Download Fluid compartments of the embryonic environment

Human Reproduction Update 2000, Vol. 6 No. 3 pp. 268–278
© European Society of Human Reproduction and Embryology
Fluid compartments of the embryonic environment
Eric Jauniaux1,* and Beatrice Gulbis2
1
Academic Departments of Obstetrics and Gynaecology, Royal Free and University College London Medical School, UCL campus, London,
UK and 2Academic Department of Clinical Chemistry, Academic Hospital Erasme, Université Libre de Bruxelles, Brussels, Belgium
Received on September 28, 1999; accepted on February 15, 2000
The exocoelomic cavity was probably the last remaining physiological body fluid cavity to be explored in the human
embryo. Its unique anatomical position has enabled us to study the protein metabolism of the early placenta and
secondary yolk sac and to explore materno–embryonic transfer pathways. The exocoelomic cavity forms inside the
extraembryonic mesoderm alongside the placental chorionic plate and is now believed to be an important transfer
interface and a reservoir of nutrients for the embryo. Maternal or placental proteins filtered in the extraembryonic
coelomic cavity are probably absorbed by the secondary yolk sac which is directly connected with the primitive
digestive system throughout embryonic development. Protein electrophoresis has shown that the coelomic fluid
results from an ultrafiltrate of maternal serum with the addition of specific placental and secondary yolk sac
bioproducts demonstrating that the exocoelomic cavity is a physiological liquid extension of the early placenta. The
selective sampling of fluid from the exocoelomic cavity has also offered a novel approach to the study of drug and
toxin transfer across the early human placenta and as a unique tool to explore embryonic physiology in vivo. Further
investigation should include a comparison between the coelomic fluid values of a molecule and its quantifiable
presence in decidual, placental and fetal tissues.
Key words: amniotic fluid/coelomic fluid/first trimester of pregnancy/placenta/yolk sac
TABLE OF CONTENTS
Introduction
Embryology of the human placenta and adnexae
Embryonic physiology
Biology of the extraembryonic coelom
Biology of the early amniotic cavity
Prospects in the investigation of the biology of
embryonic fluids
References
268
269
270
271
275
276
277
Introduction
With the development of amniocentesis for genetic investigation,
the amniotic fluid (AF) became the first fluid available to study
the fetal environment in utero. AF analyses have been performed
from the third month of gestation onwards and have demonstrated
important variation in gas tension, acid–base status and
biochemical composition with gestational age and differences
compared with maternal blood. During the 1970s, first trimester
AF biochemistry was investigated (Sinha and Carlton, 1970;
Johnell and Nilsson, 1971).
Due to technical difficulties in recognizing accurately the
different anatomical structures of the early gestational sac in
utero, it is likely that most of the few samples obtained in these
studies, at <12 weeks gestation, were a mixture of coelomic fluid
(CF) and AF. In fact the original anatomical finding that the
extraembryonic coelom is a fluid cavity, which surrounds the
embryo and fetus during most of the first trimester (Boyd and
Hamilton, 1970), was completely ignored by most authors in the
1960s and 70s. Furthermore, some authors believed that it was a
thin virtual space containing a gelatinous substance that could not
be aspirated (MacCarthy and Saunders, 1978).
The advent of high resolution transvaginal ultrasound
transducers at the end of the 1980s has enabled a more detailed
morphological assessment of the early gestational sac in utero. In
particular, the membrane separating the exocoelomic and
amniotic cavities can now be clearly identified and CF can be
selectively aspirated from 5 weeks gestation (Figure 1). In 1991,
two independent teams, based at King’s College Hospital Medical
School (Jauniaux et al., 1991) and St Bartholomew’s Hospital
Medical College (Wathen et al., 1991), reported what they
believed were the first data on the biochemistry of the
extraembryonic coelom. However, in 1958, McKay et al. had
already published a study on the protein content of the coelomic
and amniotic fluids of five normal first-trimester pregnancies,
obtained during hysterotomy (McKay et al., 1958). Although the
number of samples studied was extremely small, the total protein
concentration found by these authors in chorionic or CF was very
similar to that found in our studies, >30 years later (Jauniaux et al.,
1991, 1993; Gulbis et al., 1992).
*To whom correspondence should be addressed at The Academic Department of Obstetrics and Gynaecology, University College London, 86–96 Chenies Mews,
London WC1E 6HX, UK. Phone: +44 207 2096057; Fax: +44 207 3837429; E-mail: [email protected]
Embryonic fluid compartments
269
Figure 1. Coelomic fluid aspiration procedure. (A) The exocoelomic fluid cavity (EEC) is located by means of transvaginal ultrasound. (B–D) The needle (20-gauge)
is inserted between the placenta (P) and the amniotic cavity (AC); the procedure takes between 30 s and 1 min. YS = yolk sac; D = decidua; M = maternal tissue.
Coelocentesis has a success rate of >95% at 6–11 weeks
gestation. In theory, it is the ideal alternative to early
amniocentesis and chorion villous sampling (CVS), because the
risk of directly injuring the growing embryo or damaging its
placenta is very low (Jurkovic et al., 1993). Furthermore, the
procedure is easy to learn, induces only minimal discomfort to the
mother and is associated with a very low rate of contamination of
the sample by maternal cells (Jurkovic et al., 1995). However, the
only study, so far, evaluating the safety of CF aspiration in
ongoing pregnancies, has shown that the risk of miscarriage after
coelocentesis is ~25% (Ross et al., 1997). This finding and the
high failure rate of cell growth from CF currently limits the
application of coelocentesis to exploring the biology of materno–
embryonic exchanges at a time of gestation when fetal blood can
not be obtained. The main findings of these studies and their
contribution to our understanding of embryonic physiology are the
basis of this review.
Embryology of the human placenta and adnexae
The formation of the placenta begins 13–15 days after ovulation,
corresponding to stage 6 of embryonic development and to the end
of the fourth week after the last menstrual period (Boyd and
Hamilton, 1970). The primary villi are composed of a central mass
of cytotrophoblast surrounded by a thick layer of
syncytiotrophoblast. During the following week of gestation, they
acquire a central mesenchymal core from the extraembryonic
mesoderm and become branched, forming the secondary villi. The
appearance of embryonic blood vessels within the mesenchymal
core transforms the secondary villi into tertiary villi. At the end of
the fifth gestational week, all three primitive types of placental
villi can be found but tertiary villi progressively predominate. Up
to the 9–10th week post-menstruation, which corresponds to the
last week of the embryonic period (stages 19–23), villi cover the
entire surface of the chorionic sac (Figure 2). As the gestational
sac grows during fetal life, the villi associated with the decidua
capsularis (surrounding the amniotic sac) degenerate to form the
chorion laeve, whereas the villi associated with the decidua basalis
proliferate, forming the chorion frondosum or definitive placenta
(Jauniaux et al., 1992).
