Chapter 115
Amniotic Fluid and the Fetal Mucosal
Immune System
Stella Nowicki
Department of Microbiology and Immunology, Meharry Medical College, Nashville, TN, USA
Randall M. Goldblum
Pediatric Child Health Research Center, University of Texas Medical Branch, Galveston, TX, USA
Chapter Outline
Introduction2251
Development of the Amniotic and Chorionic
Cavities and the Placenta 2252
Production of Chorionic and Amniotic Fluids 2254
Metabolism of Amniotic Fluid 2256
Immunological Factors in Amniotic Fluid 2256
Innate Immunity 2257
Complement and Complement Inhibitors 2257
Cytokines, Chemokines, and Other Modulatory and
Growth Factors 2258
Cytokines2259
INTRODUCTION
Amniotic fluid, the solution contained within the amniotic
membranes, surrounds the developing fetus from about
the sixth week of gestation until immediately before birth,
when the fluid is released. A major function of the amniotic
fluid is to protect the infant from physical trauma due to
forces that might be applied to the mother’s abdomen. A
less conspicuous but important function of amniotic fluid is
its role in protecting the fetus from microbial colonization.
This chapter reviews the development of the chorionic and
amniotic cavities and the synthesis, composition, and fate
of amniotic fluid. Evidence concerning the role of amni-
otic fluid in protecting the infant from immunological or
infectious injury will then be addressed. Furthermore, since
amniotic fluid is produced by or at least transferred across
epithelial surfaces and bathes many of the mucosal surfaces
of the fetus, it should be considered part of the mucosal
immune system. This important concept is also explored.
Early exposure of the fetal respiratory and gastrointestinal
tracts to amniotic fluid might help shape mucosal immune
Mucosal Immunology. http://dx.doi.org/10.1016/B978-0-12-415847-4.00115-4
Copyright © 2015 Elsevier Inc. All rights reserved.
Nonspecific Antimicrobial Factors 2260
Humoral Immunity 2261
Immunoglobulins and Immunoglobulin
Transporters2261
Increased Activity of the Immune System in
African Americans 2263
Pathological Conditions Involving Amniotic Fluid 2264
Chorioamnionitis2264
Fetal Growth Restriction 2265
Antiretroviral Drugs and Amniotic Fluid 2265
References2265
responses at these sites. In this respect, amniotic fluid might
be considered a precursor of human milk feedings as an
exogenous source of immunological factors that protect the
infant directly and shape the responses of the infant’s own
immune system. Conversely, since some of the volume and
perhaps the solute content of amniotic fluid are produced
by the mucosal surfaces of the fetus, it can also be consid-
ered an early product of the fetus’s mucosal immune sys-
tem. This chapter is largely limited to human amniotic fluid,
since there are major differences between the structure and
the function of hemochorial placentas of primates and those
of other mammals and since there are very few studies on
amniotic fluid in nonhuman primates.
Among the body fluids, amniotic fluid is perhaps one
of the least well studied. This may be due in part to the
limited opportunities to access this fluid. Amniotic fluid is
frequently sampled by amniocentesis for genetic analysis
of the fetus between 10 weeks and the end of the first half
of pregnancy. Fluid samples are also removed for studies on
fetal maturity, usually during the last 10 weeks of normal
2251
2252  SECTION | I  Genitourinary Tract and Mammary Gland
gestation. Finally, large amounts of fluid are available near
the time of delivery. However, the later samples have the
potential to be contaminated with maternal cervical and
vaginal fluid unless they are collected during cesarean sec-
tion or after insertion of monitoring devices into the amni-
otic cavity. Thus, studies on the development of various
constituents in the amniotic fluid often have temporal gaps
because of the unavailability of samples during some stages
of gestation.
The diagnostic usefulness of amniotic fluid became
broadly recognized when intrauterine treatments were
developed for isoimmune sensitization against the infant’s
rhesus antigens. Amniotic fluid has also been used as a win-
dow into the physiological events that lead up to both nor-
mal and premature parturition. The latter studies have often
focused on cytokines and prostaglandins in the amniotic
fluid that might also impact the development of the immune
system of the fetus.
