05) and inhibin A levels (r=20.5,
P=0.01) in PB. PlGF, sEng and sFLT-1 were all significantly
associated with each other in PB. In the MS Activin A was
positively associated to PlGF and sEng.
The results of the present study confirms that in vivo PaO2in
the uterine decidua under the placental bed (PB) increases after
10 weeks gestation as shown in our previous study  and
suggest that the variations in the utero-placental O2 gradient
during the first trimester of human pregnancy could have a major
impact on the production of TGFb proteins and thus modulate
indirectly placentation and early foeto-placental angiogenesis in
Table 1. Mean6SEM concentration of activin A, inhibin A, sEndoglin, PlGF, sFLT-1 and VEGF in the different gestational fluids in
the first trimester (6–12 weeks).
Coelomic fluid Maternal SerumPlacental Blood Placental Extract
Activin A (pg/ml) 1033.66198 770.8696.7 1655.46282 19336297
Inhibin A (pg/ml) 354.5645.2 192.8619.3 674.661421540.7661.7
sEndoglin (ng/ml)41.2660.7 726289.764.359.767.3
PlGF (pg/ml)1462.2625.1165.11208.9627 5869
Figure 1. a: Placental bed oxygen concentration in early
pregnancy. Mean6SEM values of placental bed blood concentrations
in the different fluids measured. Group 1=6–8 weeks, group 2=8–
10 weeks, group 3=10–12 weeks. P=0.02, ANOVA. b: Relationship
between gestation and oxygen concentration in the placental
bed. Linear regression curve for PB PaO2(log) vs gestation in days. 95%
confidence interval lines are above and below the regression line.
R=regression co-efficient and P=significance level. N=37.
Oxygen and Placental Proteins
PLoS ONE | www.plosone.org3 June 2008 | Volume 3 | Issue 6 | e2302
We found lower concentrations of sEng and PIGF and higher
concentrations of s-Flt-1 in the exocoelomic cavity than in the
other compartments whereas activin A and inhibin A concentra-
tions were similar in CF and MS but lower than in PB and
placental extract. Free VEGF was not detectable in CF samples.
The exocoelomic cavity is the largest fluid space inside the first-
trimester gestational sac . It disappears at the end of the first
trimester when the amniotic cavity reaches the chorionic plate of
the placenta. Biochemical investigations of early fetal fluid have
indicated that the CF results from an ultrafiltrate of MS and
uterine gland secretions with the addition of specific placental and
secondary yolk sac bio products . The higher concentrations of
human chorionic gonadotrophin (hCG), oestradiol, oestriol and
progesterone found in the CF compared to MS indicate the
presence of a direct pathway between the trophoblast and the
early fetus .
VEGF and P1GF are vascular endothelial growth factors that
play a key role in angiogenesis and vasculogenesis in a number of
physiologic situations and in particular they play an important role
during embryogenesis .The main source of VEGF molecules
and P1GF during pregnancy is the placental trophoblast. sFlt-1 is a
splice variant of VEGF receptor 1 (Flt1) and is produced by a
variety of tissues including the placenta. SFlt-1 binds to both
VEGF and PlGF in MS reducing their bioavailability .
Unbound PlGF, and VEGFs exist as free molecules and have a
soluble receptor which facilitates its transport across membranes
. This could explain the higher total concentration of sFlt-1 in
Endoglin is a trans-membrane glycoprotein found on cell
surfaces highly expressed in endothelial cells and syncytiotropho-
blasts . sEng is the soluble form of Endoglin found in serum.
Its level is increased in the blood circulation of patients with
angiogenic tumours, neovascularisation and myeloid malignancies
and of pregnant women . The presence of these pro and anti-
angiogenic proteins in the CF in early pregnancy suggests that the
exocoelomic cavity is also a reservoir for molecules with a direct
role in the regulation of early fetal and placental angiogenesis.
Activin A and inhibin A are both glyco proteins, which have
been identified in a wide variety of tissues including the placenta,
adrenal glands, pituitary gland, bone marrow, kidneys, spinal cord
and brain . MS concentrations of activin A and inhibin A fall
rapidly in the puerperium suggesting that the placenta is the
primary source of these proteins throughout most of pregnancy. In
the present study, the distribution of these proteins within the
different compartments is consistent with this concept. It has been
shown that inhibin and activin increase the production of GnRH
and progesterone by the placenta, suggesting that they may play a
role in the hormonal support of pregnancy . It has also been
shown that activin A promotes trophoblast invasion between 6–
10 weeks in vitro . Additionally, in bovine arterial cells, activin
A has been shown to promote angiogenesis . We found that in
blood samples from the PB, activin A levels were positively related
to PlGF, sFlt-1 and sEng whereas, in MS activin A levels were
positively related to PlGF and sEng. These relationships suggest
that activin A may also play a role in foeto-placental angiogenesis
in early human pregnancy.