The extraembryonic coelom or exocoelomic cavity develops
during the fourth week after the last menstrual period (Boyd and
Hamilton, 1970). It surrounds the blastocyst which is composed of
two cavities separated by the bilaminar embryonic disk, i.e. the
amniotic cavity and the primary yolk sac (Figure 3). At the end of
the fourth week of gestation, the developing exocoelomic cavity
270
E.Jauniaux and B.Gulbis
Figure 2. Diagrams showing the different anatomical barriers inside the first
trimester gestational sacs. U = uterus; P = placenta; UC = umbilical cord;
ECC = exocoelomic cavity; SYS = secondary yolk sac; AC = amniotic cavity;
AM = amniotic membrane.
placental chorionic plate at the end of the first trimester (Boyd and
Hamilton, 1970). The amniotic cavity is smaller than the
exocoelomic cavity up to 9 weeks gestation. Thus, during the
second and third month of pregnancy, the embryo and
subsequently the fetus are surrounded by the amniotic cavity
which is, in turn, surrounded by the exocoelomic cavity
containing the secondary yolk sac.
The secondary yolk sac is an independent organ floating inside
the exocoelomic cavity (Figure 3). It forms at the beginning of the
fifth week post-menstruation and develops rapidly so that by the
37th menstrual day it is larger than the amniotic cavity (Boyd and
Hamilton, 1970). From the sixth week of gestation it appears as a
spherical and cystic structure covered by numerous superficial
small vessels merging at the basis of the vitelline duct. This
connects the yolk sac to the ventral part of the embryo, the gut and
main blood circulation (Figure 4). The wall of the secondary yolk
sac is formed by an external mesothelial layer facing the
extraembryonic coelom, a vascular mesenchyme and an
endodermal layer facing the yolk sac cavity. The extraembryonic
human circulation is first established within the vitelline duct
artery via the dorsal aorta (Jones and Jauniaux, 1995). During the
10th week of gestation the yolk sac starts to degenerate and
rapidly ceases to function (Jones and Jauniaux, 1995).
Embryonic physiology
Figure 3. Schematic representations of human pregnancies at the beginning
(A) and at the end (B) of the 4th menstrual week and during the fifth (C) and
the sixth (D) menstrual week.
splits the extraembryonic mesoderm into two layers, the somatic
mesoderm, lining the trophoblast and the splanchnic mesoderm
covering the secondary yolk sac and the embryo (Figure 3). There
is no anatomical barrier between the mesenchyme of the placental
fetal plate and the exocoelomic or chorionic cavity (Jones and
Jauniaux, 1995). At ~31 days menstrual age, the gestational sac is
2–3 mm in diameter and can be detected by means of transvaginal
ultrasound imaging.
The amniotic cavity develops during the third week of
pregnancy from the inner cell mass of the implanted blastocyst
and grows inside the extraembryonic coelom fusing with the
Human embryonic physiology and developmental biology are
treated only incidentally in classical embryology textbooks and
have been overshadowed by the extraordinary profusion of
anatomical descriptions of embryos and early fetuses (Jauniaux
and Gulbis, 1997). Over the last decade, developmental biology
has become one of the leading fields of fundamental research.
However, the developmental physiology of most embryonic
organs remains largely unknown because of the limited access to
these organs for in-vivo and in-vitro experimentation. Since the
placenta and its membranes are larger than the fetus up to midpregnancy and, therefore, more accessible for research, it is not
surprising that most of our knowledge on the physiology of the
embryo and early fetus is essentially that of its adnexae which
provide the habitat in which the embryo and its functions develop.
Human placentation is theoretically haemochorial and mainly
characterized by diffuse infiltration by extravillous trophoblastic
cells of the uterine endometrium and superficial myometrium.
Classically, immediately after implantation, a number of
endometrial vessels are opened by the phagocytic activity of the
extravillous trophoblastic cells and the maternal circulation starts
in the intervillous space (Ramsey and Donner, 1980). This dogma
has been challenged by the data of Hustin et al. which show that
during the first trimester of pregnancy, the intervillous space of
the definitive placenta is separated from the uterine circulation by
trophoblastic plugs obliterating the tip of the uteroplacental
arteries (Hustin and Schaaps, 1987; Hustin et al., 1988). At the
end of the first trimester, these plugs are progressively dislocated
allowing maternal blood to flow freely and continuously into the
intervillous space.
In-vivo measurements of oxygen concentrations in early human
pregnancy have shown that the placental oxygen pressure is 2–3
times lower at 8–10 weeks than after 12 weeks (Rodesch et al.,
Embryonic fluid compartments
271
Figure 4. Photograph of an embryo and its yolk sac at 8 + 4 weeks gestation. The yolk sac shows a honeycomb pattern and is covered by numerous small vessels
merging at the basis of the vitelline duct.
1992). Furthermore, there is mounting evidence that early
placental trophoblast function and development is influenced by
oxygen tension (Genbacev et al., 1996, 1997). The results of our
recent experiments on trophoblast antioxidant defence are
consistent with the proposal that the placenta develops in a
physiologically low oxygen environment during the early part of
gestation (Watson et al., 1997, 1998a,b). The investigation of the
effect of oxygen metabolites on embryonic development is likely
to become an important field of research during the next decade
(Umaoka et al., 1992; Parman et al., 1999).
Biology of the extraembryonic coelom
Coelomic fluid (CF) has a lower pH, base excess and bicarbonate
value than maternal venous blood and has a higher carbon dioxide
pressure, higher lactate and phosphate concentrations and lower
protein concentrations than the maternal serum. These findings
are consistent with a metabolic anaerobic acidosis status which is
mainly due to the accumulation of acidic byproducts from the
placental metabolism in the exocoelomic cavity (Jauniaux et al.,
1994a). As a consequence of respiratory alkalosis of pregnancy,
maternal renal excretion of bicarbonate secondarily increases and
the overall maternal blood pH remains relatively unchanged
(Blackburn and Loper, 1992). Except for the total protein and
lactate concentrations, there is no significant variation in the
coelomic fluid biochemical composition at 7–11 weeks gestation
and no correlation of the different variables between coelomic
fluid and maternal serum, suggesting that the coelomic fluid acid–
base regulation is not directly influenced by maternal
physiological changes.