DEVELOPMENT OF THE AMNIOTIC AND
CHORIONIC CAVITIES AND THE PLACENTA
The amniotic cavity begins its development during the
bilaminar germ disk stage, in the second week of gestation,
as a cleft within the ectoderm of the inner mass of embry-
onic cells (Langman, 1981). At first, the cavity is a simple
(a) Trophoblastic lacunae Maternal sinusoids
Amniotic
Endoderm
cavity
cells
Extra-embryonic
coelom
Extra-embryonic
splanchnopleuric
mesoderm
Extra-embroyonic
Exocoelomic cavity somatopleuric mesoderm
Exocoelomic
(primitive yolk sac)
membrane
FIGURE 1  Embryologic development of the chorionic and amniotic cavities. (a) Human blastocyst at about 12 days of gestation. The amniotic cav-
ity is developing between the columnar ectodermal plate and the small ectodermal cells migrating from the edges of the plate. (b) Human blastocyst at
about 13 days. The extraembryonic coelom has developed into the chorionic cavity, and the amniotic cavity has enlarged. Modified with permission from
Langman (1981).
cleft lined on one side by the ectodermal plate and on the
other side by cells (amnioblasts) that grow from the edge of
the ectodermal layer and migrate along the overlying cyto-
trophoblast layer. A similar process occurring on the ventral
or endodermal surface of the blastocyst results in formation
of the primitive yolk sac (Figure 1(a)). The chorionic cav-
ity, which develops shortly thereafter from extraembryonic
mesoderm, surrounds the developing embryo, the amniotic
cavity, and the secondary yolk sac by 13 days of gestation
(Figure 1(b)).
As the growing embryo causes the surface of the decidua
(modified uterine endometrium) to protrude into the uter-
ine cavity during the second month of gestation, the cho-
rionic and amniotic cavities are pushed closer together as
they approach from the opposite side of the uterine cavity
(Figure 2(a)). By the end of the third month of gestation,
the chorionic and amniotic membranes fuse with each other
(Figure 2(b)). The outer surface of the expanding chorionic
membrane also fuses to the surrounding decidua parietalis,
forming a single membrane which surrounds the growing
fetus. From the end of the third month to the end of ges-
tation, this fluid-filled chorioamniotic sac containing the
fetus fills the entire uterine cavity not occupied by the pla-
centa. At the cervical os, this membrane is modified in that
the chorioamniotic membrane is not fused to the decidua.
The fetal surface of the placenta and the outer surface
(b) Prochordal
plate
Primary stem
villi
Trophoblastic
lacunae
Maternal
sinusoid
Connecting
stalk
Amniotic
cavity
Secondary
yolk sac
Extra-embryonic
somatopleuric
mesoderm
(chorionic plate)
Extra-embryonic
coelom
(chorionic cavity
Exocoelomic cyst
 Amniotic Fluid and the Fetal Mucosal Immune System Chapter | 115  2253

of the umbilical cord are also covered by the amniotic mem-
brane. Thus, the chorioamniotic membrane, derived from
fetal mesoderm and ectoderm, forms a large area of inter-
face with maternal uterine decidua.
Detailed histological examination of the chorioamniotic
membranes at term (Danforth and Hull, 1958) revealed that
the amnion consisted of a single layer of epithelial cells,
superimposed and tightly attached to an underlying layer of
fibrous connective tissue about twice as thick as the cellular
layer. There is no organized basement membrane separating
these two layers of the amnion. The amniotic cells seem to
have a unique morphology that differs for those lining the
inner surface of the reflected membrane and those overly-
ing the fetal surface of the placenta. The amniotic epithelial
cells covering the placenta are more complex in appearance,
being columnar in shape and containing a canalicular appa-
ratus in the Golgi zone, secretory granules, and brush-type
borders at the basal surface (Danforth and Hull, 1958). The
latter are tightly applied to the underlying fibrous connec-
tive tissue. The epithelial cells of the reflected amniotic
membrane are cuboid, having fewer cellular elements and
a more mature appearance. Given the ectodermal origin,
antigenic similarity, and continuity between fetal skin and
amniotic epithelial cells, it is interesting to speculate that
the cells closest to the fetus are less mature but more meta-
bolically active than those that have migrated farther from
the fetus. The potential ability of amniotic epithelial cells to
secrete and/or transport water and immunologically active
agents will be considered later. In addition, the more cuboi-
dal amniotic epithelium of the reflected membranes may be
important in the more passive transport of fluid and solutes
to and from the maternal decidua through the chorionic
membrane.