Anatomic and in vivo studies have shown that human
placentation is in fact not truly haemochorial in early pregnancy
[1,3]. From the time of implantation, the extravillous trophoblast
not only invades the uterine tissues but also forms a continuous
shell at the level of the decidua and plugs in the tips of the utero-
placental arteries. The shell and the plugs act like a labyrinthine
interface that filters maternal blood, permitting a slow seepage of
maternal plasma but no true blood flow into the intervillous space.
This mechanical barrier results in intra placental PaO2which are
2 to 3 times lower at 8–10 weeks than after 12 weeks. During
pregnancy there is a progressive, but independent, increase in
decidual PO2 advances between 7 and 16 weeks, which most
probably reflects the increase in maternal blood flow volume
within the uterine circulation. Biochemical analysis has also shown
that the CF contributes to the redox potential of the gestational sac
in a low oxygen environment  and in the fetal tissues
antioxidant capacity at a time when the fetus is most vulnerable to
oxidative stress .
Our data indicate a significant increase in HSP 70 expression in
placental extracts between early and late first-trimester. In normal
pregnancies, we have previously shown using immunohistochem-
istry that there is a physiological oxidative stress at around 9 weeks
which is associated with an increased expression of heat shock
proteins. This increased expression is particularly marked in the
periphery of the placenta which is where the utero-placental
circulation is first established [2,3] SFlt-1 expression in placental
villous explant from early first-trimester pregnancies (5–9 weeks)
Figure 2. Inhibin A and Activin A in early pregnancy. Mean+SEM
values of inhibin A and activin A concentrations in the different fluids
measured. Group 1=6–9 weeks, group 2=9–12 weeks. ***=P,0.001,
unpaired Student’s T-test between the different groups.
Oxygen and Placental Proteins
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 and in vitro cytotrophoblast cell culture  is significantly
increased under physiological low oxygen (,10%) conditions. In
cytotrophoblast culture media, VEGF is not detected regardless of
oxygen concentration whereas free PlGF is diminished by reduced
oxygen (48). These results suggests that low oxygen levels play an
important role in regulating TGF b proteins expression in the
developing human placenta, however further interpretation of in
vitro data is limited by laboratory experimental conditions. In our
study, we found that in vivo placental bed serum inhibin A and
sFlt-1 levels are inversely proportionate to the PaO2measured in
vivo at the same anatomical level. Recently it has been suggested
that maternal endothelial cell dysfunction mediated by excess sFlt-
1 is a major cause of the onset of the disease . The inverse
relationship that we have observed between increasing O2
concentration, inhibin A and sFLT-1 in blood samples from the
PB could explain the increased concentrations of these proteins
found in women presenting with pre-eclampsia . Although
sEng is a TGF beta receptor binding protein, the affinity of sEng
to PlGF or VEGF is unknown.
In placental-related disorders of pregnancy such as pre-
eclampsia, it has been reported that sFlt-1[18,29,30] and inhibin
A serum levels are higher in patients weeks before they develop the
clinical symptoms. Similar to the in vitro experiment at low O2
, PlGF levels have been reported to decrease in women
presenting later in pregnancy with pre-eclampsia [30,31], whilst
sEng levels were found to increase [22,32]. In addition, a recent
systematic review has suggested that only in the third trimester
sFLT-1 consistently increases and PlGF decreases in serum of
women who subsequently develop pre-eclampsia, reaching a
statistical significance in the early onset cases . These results
Figure 3. Pro and Anti angiogenic factors in early pregnancy. Mean+SEM values of soluble Endoglin, placental growth factor (PLGF), soluble
VEGF receptor 1 (sFLT-1) and ‘‘free’’ vascular endothelial growth factor (VEGF) concentrations in the different fluids measured. Group 1=6–9 weeks,
group 2=9–12 weeks ***=P,0.001, *=P,0.05 Unpaired Student’s T-test between the different groups.