For the duration of the first trimester, the CF is yellow coloured
and more viscous than the AF, which is always clear. This is
mainly due to a higher protein concentration in CF than in AF. We
found that at 6–12 weeks gestation, the mean total protein
concentration is 18 times lower in the CF than in maternal serum
but 54 times higher in CF than in AF (Jauniaux et al., 1993). In
fact, the concentration of almost every protein is higher in CF than
in AF, ranging between 2 and 50 times depending on the
corresponding molecular weight of the protein investigated
(Table I). Small molecules such as urea can easily cross most
plasma membrane or tissue and it is, therefore, not surprising that
no significant difference was observed between urea
concentration in maternal serum and both embryonic
compartments (Table I). However, a gradient is observed for
slightly larger molecules, e.g. creatinine. These findings indicate
that transfer through the amniotic membrane separating the two
embryonic fluid cavities is limited.
There is an increase of most protein concentrations observed in
the exocoelomic cavity during the first trimester (Figure 5). This
can be explained by the slow turnover of the coelomic fluid and/or
the increased production of these proteins by fetal organs. In
contrast, there is a physiological decrease in total maternal serum
protein which occurs mainly during the first 3 months of gestation,
ranging from 10 to 14% of non-pregnant values (Blackburn and
Loper, 1992). During the first trimester, albumin concentration in
maternal serum demonstrates a relative decrease due to increased
maternal blood volume and haemodilution, while globulin and
fibrinogen concentrations demonstrate both absolute and relative
increases. During the same period, the amounts of total protein
and pre-albumin, increase in CF whereas the amounts of albumin
do not change significantly (Jauniaux et al., 1994b). There is no
difference in crown–rump length (CRL), yolk sac volume and the
concentration of protein in the CF between mothers with low
serum pre-albumin concentrations and mothers with high serum
272
E.Jauniaux and B.Gulbis
Table I. Concentration of various proteins and other molecules in embryonic fluids and maternal
serum according to their main site of production or origin during the first trimester of pregnancy
Molecules
Maternal serum
Coelomic fluid
Amniotic fluid
Total protein (g/l)a
71.3
3.5
0.2
Creatinine (µmol/l)a
50.1
43.6
27.7
Urea (mmol/l)a
7.2
8.3
7.2
Albumin (g/l)b
45.5
1.7
ND
Pre-albumin (g/l)b
1.14
0.04
ND
Tyroxine (nmol/l)c
180
0.9
0.02
Relaxin (ng/l)d
1000
122
9
Immunoglobulin G (mg/dl)e
907
32
3
e
ND
Mother
122
1
Complement factors 3 (mg/dl)e
114
ND
ND
Complement factors 4 (mg/dl)e
21
ND
ND
Iron (µmol/l) f
21
4.8
1.8
Glucose (mmol/l)g
3.4
2.7
2.8
IGF-I (µg/l)h
233
41
38
1057
Immunoglobulin A (mg/dl)
Villous tissue
Intact HCG (mIU/ml)i
80193
105605
(mIU/ml)i
70
11200
169
Free β-HCG (mIU/ml)i
45
1478
20
hPL (ng/ml)j
210
80
30
Progesterone (pg/ml)k
17
240
8
Oestradiol (pg/ml)k
917
8469
1898
Activin A (ng/ml)l
0.68
0.98
0.09
Inhibin B (pg/ml)l
5.9
24.3
6.3
β2-microglobulin (mg/l)m
0.9
4.7
N/D
0.3
0.6
0.9
687
199
40
Vitamin B12 (ng/l)n
405
3680
987
Prolactin (mIU/l)
709
371
40
Placental protein 14 (µ g/l)j
642
4416
77
Interleukin-6 (ng/ml)p
40
88
17
IGFBP-1 (µg/l)h
76
150
16
123
167
49
AFP (kIU/l)k
1.4
21816
27096
Erythropoietin (mIU/ml)q
15.4
15.5
5.0
τ-glutamyltransferase (IU/l)m
9
2
25
Ferritin (µg/l)f
49
287
2.0
Cancer antigen 125 (IU/ml)r
35
35
496
Free α-HCG
Lactate (mmol/l)
g
IGF-II (µg/l)h
Decidua
IGFBP-2 (µg/l)
h
Secondary yolk sac
Embryo/fetus
ND = not detectable; IGF = insulin-like growth factors; IGFBP = insulin-like growth factor binding
proteins; HCG = human chorionic gonadotrophin; AFP = α-fetoprotein.
a
Mean value (Jauniaux et al., 1991); bmean value (Jauniaux et al., 1994b); cmean value
(Contempre et al., 1993); dmedian value (Johnson et al., 1994); emedian value (Jauniaux et al.,
1995a); fmedian value (Gulbis et al., 1994); gmean value (Jauniaux et al., 1994a); hmean value
(Miell et al., 1997); imean value (Jauniaux et al., 1995b); jmedian value (Wathen et al., 1992);
kmean value (Jauniaux et al., 1993); lmedian value (Luisi et al., 1998); mmedian value (Gulbis
et al., 1996); nmedian value (Campbell et al., 1992a); omedian value (Wathen et al., 1993);
pmedian value (Jauniaux et al., 1996a); qmedian value (Campbell et al. , 1992b); rmedian value
(Campbell et al., 1992c).
Embryonic fluid compartments
Figure 5. Diagram showing the changes of total protein concentration with
gestational age in coelomic fluid (squares) and amniotic fluid (triangles). Note
the linear increase in coelomic fluid concentration at 8–12 weeks and the
exponential increase in amniotic fluid concentration after 12 weeks.
pre-albumin concentrations. These results suggest that the total
protein concentration in CF is not directly influenced by changes
in maternal serum protein concentrations during the first trimester.
Fetal growth and development are closely dependent on the
availability of a constant supply of amino acids from the mother
for protein synthesis (Blackburn and Loper, 1992). The
trophoblast functions of nutrient transport and protein synthesis
generate high concentrations of amino acids in the placenta and in
fetal blood during the second half of pregnancy (Sibley and Boyd,
1992). Significant positive relationships between maternal serum
and placental tissue are found for several amino acids indicating
that active amino acid transport and accumulation by the human
syncytiotrophoblast occurs as early as 7 weeks gestation (Jauniaux
et al., 1998a). The transplacental flux of most amino acid transport
from the maternal blood to the exocoelomic cavity is against a
concentration gradient (Jauniaux et al., 1994c). The concentration
distribution of individual amino acids in coelomic and amniotic
fluid are related indicating a passive transfer through the amniotic
membrane for these small molecules. A coelomic–maternal
gradient is observed for most amino acids measured and positive
correlations are found between maternal serum and CF for
concentration of α-aminobutyric acid, tyrosine and histidine
suggesting that these amino acids are only partially retained and/
or are transferred more rapidly by the early placenta.