The placenta is formed from the outer cell mass of the
embryo, which differentiates into individual cytotropho-
blasts and a multinucleate mass of syncytiotrophoblasts,
which invade the endometrial stroma after implantation. By
the ninth day of gestation, the syncytiotrophoblasts begin
to form intercommunicating clefts or trophoblastic lacunae.
The syncytiotrophoblasts go on to invade the uterine vessels,
allowing maternal blood to course through the lacunae and
establish the uteroplacental circulation. The cytotrophoblasts
then begin to form villi that protrude into these trophoblastic
lacunae. These villi are themselves invaded centrally by fetal
mesoderm, which differentiates into blood vessels by the
third week of gestation. Thus, the mature villi, or fingerlike
projections, which interface with maternal blood are formed
by three cellular layers: a syncytiotrophoblastic outer layer,

(a) (b)
Fused decidua parietalis, Placenta
chorion laeve and amnion
Decidua basalis

Chorion
Decidua frondosum
parietalis

Chorionic
cavity Amniotic
cavity

Yolk sac

Decidua
capsularis
Uterine
cavity

Chorion laeve
Amniotic cavity

FIGURE 2  Embryologic development of the fetus, placenta, and chorionic and amniotic cavities. (a) Human fetus at the end of the second month.
Note that the amniotic cavity is still separate, though within the chorionic cavity. (b) Human fetus at the end of the third month. Note that the chorionic
and amniotic cavities have now fused and that the uterine cavity is essentially obliterated by the fusion of the chorion laeve and decidua parietalis. The
small cavity that remains near the cervical os is a potential site for transport of maternal cervicovaginal secretions into the chorioamniotic sac. Modified
with permission from Langman (1981).
2254  SECTION | I  Genitourinary Tract and Mammary Gland
a cytotrophoblastic middle layer, and a mesodermal inner
or vascular layer. As the villi develop, they lengthen and
branch. The ends of these branches are the terminal villi. It
is these terminal chorionic villi, which protrude into the tro-
phoblastic lacunae filled with maternal blood, that form the
major anatomical site for the exchange of gases and nutri-
ents between the fetus and the mother. This is also a site
for active transport of immunological factors, such as IgG,
directly from the maternal to the fetal circulation without
transiting the amniotic cavity.
PRODUCTION OF CHORIONIC AND
AMNIOTIC FLUIDS
The sources of the numerous constituents of chorionic and
amniotic fluids throughout gestation are not known in detail.
Since the fluids in these compartments are in contact only
with fetal surfaces, the proximal sources are predominantly
fetal. However, maternal contributions can also reach these
spaces, either indirectly via the maternofetal circulation or
more directly by diffusion or active transport by the fetal
membranes of constituents that diffuse from the decidua or
intervillus spaces and across the chorionic plate of the pla-
centa. The potential sources of chorionic and amniotic fluids
thus include fetally derived constituents from the external
and mucosal surfaces of the fetus, including the definitive
yolk sac, the umbilical cord, and the fetal circulation to the
chorionic and amniotic membranes; maternally derived
constituents from the decidua that diffuse or are transported
across one or more fetal membranes into the cavities; and
the maternal blood in the intervillus spaces of the placenta,
across the overlying chorioamniotic membranes.