Table 2. Pearson correlation analysis data showing significant
relationships between the proteins and PaO2concentration in
the different compartments of the early gestational sac.
Placental bed blood (PB) Activin A vs PlGF (r=0.57, P,0.02)
PlGF vs sFlt-1 (r=0.57, P,0.01)
PlGF vs sEng (r=0.69, P=0.001)
sFlt-1 vs sEng (r=0.42, P,0.05)
sFlt-1 vs Po2 (r=20.44, P,0.032)
Maternal serum (MS) Activin A vs PlGF (r=0.52, P=0.005)
Activin A vs sEng (r=0.38, P=0.05)
Coelomic Fluid (CF)Inhibin A vs activin A (r=0.6, P=0.02)
Placental Extracts (PEx)Inhibin A vs activin A (r=0.73, P,0.001)
VEGF vs PlGF (r=0.67, P,0.001)
VEGF vs sEng (r=0.63, P,0.001)
PlGF vs sEng (r=0.54, P=0.001)
Inhibin AGestation vs PB (r=20.45, P=0.02),
PaO2 vs PB (r=20.5, P=0.01)
Activin ACF vs MS (r=0.8, P,0.001)
CF vs gestation (r=0.74, P,0.001)
MS vs gestation (r=0.54, P=0.002)
sFlt-1 PaO2 vs PB (r=20.44, P,0.05)
CF vs gestation (r=0.45, P,0.05)
PlGFCF vs MS (r=20.5, P=0.025)
MS vs gestation (r=0.61, P,0.001)
Endoglin MS vs PB (r=0.56, P,0.01)
Oxygen and Placental Proteins
PLoS ONE | www.plosone.org5 June 2008 | Volume 3 | Issue 6 | e2302
suggest that placental secretion of sFLT-1 could increase during
the second trimester in women who subsequently present with
clinical pre-eclampsia in the third trimester, long before these
changes are detectable in maternal serum. These finding suggest
that the molecules that are altered by PaO2 could be useful
markers of the disease prior to the onset of the clinical symptoms
as placental hypoxia in early pregnancy is one of the primary
causes of the multisystem dysfunction later in pre-eclampsia.
In the present study, MS PlGF levels increased almost 2 fold
from 6–9 weeks to 9–12 weeks gestation whereas sFlt-1 in the PB
serum decreased by 75%. SFlt-1 has a high affinity binding to pro
angiogenic proteins VEGF and PlGF . VEGF was below the
detection limit in CF, MS and PB samples measured in our study
because the assay measures ‘free’ rather than ‘total’ VEGF.
However, in placental extracts VEGF levels were above detection
limit and it was positively correlated to PlGF and sEng levels. We
also found that sFLT-1 levels in samples from the PB decreased
with advancing gestation in the first trimester suggesting that as
placental vascularisation increases with gestation, sFLT-1, an anti-
angiogenic protein that controls the availability of pro angiogenic
factors such as VEGF and PlGF are diminished contributing to an
optimal development of the placenta vascularisation.
Our data indicate that inhibin A levels in samples from the PB
decrease significantly (,75%) between 6 to 12 weeks of gestation.
These changes are similar to that reported previously in maternal
serum for inhibin A and activin A between 8 to 12 weeks [25,34].
Our correlation analysis showed that in samples from the PB,
inhibin A levels were negatively correlated to PaO2. Inhibin A is
also increased in the early second trimester in the serum of women
who subsequently develop preeclampsia . Furthermore, the
significant inverse relationships found in samples from the PB
between PaO2 and inhibin A and sFlt-1 concentration (table 2)
suggest a probable mechanism for the rise in these molecules in
placenta-related oxidative disorders of pregnancy. The observa-
tion of a similar trend in PB samples between PaO2, sFlt-1 and
inhibin A suggests that these molecules could be used in
combination for the screening of pre-eclampsia.
In conclusion this study showed an association between
intrauterine oxygen tension and inhibin A and sFLT-1 confirming
our hypothesis that oxygen tension may regulate placental protein
secretion. The association between activin A, PlGF, sEng and
sFLT-1 provides further evidence for activin A to be associated in
The authors wish to thank Katherine Rogers for her help in this study.
Conceived and designed the experiments: EJ SM. Performed the
experiments: SM. Analyzed the data: SM. Contributed reagents/
materials/analysis tools: EJ SS NG. Wrote the paper: SM. Other: Also
contributed to the writing: EJ.
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