Molecules such as thyroid hormones, immunoglobulins (Ig),
complement factors, relaxin or iron are not synthesized by the
feto–placental unit during the first trimester but play an essential
role in fetal development. These molecules are detectable in CF
indicating materno–embryonic transfer, probably from when the
tertiary placental villi are formed. In particular, thyroxine (T4) and
3′,5′,3-triiodothyronine (rT3) have been found in CF samples
suggesting that maternal thyroid hormones are potentially
available to the embryo as early as 5 weeks gestation (Contempre
et al., 1993). Another example are the immunoglobulins; trace
values of IgG and IgM have only been found in cultures of human
fetal liver and spleen from the end of the third trimester and IgA
synthesis has not been demonstrated in vitro until after 30 weeks
gestation (Gitlin and Biasucci, 1969). Only very low amounts of
IgG and IgM can be detected in the plasma of 12–14 week fetuses
(Gitlin, 1984). The concentration of IgG, including IgG specific
273
against Toxoplasma gondii, cytomegalovirus and rubella virus and
IgA values are measurable in CF samples from 6 weeks gestation
whereas IgM is not (Jauniaux et al., 1995a). This suggests that the
placental transfer of IgG and IgA begins very early in pregnancy.
IgG and IgA molecules in CF could play a role in the initial
antigenic challenge of blood cell precursors to early congenital
infection and in limiting the potentially devastating effects of
congenital infections. Using the same methodology, placental iron
transfer has been demonstrated from 7 weeks gestation and iron
was found to accumulate in the exocoelomic cavity (Gulbis et al.,
1994). The distribution of iron and iron-binding proteins between
the maternal and embryo–placental compartments in the first
trimester is comparable with that found later in gestation
(Figure 6).
The trophoblast produces a variety of specific proteins such as
human chorionic gonadotrophin (HCG), human placental lactogen
(hPL), activin A or inhibin which are excreted in both maternal
and embryonic fluid compartments (Table I). The higher
concentrations of these molecules found in CF compared with
maternal serum can be explained by the close anatomical
relationship existing between the exocoelomic cavity and the
trophoblast as both structures are only separated by the loose
mesenchymal tissue of the chorionic plate (Jones and Jauniaux,
1995). An exception to this principle is pregnancy-associated
protein A (PAPP-A) which is theoretically synthesized by the
villous tissue but found in higher concentration in maternal serum
than in CF (Iles et al., 1994). Free α-HCG and free β-HCG
concentrations are 185 and 33 times higher respectively in the CF
of normal pregnancy than in the corresponding maternal serum
samples (Jauniaux et al., 1995b). This finding supports the
hypothesis that, in the first trimester, there is an excess of α- over
β-subunit secretion by the villous trophoblast (Nagy et al., 1994)
and confirms that the HCG clearance rate is slower in the
exocoelomic cavity than in maternal circulation.
It is likely that maternal serum HCG concentrations are
influenced by both villous and extravillous trophoblastic synthesis
whereas (the exocoelomic cavity being completely surrounded by
villous tissue), coelomic HCG concentrations are only influenced
by villous trophoblastic secretion. In view of the large amount of
free α-HCG that is present in CF and the observation that free αHCG can stimulate decidual prolactin secretion in vitro (Blithe
and Iles, 1995), it is likely that this high concentration of placental
protein in CF has a regulatory effect on the endocrine function of
the materno–placental interface. In contrast to maternal serum, CF
concentrations of intact HCG and free α-HCG decrease
progressively at 8–12 weeks gestation and free β-HCG values do
not vary (Jauniaux et al., 1995b). Oestradiol and progesterone
concentrations also decrease in coelomic fluid between the second
and the third month of gestation whereas the opposite was true in
maternal serum. These changes in CF hormonal concentrations
are probably secondary to a decrease in the exchange surface, as
two thirds of the primitive placental ring start to degenerate during
the third month of gestation (Jauniaux et al., 1993). The decrease
in intact HCG and free α-HCG coelomic values with advancing
gestation may also be secondary to a simultaneous decline in the
number of differentiating cytotrophoblastic cells and/or to the
disappearance of two thirds of the original placental tissue which
takes place during the same period.
274
E.Jauniaux and B.Gulbis
Figure 6. Distribution of iron and iron-binding proteins between the maternal and embryo–placental compartments during the first trimester of pregnancy
(modified from Gulbis et al., 1994). MS = maternal serum; ECF = extracoelomic fluid; AF = amniotic fluid. T bars indicate SD.
Molecules, e.g. vitamin B12, prolactin and placental protein 14
(PP14), are known to be mainly produced by the uterine decidua
(Table I). They are often found at higher concentrations in CF than
in maternal serum, suggesting preferential pathways between the
decidual tissue and the embryonic fluid cavities via the villous
trophoblast. This mechanism may be a pivotal in providing the
developing embryo with sufficient nutrient before the intervillous
circulation becomes established. Molecules such as insulin-like
growth factors (IGFs) and their binding proteins (IGFBPs) are
also important in fetal growth and are produced by various
maternal and fetal tissues. IGF-I and II concentrations are highest
in maternal serum and low in CF and AF (Miell et al., 1997).
IGFBP-1 concentrations are higher in CF than either maternal
serum or AF and show a significant correlation to gestational age.
Analysis of IGFBP-1 phosphoforms show clear differences in
phosphorylation of IGFBP-1 between compartments with
maternal serum containing predominantly the phosphorylated
forms and CF almost exclusively the non-phosphorylated forms.
These findings suggest that the high IGF-II concentrations and
lack of inhibitory phosphoforms of IGFBP-1 in CF could
potentially enhance mitogenic activity in the early human
gestational sac (Miell et al., 1997).
The endodermal layer of the secondary yolk sac is known to
synthesize several serum proteins in common with the fetal liver,
e.g. α-fetoprotein (AFP), α1-antitrypsin, albumin, pre-albumin
and transferrin (Gitlin and Perricelli, 1970; Shi et al., 1985). With
rare exceptions, the synthesis of most of these proteins is confined
to embryonic compartments and the contribution of the yolk sac to
the maternal protein pool is limited. This can explain why their
concentration is always higher in embryonic fluids than in
maternal serum (Tables I and II). AFP is also produced by the
embryonic liver from 6 weeks until delivery, has a high molecular
Table II. Embryonic fluids and maternal blood gases, acid–base values
and electrolyte concentration (modified from Jauniaux et al., 1994)
Variable
Maternal blood
Coelomic fluid Amniotic fluid
pH
7.38
7.18
7.42
CO2 pressure (mmHg)
43
56
53
Base excess (mmol/l)
–2.6
–7.8
10.2
Bicarbonate (mmol/l)
22
18
38
Chloride (mmol/l)
105
105
92
Potassium (mmol/l)
4.0
3.9
3.6
Sodium (mmol/l)
134
134
131
Phosphate (mmol/l)
1.01
2.1
0.71
weight (±70 kDa) and conversely to HCG was found in similar
amounts on both sides of the amniotic membrane (Table I).