In the early stages of development, the amniotic cleft
must be filled with fluid produced by the fetal ectoder-
mal cells surrounding it (Figure 1(a)). However, as the
cleft enlarges into a cavity and the lining ectoderm thins,
it becomes possible for water or specific solutes to be
transported from the surrounding chorionic cavity into the
amniotic cavity (Figure 1(b)). Studies of the transport of
fluid and various solutes into and out of the chorionic and
amniotic cavities of humans are predominantly restricted to
the later period of gestation, when the single chorioamni-
otic sac becomes accessible by amniocentesis. Studies of
chronically cannulated fetuses in experimental animals,
predominantly sheep, have also provided data concerning
the production and metabolism of fetal fluids.
However, Jauniaux et al. (1994) selectively sampled the
chorionic and amniotic sacs of human fetuses under ultra-
sound guidance before elective abortions at 6–12 weeks of
gestation. They determined the total protein and albumin in
maternal serum, chorionic fluid, and amniotic fluid, as well
as the concentration of α-fetoprotein in maternal serum and
chorionic fluid. The total protein increased linearly in the
chorionic cavity and exponentially in the amniotic cavity
during the first 12 and 14 weeks of gestation, respectively.
These results also indicated a marked gradient of total
protein concentration between maternal blood and cho-
rionic fluid (18 times) and chorionic and amniotic fluids
(54 times), suggesting that the amniotic membrane is rela-
tively impermeable to proteins during the first trimester of
gestation. They concluded that the major sources of the low
concentration of proteins in the amniotic cavity during the
first half of gestation are the embryo and its yolk sac and,
subsequently, the fetus. However, the concentration of pro-
teins in the amniotic cavity increases dramatically at about
11 weeks of gestation. This is about the time of fusion of
the chorionic and amniotic membranes, which allows much
more rapid diffusion of solutes from the maternal decidua
across the fused fetal membranes (Jauniaux et al., 1994).
Jauniaux et al. (1995) subsequently measured IgG, IgA,
IgM, C3, C4, and specific IgG antibodies to potential intra-
uterine pathogens in the chorionic and amniotic fluids and in
the mother’s serum during the same early (6–12 weeks) ges-
tation period. These are all proteins that are either absent or
present in extremely low concentrations, in the early fetus.
Their assays for C3 and C4 were sensitive enough to detect
only concentrations greater than 10% of serum levels, elimi-
nating their usefulness in assessing the transport of these
proteins into the fetal cavities. However, data from Gitlin
et al. (1972) suggest that IgG and IgA, in amounts adequate
to achieve μg/mL concentrations, reach the chorionic sac
during early gestation by either size-dependent, passive dif-
fusion or active transport of selected molecules from the
maternal decidua parietalis or intervillus spaces of the pla-
centa. The difference between the maternal serum to chori-
onic fluid gradients for IgG (28-fold) and monomeric IgA
(128-fold) suggests some selectivity of transport for these
proteins of similar size and structure. However, the concen-
tration of both of these immunoglobulin classes in chorionic
fluid correlated with increasing gestational age and with
each other. No higher-molecular-weight IgM was detectable
in the chorionic fluid. Significantly, of the proteins quanti-
fied, only IgG (the analyte of highest concentration in the
chorionic fluid) was detected in the amniotic fluid, and in
only two of 34 fetuses. This is most consistent with limited
diffusion of these proteins from the chorionic fluid across the
amniotic membrane. Given the sensitivity of the assays for
IgG and IgA, a gradient of greater than 30-fold and 10-fold,
respectively, across the amniotic membrane would have pre-
cluded detection of these proteins in amniotic fluid at about
11 weeks of gestation. The sources and potential function of
the immunoglobulins and antibodies in the chorionic and
amniotic cavities are considered further later.
The volume of amniotic fluid increases steadily until
approximately 30 weeks, when it peaks at about 900 mL.