Analysis of concanavalin A affinity molecular variants of AFP
have demonstrated that both exocoelomic fluid and amniotic fluid
AFP molecules originated mainly from albumin the yolk sac while
maternal serum AFP molecules came mainly from the fetal liver
(Jauniaux et al., 1993). These results suggest that the human
secondary yolk sac also has an excretory function and secretes
AFP into the embryonic and extraembryonic compartments
(Figure 7).
The potential absorptive role of the yolk sac membrane has been
recently evaluated by examining the distribution of proteins and
enzymes between CF and yolk sac fluid and by comparing the
synthesising capacity of secondary yolk sac, fetal liver and
placenta for HCG and AFP (Gulbis et al., 1998). The distribution
Embryonic fluid compartments
Figure 7. Percentage of Con A non-reactive α-fetoprotein (AFP) [% Con A(–)]
from exocoelomic fluid (ECF), amniotic fluid (AF), maternal serum (MS), fetal
liver, and yolk sac collected during the first trimester of gestation (From Jauniaux
et al., 1993).
of the placental-specific protein, HCG, in yolk sac fluid and CF
and the absence of HCG mRNA expression in yolk sac tissue has
provided the first biological evidence of its absorptive function
(Figure 8). Similarities in the composition of the yolk sac and CF
fluid suggest that there is a free transfer for most molecules
between the two compartments through the layers of the dividing
wall. The yolk sac lumen contains digestive enzymes which were
not found inside CF but are present (in increasing concentrations
as pregnancy advances) in the amniotic cavity. These findings
suggest that the yolk sac membrane is an important zone of
transfer between the extraembryonic and embryonic
compartments and that the main flux of molecules occurs from
outside the yolk sac, i.e. from the exocoelomic in the direction to
the lumen and subsequently to the embryonic gut and circulation.
When, after 10 weeks gestation, the cellular components of the
wall of the secondary yolk start to degenerate, this route of transfer
is no longer functional and most exchanges between the
exocoelomic cavity and the fetal circulation must then take place
at the level of the chorionic plate.
Biology of the early amniotic cavity
During the first trimester, the amniotic membrane floats freely
between the embryonic cavities. Despite its apparent simplicity
(Jones and Jauniaux, 1995), direct transfer from the exocoelomic
to the amniotic cavity via the amniotic membrane is limited and
the AF contains very low protein concentrations (Gulbis et al.,
1992, Jauniaux et al., 1993). The total AF protein concentration is
50 and 900 times lower than in CF and maternal serum (Table I)
respectively. Almost all individual proteins, except AFP, are
present at very low concentrations in the AF. The vitelline duct
has the same cellular constitution as the secondary yolk sac (Jones
and Jauniaux, 1995). AFP and other yolk sac proteins found in the
early AF could be excreted at the level where the duct fuses with
the primitive umbilical cord. Yolk sac AFP could also be moved
in AF via the vitelline duct and from 10 weeks post-menstruation
when the anal membranes break down, intestinal AFP is also
found in AF (Boyd and Hamilton, 1970). During the second and
275
Figure 8. Expression of β-human chorionic gonadotrophin (β-HCG),
α-fetoprotein (αFP) and β-actin mRNAs in four series of placenta (P), yolk sac
(Y), and fetal liver (L) matched tissue samples. (1 = 8 weeks gestation; 2 = 9
ωεεκσ gestation; 3 = 7 weeks gestation; 4 = 12 weeks gestation) (from Gulbis
et al., 1998).
third trimester, the pool of AF is subject to a constant turnover,
with the accumulation of fetal lung fluid and urine and removal by
fetal swallowing (MacCarthy and Saunders, 1978). When the
definitive placenta has formed, movement of water and
electrolytes also takes place across the free placental membranes.
AF samples, collected before 11 weeks gestation, have a higher
pH and base excess, higher concentrations of lactate and
bicarbonate and lower concentrations of total protein, phosphate,
chloride, sodium and potassium than the CF collected during the
same period of gestation (Jauniaux et al., 1994a). Of these
biological parameters, only the pH and the bicarbonate levels
varied at 7–11 weeks gestation (Table II). The metabolic alkalosis
of first-trimester AF probably results from the accumulation of
bicarbonate and the increased consumption of organic anions such
as lactate by the embryonic tissue. The embryonic skin, which
only becomes keratinized during the second trimester, is probably
the major source of AF in early pregnancy (MacCarthy and
Saunders, 1978). These results indicate that in contrast to the CF
composition which is mainly influenced by placental and yolk sac
bioproducts, the first trimester AF composition is mainly
influenced by fetal bioproducts which may diffuse through the
fetal skin or through the oropharyngeal and cloacal membranes
(Jauniaux et al., 1994a; Gulbis et al., 1996). The latter rupture
around the end of the fifth and seventh week of gestation
respectively, allowing free circulation between fetal digestive and
respiratory tracts and the amniotic cavity (O’Rahilly and Muller
1992).
Fetal urine is a major source of AF in the latter half of
pregnancy. AF electrolyte composition, protein patterns and acid–
base balance change rapidly at the end of the first trimester
(Gulbis et al., 1992, 1996; Jauniaux et al., 1993, 1994a). Various
mechanisms should be considered in order to explain these
changes, in particular, the metabolic activity of the definitive
kidneys, the lungs and the digestive tract. The development of
nephrons starts around the beginning of the third month of
276
E.Jauniaux and B.Gulbis
Table III. Mean concentration of various drugs in maternal serum and embryonic fluids 5–25 min after a
single i.v. bolus (Diazepam 0.1 mg/kg, Fentanyl 1.5 µg/kg, Propofol 3 mg/kg, Inulin 5 mg/kg) or after chronic
intake (Cotinine)
Drugs
Reference
Maternal serum
Coelomic fluid
Amniotic fluid
Diazepam (ng/ml)
Jauniaux et al., 1996b
189
6.9
7.4
Fentanyl (ng/ml)
Shannon et al., 1998
1.3
ND
1.1
Propofol (µg/ml)
Jauniaux et al., 1998b
1.96
ND
ND
Inulin (mg/ml)
Jauniaux et al., 1997b
6.9
5.1
3.0
Cotinine (ng/ml)
Jauniaux et al., 1999b
72
99
108
ND = not detectable.
gestation and, theoretically, the metanephros or definitive kidney
produces urine from 10 weeks onwards (O’Rahilly and Muller
1992). At ~11 weeks, we have observed an abrupt increase in
β2-microglobulin and τ-glutamyl transferase (τGT) AF
concentrations and it is possible that this reflects the maturation of
the fetal renal glomerular function (Gulbis et al., 1996). In
particular, β2-microglobulin concentrations found in the amniotic
cavity during the second trimester are linked with the
establishment of glomerular filtration in the definitive fetal
kidneys at a time when the tubular function is still immature. The
AF β2-microglobulin concentration decreases during the third
trimester as a consequence of the increasing reabsorption capacity
of proximal tubular cells. The very low activity of τGT in CF
suggests that the placental villi are not a main source of this
enzyme.