After that it begins to decrease, at first slowly but then more
rapidly after 35 weeks of gestation (Lotgering and ­Wallenburg,
1986). The rate at which water and various solutes flow
 Amniotic Fluid and the Fetal Mucosal Immune System Chapter | 115  2255
into and out of the amniotic fluid late in gestation has been
studied with radiolabeled and stable isotope clearance tech-
niques. Water and sodium are replaced quite rapidly: 34%
and 7% every hour, respectively (Vosburge et al., 1948).
Gitlin et al. (1972) injected several isolated radiolabeled
serum proteins into the amniotic cavity via amniocentesis.
They then measured the volume of amniotic fluid by iso-
tope dilution and the rate of clearance of the proteins by
obtaining fluid samples by repeat amniocentesis and at the
time of cesarean section. Their results indicated that near
term, the amniotic fluid volume was about 600 mL ±10%.
Approximately 2.6% of all the labeled proteins tested were
cleared per hour in the absence of labor. In the presence of
labor the apparent hourly clearance of proteins increased
to 3.7%. The role of the infant in protein clearance was
investigated by comparison with two pregnancies with dead
fetuses. The rate was reduced to 12% per day, or approxi-
mately 0.5% per hour. Gitlin’s results indicate that 60–90%
of amniotic fluid proteins are replenished each day. The
volume of maternal serum required to maintain the protein
levels in the fluid varied from 12 to 18 mL for albumin and
transferrin, respectively, to as little as 6.3 and 1.4 mL for
IgG and IgA, respectively. Whereas the differences between
the transfer of immunoglobulins and smaller serum proteins
might be explained by their concentrations in the decidua,
the differences might also suggest that the immunoglobu-
lins are actively transported into the chorioamniotic cavity
late in gestation.
Under the conditions of mass removal of amniotic fluid
(indicated by Gitlin et al., 1972), the rates of clearance of
all amniotic fluid proteins—and thus their rate of formation
(when the volumes and concentrations are stable)—can be
ascertained from the rate of disappearance of any labeled
protein and the endogenous concentration of the constitu-
ent of interest. Total protein accretion was found to be 0.79
and 1.06 g/day for patients prior to and during labor, respec-
tively (Gitlin et al., 1972). They also found that approxi-
mately one-half of the protein in amniotic fluid is albumin.
The number of potential sites for secretion of various
constituents into the amniotic fluid is expanded after organ-
ogenesis. Each of the fetal and maternal sites is considered
in the following discussion. During the first half of gestation
the fetal skin is very thin, consisting of only a few cell lay-
ers, and lacks the keratinified epithelium that prevents fluid
loss later in gestation and in postnatal life. The superficial
layer or periderm cells contain structures suggesting that
these cells are metabolically active (Brace, 1986). In vitro
and in vivo studies indicate the permeability of the periderm
to water and sodium and transport of urea and electrolytes
(Lind et al., 1972). Thus, early in gestation, the amniotic
fluid water and electrolytes are believed to pass relatively
freely from the infant’s skin to the amniotic fluid. That
this exchange process may continue, to some extent, later
in gestation is suggested by observations that premature
infants born after 28–30 weeks of gestation have transepi-
thelial water losses during the first day of life that are up
to 15 times higher than those of infants born at term (Sedin
et al., 1981). Although the fetal skin has a high density of
sweat glands, their contribution to amniotic fluid volume
and content is not well established.