The changes observed in AF composition at the end of the first
trimester are characterized by a decrease in pH, base excess and
bicarbonate values and an increase in carbon dioxide pressure and
chloride concentrations. It is of interest that, except for the total
protein values which remained lower, the mean value of the other
amniotic biological parameters obtained at >11 weeks gestation
were similar to those found in CF at <11 weeks (Jauniaux et al.
1994a). Similar changes occur during the second and third
trimesters (Sinha and Carlton, 1970) and probably reflect the
increasing contribution of the different fetal organs to AF
composition.
Prospects in the investigation of the biology of
embryonic fluids
The importance of the materno–embryonic exchange system in
humans has previously been difficult to assess, due to the
difficulties involved in accessing embryonic fluid or blood.
Investigation of the various biochemical constituents in
embryonic fluids have demonstrated that the exocoelomic cavity
is not a virtual space, and that it is the site of important molecular
exchanges between the mother and the embryo. Thus the study of
the different molecular concentrations in embryonic fluid has
provided useful information for understanding the transfer
pathways between the mother and the embryo (Table I). However,
these studies were purely descriptive and more studies comparing
the CF concentrations of a molecule with its quantifiable presence
in decidual, placental and fetal tissues (Gulbis et al., 1994, 1998;
Jauniaux et al., 1996a; 1998b; Riley, 1999) are needed.
CF can be collected in cases of early pregnancy failure. The CF
concentration of total protein is extremely low in all missed
abortions with advanced trophoblastic necrosis, whereas the HCG
concentration is low only when there are no embryonic remnants
on ultrasound (Jauniaux et al., 1995c). Normal or high maternal
serum AFP values and AFP molecules (predominantly of yolk sac
origin in the CF of pregnancies with an empty gestational sac on
ultrasound) provide further evidence that the most likely
explanation for this feature is the early death of the embryo with
persistence of the placental tissue (Jauniaux et al., 1995c). Thus,
the vast majority of pregnancies traditionally classified as
anembryonic gestation, in fact result from early embryonic
demise. The embryo having developed for at least 14 days after
ovulation corresponds to the stage of embryonic life when the
secondary yolk sac starts to form. Similar studies are needed to
better understand the pathophysiology of early pregnancy failure.
Placental physiology is notoriously difficult to study in vivo.
In-vitro models have been extensively used and most information
has been obtained from the placenta at term. The presence of the
exocoelomic cavity between the placenta and the amniotic sac
containing the fetus, the disappearance of two thirds of the
placental tissue mass and the major changes in both maternal and
fetal circulations at the end of the first trimester, make
comparisons between first and third trimester placental
physiology almost impossible. The unique anatomical position of
the exocoelomic cavity, which in primates is in direct contact with
the mesenchyme of the placental villi, opens new possibilities to
perform in vivo, physiological experiments traditionally
performed in vitro. Catheters (Ward et al., 1998) and sensors
(Jauniaux et al., 1999a) can be inserted, under ultrasound
guidance, and used to gain some insight into in-vivo first-trimester
placental physiology.
Placental transfers have been mainly studied in experimental
animals such as guinea pigs and monkeys because they have
haemochorial placentas similar to those of humans (Burton,
1992). In lower vertebrates, the yolk sac serves as the principal
membrane for placental exchanges. In particular, rodents have a
subsidiary yolk sac placenta which completely envelops the fetus
throughout the whole gestation and serves as the principal site for
the acquisition of protein by the fetus. Due to the complexity of
the experimental situation in the intact animal and the
Embryonic fluid compartments
considerable embryological differences between human and some
animal species, the precise mechanism of transfer of many
substances across the human placenta has not been elucidated.
However, the selective sampling of fluid from the exocoelomic
cavity has also offered a novel approach to the study of drug
transfer across the early human placenta (Table III). So far, these
studies have demonstrated that the permeability of the placenta is
greater in early pregnancy than at term but also because of the
slow turnover of CF substances to which the mother is chronically
exposed are likely to accumulate inside the exocoelomic cavity
(Jauniaux and Gulbis, 1998). This prolonged exposure to toxins
such as tobacco carcinogens (Jauniaux et al., 1999b,c) has
important teratological implications and should be further
explored.
References
Blackburn, S.T. and Loper, D.L. (1992) Maternal, Fetal and Neonatal
Physiology: A Clinical Perspective. Saunders, Philadelphia, USA.
Blithe, D.L. and Iles, R.K. (1995) The role of glycosylation in regulating the
glycoprotein hormone free alpha-subunit and free beta-subunit
combination in the extraembryonic coelomic fluid of early pregnancy.
Endocrinology, 136, 903–910.
Boyd, J.D. and Hamilton, W.J. (1970) The Human Placenta. Heffer,
Cambridge, UK.
Burton, G.J. (1992) Human and animal models: Limitations and comparisons.
In Barnea, E., Hustin, J. and Jauniaux, E. (eds), The First Twelve Weeks of
Gestation. Springer-Verlag, Heidelberg, Germany, pp. 469–485.
Campbell, J., Wathen, N., Perry, G. et al. (1992a) The coelomic cavity: an
important site of materno–fetal nutrient exchange in the first trimester of
pregnancy. Br. J. Obstet. Gynaecol., 100, 765–767.
Campbell, J., Wathen, N., Lewis, M. et al. (1992b) Erythropoietin levels in
amniotic fluid and extraembryonic coelomic fluid in the first trimester of
pregnancy. Br. J. Obstet. Gynaecol., 9, 974–976.
Campbell, J., Kitau, M., Cass, P. et al. (1992c) CA 125 in matched samples of
amniotic fluid, extra-embryonic coelomic fluid and maternal serum in the
first trimester of pregnancy. J. Obstet. Gynaecol., 100, 563–565.
Contempre, B., Jauniaux, E., Calvo, R. et al. (1993) Detection of thyroid
hormones in human embryonic cavities during the first trimester of
pregnancy. J. Clin. Endocrinol. Metab., 77, 1719–1722.
Genbacev, O., Joslin, R., Damsky, C.H. et al. (1996) Hypoxia alters early
gestation human cytotrophoblast differenciation/invasion in vitro and
models the placental defects that occur in preeclampsia. J. Clin. Invest.,
97, 540–550.
Genbacev, O., Zhou, Y., Ludlow, J.W. and Fisher, S.J. (1997) Regulation of
human placental development by oxygen tension. Science, 277, 1669–
1672.
Gitlin, D. (1984) Protein transport across the placenta and protein turnover
between amniotic fluid, maternal and fetal circulation. In Moghissi, K.S.
and Hafez, E.S.E. (eds), The Placenta. Charles C.Thomas, Springfield,
USA, pp. 151–72.
Gitlin, D. and Biasucci, A. (1969) Development of τG, τA, τM, β1C/β1a, C′1
esterase inhibitor, ceruloplasmin, haptoglobin, fibrinogen, plasminogen,
α1-antitrypsin, orosomucoid, β-lipoprotein, α2-macroglobulin and
prealbumin in the human conceptus. J. Clin. Invest., 48, 1433–1446.
Gitlin, D. and Perricelli, A. (1970) Synthesis of serum albumin, prealbumin,
alphafetoprotein, alpha1-antitrypsin and transferrin by the human yolk
sac. Nature, 228, 995–997.
Gulbis, B., Jauniaux, E., Jurkovic, D. et al. (1992) Determination of protein
pattern in embryonic cavities of early human pregnancies: A model to
understand materno–embryonic exchanges. Hum. Reprod., 7, 886–889.
Gulbis, B., Jauniaux, E., Decuyper, J. et al. (1994) Distribution of iron and
iron-binding proteins in first trimester human pregnancies. Obstet.
Gynecol., 84, 289–293.
Gulbis, B., Jauniaux, E., Gervy, C. et al. (1996) Biochemical investigation of
kidney functional maturation in early human pregnancy. Pediatr. Res., 39,
1–5.
Gulbis, B., Jauniaux, E., Cotton, F. and Stordeur, P. (1998) Protein and
enzyme patterns in the fluid cavities of the first trimester gestational sac:
277
relevance to the absorptive role of secondary yolk sac. Mol. Hum.
Reprod., 4, 857–862.
Hustin, J, and Schaaps, J.P. (1987) Echographic and anatomic studies of the
maternotrophoblastic border during the first trimester of pregnancy. Am.
J. Obstet. Gynecol., 157, 162–168.
Hustin, J., Schaaps, J.P., and Lambotte, R. (1988) Anatomical studies of the
utero–placental vascularization in the first trimester of pregnancy.
Trophoblast Res., 3, 49–60.
Iles, R.K., Wathen, N.C., Sharma, K.B. et al. (1994) Pregnancy-associated
plasma protein A levels in maternal serum, extraembryonic coelomic and
amniotic fluids in the first trimester. Placenta, 15, 693–699.
Jauniaux, E. and Gulbis, B. (1997) Embryonal physiology. In Jauniaux, E.,
Barnea, R., Edwards, R. (eds) Embryonic Medicine and Therapy. Oxford
University Press, Oxford, UK, pp. 223–243.
Jauniaux, E. and Gulbis, B. (1998) In-vivo study of placental drug transfer
during the first trimester of human pregnancy. Trophoblast Res., 12, 257–
264.
Jauniaux, E., Jurkovic, D., Gulbis, B. et al. (1991) Biochemical composition of
coelomic fluid in early human pregnancy. Obstet. Gynecol., 78, 1124–
1128.
Jauniaux, E., Burton, G.J. and Jones, C.P.J. (1992) Early human placental
morphology. In Barnea, E., Hustin, J. and Jauniaux, E. (eds), The First
Twelve Weeks of Gestation. Springer-Verlag, Heidelberg, Germany, pp.
45–64.
Jauniaux, E., Gulbis, B., Jurkovic, D. et al. (1993) Protein and steroid levels in
embryonic cavities of early human pregnancy. Hum. Reprod., 8, 782–787.
Jauniaux, E., Jurkovic, D., Gulbis, B. et al. (1994a) Investigation of the acid–
base balance of coelomic and amniotic fluids in early human pregnancy.
Am. J. Obstet. Gynecol., 170, 1359–1365.
Jauniaux, E., Gulbis, B., Jurkovic, D. et al. (1994b) Relationship between
protein levels in embryological fluids and maternal serum and yolk sac
size during early human pregnancy. Hum. Reprod., 9, 161–166.
Jauniaux, E., Sherwood, R., Jurkovic, D. et al. (1994c) Amino acids
concentration in human embryological fluids. Hum. Reprod., 9, 1175–
1179.
Jauniaux, E., Jurkovic, D., Gulbis, B. et al. (1995a) Materno–fetal
immunoglobulin transfer and passive immunity during the first trimester
of human pregnancy. Hum. Reprod., 10, 3297–3300.
Jauniaux, E., Gulbis, B., Nagy, A.M. et al. (1995b) Coelomic fluid chorionic
gonadotropin and protein levels in normal and complicated first trimester
human pregnancies. Hum. Reprod., 10, 214–220.
Jauniaux, E., Gulbis, B., Jurkovic, D. et al. (1995c) The origin of
alphafetoprotein in first trimester anembryonic pregnancies. Am. J.
Obstet. Gynecol., 173, 1749–1753.
Jauniaux, E., Gulbis, B., Schandene, L. et al. (1996a) Distribution of
interleukin-6 in maternal and embryonic tissues during the first trimester.
Mol. Hum. Reprod., 2, 239–243.
Jauniaux, E., Jurkovic, D., Lees, C. et al. (1996b) In vivo study of diazepam
transfer across the first trimester human placenta. Hum. Reprod., 11, 889–
892.
Jauniaux, E., Gulbis, B., Gerlo, E. and Rodeck, C.H. (1998a) Free amino acid
distribution in the first trimester human gestational sac. Early Hum. Dev.,
51, 159–169.
Jauniaux, E., Gulbis, B., Shannon, C. et al. (1998b) Placental propofol transfer
and fetal sedation during maternal general anaesthesia in early pregnancy.
Lancet, 352, 290–291.
Jauniaux, E., Watson, A., Ozturk, O. et al. (1999a) In vivo measurement of
intrauterine gases and acid–base values in early human pregnancy. Hum.
Reprod., 14, 2901–2904.
Jauniaux, E., Gulbis, B., Acharya, G. et al. (1999b) Maternal tobacco exposure
and cotinine levels in fetal fluids in the first half of pregnancy. Obstet.
Gynecol., 93, 25–29.