The fetal kidney is known to produce urine during the
second half of gestation (Langman, 1981), and urine has
been detected in the fetal bladder as early as 11 weeks of
gestation (Wladimiroff and Campbell, 1974). The volume
of urine produced by the human fetus increases in propor-
tion to fetal size, from about 110 mL/kg/day at 25 weeks
to 190 mL/kg/day at 39 weeks of gestation. Since the vol-
ume of amniotic fluid is stable or decreasing during this
period, the increasing output of urine must be matched by
an increase in the fluid clearance rate. One effect of the
increasing urine output from the fetus is a decrease in the
osmolarity and sodium concentration, from about 98% to
91% of that in maternal or fetal plasma. This is accompa-
nied by an increase in urea, from 100% to 168% of that
in maternal plasma. However, measurement of albumin in
the first urine after birth indicated that fetal urine could
account for only a small proportion of the production of
amniotic fluid proteins at term (Gitlin et al., 1972). The
urinary contribution to amniotic fluid IgA and transferrin
was also small. However, according to Gitlin’s calculations,
IgG in amniotic fluid could be derived largely from fetal
urine. Nonetheless, even for IgG, the majority of this IgG
is probably produced by the mother and transferred via the
placenta to the infant’s circulation prior to excretion in the
fetal urine. The amount of water and individual proteins
released from other mucosal surfaces of the fetus cannot be
ascertained by this type of study. By examining the levels of
specific proteins in the blood of the newborn infant, inves-
tigators suggested that little or none of the IgA could come
from the fetus (Gitlin et al., 1972). However, this conclu-
sion ignores the possibility that the IgA is produced locally
and excreted from the mucosal surfaces (other than urinary
tract) of the fetus.
The concentrations of a product of secretory epithelium,
free secretory component (fSC), in the amniotic fluid and
first urine samples voided by infants have been investi-
gated (Cleveland et al., 1991b). The mean concentrations
(7.5 and 6.8 μg/mL) were similar in term amniotic fluid and
first voided urine. However, with the turnover rates for pro-
teins noted by Gitlin et al. (1972), the full-term fetus would
have to have voided from 3.5 to 48 L of urine to account
for all the fSC in the amniotic fluid, an unlikely event for a
2 kg fetus. Thus, some other secretory epithelia of the fetus
or the chorioamniotic membranes must account for at least
90% of the fSC in the amniotic fluid of term infants. This
proposition is consistent with the observation of Tourville
et al. (1969) that SC can be detected in amniotic epithelial
cells by immunofluorescence.
2256  SECTION | I  Genitourinary Tract and Mammary Gland
The fetal respiratory tract is another site of fluid produc-
tion. In fetal sheep, 30–50 mL/kg/day of fluid flows from
the trachea. However, it is not clear how much of this fluid
is immediately swallowed by the fetus. The observation that
surface-active phospholipids (surfactants) appear in human
amniotic fluid during the third trimester of gestation sug-
gests that some respiratory secretions reach the amniotic
cavity.
Saliva and nasal secretions derived from the salivary
glands and nasal mucosa, respectively, may also contribute
to the volume and content of the amniotic fluid. In the fetal
lamb, this is estimated to represent only about 30 mL/day,
but this excludes the portions of the secretions that are swal-
lowed (Brace, 1986). These secretions may contribute to the
mucoid nature of the amniotic fluid at term.
It is important to point out that each of these fetal sources
of amniotic fluid also contains components of the muco-
sal immune system. Thus, amniotic fluid may represent, to
some extent, a mixture of the fetal mucosal secretions.
There are also several potential avenues by which mater-
nally derived fluid and specific solutes can reach the amni-
otic fluid. These include the fetal surface of the placenta
and the uterine decidua. The contribution of the latter is
thought to be limited by the limited maternal vascularity
of the decidua in proximity to the chorion laeve and the
mature appearance of the amniotic epithelium at that site
(­Lotgering and Wallenburg, 1986). By contrast, the amniotic
epithelial cells overlying the chorionic plate have a unique,
active-appearing morphology, as described previously. In
addition, the maternal circulation in the intervillus spaces
below is rich. Two forms of water exchange across the cho-
rion and amnion have been described: fast nondiffusional
bulk flow through intercellular channels and an inherently
slower diffusion mechanism (Lotgering and Wallenburg,
1986). While these mechanisms are highly permeable to
water, small solutes cross by simple diffusion; larger com-
pounds (1000 mol wt) do not cross these membranes well, if
at all. The driving forces for this transport are differences in
hydraulic and osmotic pressures.