Jauniaux, E., Gulbis, B., Acharya, G. and Thiry, P. (1999c) Fetal amino acid
and enzymes levels with maternal smoking. Obstet. Gynecol., 93, 680–
683.
Jones, C.P.J. and Jauniaux, E. (1995) Ultrastucture of the materno–embryonic
interface in the first trimester of pregnancy. Micron, 2, 145–173.
Jurkovic, D., Jauniaux, E., Campbell, S. et al. (1993) Coelocentesis: a new
technique for early prenatal diagnosis. Lancet, 341, 1623–1624.
Jurkovic, D., Jauniaux, E., Campbell, S. et al. (1995) Detection of sickle gene
by coelocentesis in early pregnancy: A new approach to prenatal diagnosis
of single gene disorders. Hum. Reprod., 10, 1287–1289.
Johnson, M.R., Jauniaux, E., Jurkovic, D. et al. (1994) Relaxin levels in
embryonic fluids during the first trimester. Hum. Reprod., 9, 1561–1562.
278
E.Jauniaux and B.Gulbis
Johnell, H.E. and Nilsson, B.A. (1971) Oxygen tension, acid–base status and
electrolytes in human amniotic fluid. Acta Obstet. Gynecol. Scand., 50,
183–192.
Luisi, S., Battaglia, C., Florio, P. et al. (1998) Activin A and inhibin B in extraembryonic coelomic and amniotic fluids, and maternal serum in early
pregnancy. Placenta, 19, 435–438.
MacCarthy, T. and Saunders, P. (1978) The origin and circulation of the
amniotic fluid. In Fairweather, D.V.I. and Eskes, T.K.A.B. (eds),
Amniotic Fluid: Research and Clinical Application. 2nd edn. Excerpta
Medica, Amsterdam, The Netherlands, pp. 1–18.
McKay, D.J., Richardson, M.V. and Hertig, A.T. (1958) Studies of the
function of early human trophoblast: III. A study of the protein structure
of mole fluid, chorionic and amniotic fluids by paper electrophoresis. Am.
J. Obstet. Gynecol., 75, 699–707.
Miell, J.P., Jauniaux, E., Langford, K. et al. (1997) Insulin growth factor
binding proteins concentration and post translational modification in
embryological fluid. Mol. Hum. Reprod., 3, 343–349.
Nagy, A.M., Jauniaux, E., Jurkovic, D. and Meuris, S. (1994) Placental
overproduction of human chorionic gonadotropin α-subunit in early
pregnancy as evidenced in exocoelomic fluid. J. Endocrinol., 142, 511–
516.
O’Rahilly, R. and Muller, F. (1992) Human Embryology and Teratology.
Wiley-Liss, New York, USA.
Parman, T., Wiley, M.J. and Wells, P.G. (1999) Free radical-mediated
oxidative DNA damage in the mechanism of thalidomide teratogenicity.
Nature Med., 5, 582–585.
Ramsey, E.M. and Donner, N.W. (1980) Placental Vasculature and
Circulation. Georg Thieme, Stuttgart, Germany, 101 pp.
Riley, S.C., Leask, R., Chard, T. et al. (1999) Secretion of matrix
metalloproteinase-2, matrix metalloproteinase-9 and tissue inhibitor of
metalloproteinases into the intrauterine compartments during early
pregnancy. Mol. Hum. Reprod., 5, 376–381.
Rodesch, F., Simon, P., Donner, C. and Jauniaux, E. (1992) Oxygen
measurements in the maternotrophoblastic border during early pregnancy.
Obstet. Gynecol., 80, 283–285.
Ross, J., Jurkovic, D. and Nicolaides, K.H. (1997) Coelocentesis: a study of
short-term safety. Prenat. Diagn., 17, 913–917.
Shannon, C., Jauniaux, E., Gulbis, B. et al. (1998) Placental transfer of
fentanyl in early human pregnancy. Hum. Reprod., 13, 2317–2320.
Shi, W.K., Hopkins, B., Thompson, S. et al. (1985) Synthesis of
apolipoproteins, alphfoetoprotein, albumin and transferrin by the human
foetal yolk sac and other foetal organs. J. Embryol. Exp. Morphol., 85,
191–206.
Sibley, C.P. and Boyd, R.D.H. (1992) Mechanisms of transfer across the
human placenta. In Polin, R.A. and Fox, W.W. (eds), Fetal and Neonatal
Physiology. Vol. 1. Saunders, Philadelphia, USA, pp. 62–74.
Sinha, R. and Carlton, M. (1970) The volume and composition of amniotic
fluid in early pregnancy. J. Obstet. Gynaecol. Br. Commonw., 77, 211–
214.
Umaoka, Y., Noda, Y., Narimoto, K. and Mori, T. (1992) Effects of oxygen
toxicity on early development of mouse embryo. Mol. Reprod. Dev., 31,
28–33.
Ward, S., Jauniaux, E., Shannon, C. et al. (1998) Electrical potential difference
between exocoelomic fluid and maternal blood in early pregnancy. Am. J.
Physiol., 274, R1492–R1495.
Wathen, N.C., Cass, P.L., Kitau, M.J. and Chard, T. (1991) Human chorionic
gonadotrophin and alpha-fetoprotein levels in matched samples of
amniotic fluid, extraembryonic exocoelomic fluid, and maternal serum in
the first trimester of pregnancy. Prenat. Diagn., 11, 145–151.
Wathen, N.C., Cass, P.L., Campbell, J. et al. (1992) Levels of placental protein
14, human placental lactogen and unconjugated oestriol in
extraembryonic coelomic fluid. [Letter.] Placenta, 13, 195–197.
Wathen, N.C., Campbell, D.J., Patel, B. et al. (1993) Dynamics of prolactin in
amniotic fluid and extraembryonic coelomic fluid in early human
pregnancy. Early Hum. Dev., 35, 167–172.
Watson, A.L., Palmer, M.E., Jauniaux, E. and Burton, G.J. (1997) Variation in
expression of copper/zinc superoxide dismutase in villous trophoblast of
the human placenta with gestational age. Placenta, 18, 295–199.
Watson, A.L., Skepper, J.N., Jauniaux, E. and Burton, G.J. (1998a) Changes in
the concentration, localisation and activity of catalase within the human
placenta during early gestation. Placenta, 19, 27–34.
Watson, A.L., Palmer, M.E., Skepper, J.N. et al. (1998b) Susceptibility of
human placental syncytiotrophoblast mitochondria to oxygen-mediated
damage in relation to gestational age. J. Clin. Endocrinol. Metab., 83,
1697–1705.
Received on September 28, 1999; accepted on February 15, 2000