METABOLISM OF AMNIOTIC FLUID
The composition of AF changes with gestational age. In
the second half of pregnancy, there is a decrease in sodium
and chloride concentrations, an increase in urea and creati-
nine concentrations, and an overall decrease in AF osmo-
larity. Many studies suggest that AF composition is more
highly regulated than AF volume. Metabolite profiles for
second and third trimester amniotic fluid samples, using
high-resolution 1H NMR spectroscopy, showed signifi-
cant differences with gestational age (Groenen et al., 2004;
Cohn et al., 2009; Graca et al., 2010). The concentrations
of creatinine and betaine increased while concentrations of
creatine, glucose, and various amino acids decreased with
the advancing gestational age, providing excellent metabo-
lomic evidence of fetal maturation and having the potential
to improve the evaluation of fetal health and development.
The detection and quantification of creatinine may provide
new biomarkers for assessing the renal status of the fetus.
Several findings described in the preceding section sug-
gest that the process by which amniotic fluid proteins are
cleared late in gestation involves coordinated (mass) trans-
port of water and proteins (Gitlin et al., 1972). However, the
data from Gitlin et al. indicate that very little of the radio-
labeled protein injected into the chorioamniotic sac reaches
the maternal circulation. This may be due, at least in part, to
the rapidity of amniotic fluid removal by the fetus (see later
discussion), but other pressure or concentration gradients
might also be involved.
As described previously, near the end of gestation, the
water and soluble proteins in amniotic fluid are rapidly
cleared. That much of this fluid is removed by the fetus is
indicated by the relatively low rate of clearance when the
fetus was dead: 10–15% per day versus 90% for a live fetus
during labor (Gitlin et al., 1972). The low recovery of radio-
labeled proteins in either the maternal or the fetal serum at a
time when a large proportion of the label had been removed
from the chorioamniotic sac seems to eliminate absorption
of the proteins through the fetal respiratory tract, umbilical
cord, or skin, as well as the maternal decidua. This leaves
the fetal gastrointestinal tract as the most likely site for
extensive clearance of amniotic fluid. This proposition is
in keeping with the observation that atresia at several sites
in the infant’s upper gastrointestinal tract can result in an
accumulation of excessive amniotic fluid, known as poly-
hydramnios. Deglutition begins near the end of the first tri-
mester of gestation. Gitlin et al. (1972) concluded that most
of the proteins in the swallowed amniotic fluid are hydro-
lyzed and absorbed into the infant’s circulation since a large
fraction of the radiolabel was recovered in the mother’s
urine, disassociated from the proteins, 1–2 days after the
amniotic injection.
The water channel, aquaporin-8, is expressed in human
amnion, chorion, and placenta. Aquaporin-8 gene expres-
sion was demonstrated in epithelial cells of chorion and
amnion and of the syncytiotrophoblasts and outer layer
trophoblasts of placenta. These findings suggest that aqua-
porin-8 may mediate amniotic fluid resorption through the
intramembranous pathway (Wang et al., 2001). This path-
way of amniotic fluid absorption could be important in
regulating amniotic fluid volume homeostasis.
IMMUNOLOGICAL FACTORS IN AMNIOTIC
FLUID
A number of immune factors have been identified and in
some cases monitored in the amniotic fluid during the prog-
ress of pregnancy. The precise functions of most of these
 Amniotic Fluid and the Fetal Mucosal Immune System Chapter | 115  2257
factors in amniotic fluid are not known. However, on the
basis of functions of the same factors at other sites, it is
likely that immune-related factors in amniotic fluid play a
role in the defense of the fetal and uterine structures against
infection, signaling the uterus that the fetus has sustained
an infection, initiating parturition, and/or modulating the
maternal immune response to prevent rejection of the fetus.
In the following section we summarize immune factors that
participate in the innate immune system, soluble immune
modulators and growth factors, and immunoglobulins and
their receptors/transporters.
INNATE IMMUNITY
Complement and Complement Inhibitors
A fetus is semiallogeneic tissue, resembling a transplanted
allograft that must be protected from maternal complement
attack. Amniotic fluid contains a potentially functional
complement system, which gradually increases in concen-
tration during gestation. At term, hemolytic complement
activity (CH50) exceeds that of maternal serum by 60%
(Huffaker et al., 1989). While the function of complement
in amniotic fluid is presumably to provide protection from
infection, nonlethal doses of the complement membrane
attack complex can stimulate prostaglandin E2 produc-
tion in a number of cell types (Daniels et al., 1990; Hansch
et al., 1988; Rooney and Morgan, 1990a), and an increase
in prostaglandins has been implicated in the onset of both
term and preterm labor. Therefore, activation of amniotic
fluid complement could potentially participate in the nor-
mal process of parturition or precipitate the onset of pre-
mature labor.
Cytotoxic alloantibodies are present in the plasma of
some pregnant women, and IgG and IgM are present in
amniotic fluid (Quan et al., 1999). Therefore, the poten-
tial exists for activation of amniotic fluid complement
by the classical pathway. The alternative pathway could
be activated by intrauterine infection and might be caus-
ally associated with premature labor. However, human
amniotic epithelial cells express decay accelerating fac-
tor (DAF), which can protect against complement toxicity
(Rooney and Morgan, 1990b; Pacheco et al. 2011). DAF
is a complement-modulating factor that is also expressed
in the fetomaternal interface and has been implicated in
protection of the fetus from rejection (Rooney and Morgan
1990b). The expression of DAF on human trophoblast
membranes may protect them from maternal comple-
ment-mediated damage in response to microbial infection
(Nishikori et al., 1993; Jensen et al., 1995; Fenichel et al.,
1995). Our recent results support the notion that there are
inherent differences in immunological responsiveness
between individuals and that the vitamin D3 system may
be important in sustaining LPS-induced CD55 expression
(Izban at al. 2012). DAF is a key complement regulator at
the C3 level and is thought to be unique in its responsive-
ness to progesterone (Kaul et al., 1994). One hypothesis is
that a decrease in the DAF/complement ratio could release
the complement cascade. Under those circumstances, even
minor activation of complement in the fetomaternal com-
partment could result in proinflammatory responses, acti-
vation of the prostaglandin cascade, and precipitation of
labor (Nowicki et al. 2013).
Another complement-modulating factor is CD59, the
plasma membrane attack complex inhibitor protein that
has been demonstrated on the apical surface of the syn-
cytiotrophoblast, on extravillous cytotrophoblast, and in
amniotic epithelium (Vanderpuye et al., 1993). During
pregnancy the level of CD59 also increases by 50% in
maternal plasma. CD59 could help protect extraembry-
onic epithelia from damage by complement in maternal
blood and in amniotic fluid. The twofold to sixfold higher
expression in the syncytiotrophoblast than in erythrocytes
and the apical location of CD59 may reflect the immu-
nological roles and functional polarization of plasma
membranes in the syncytiotrophoblast (Vanderpuye et al.,
1993). CD59 is secreted by human amniotic epithelial
cells into amniotic fluid and could be incorporated into
target cells. This mechanism may protect fetal cells from
lysis by maternal complement.
Amniotic fluid complement factor C3 can be used in
the diagnosis of intraamniotic infection in preterm labor
with intact membranes. Assays for C3 are readily avail-
able, and their results compare favorably with other rapid
markers such as white blood cells, lactate dehydrogenase,
and glucose as an indicator of amniotic fluid infection. This
finding supports the general concept of fetal inflammatory
responses to microbial invasion of amniotic fluid (Elimian
et al., 1998).
Mannan-binding protein (MBP), as well as other mem-
bers of the collectin family, including complement protein
C1q, lung surfactant protein A, CL-43, and conglutinin,
has been shown to interact with a single C1q receptor, also
termed collectin receptor (Stuart et al., 1997) This recep-
tor, which is widely distributed in different types of cells,
together with collectins plays an important role in innate
immunity.
MBP is a serum protein that can activate the classi-
cal complement pathway. MBP has also been shown to
opsonize bacteria and enhance clearance of bacteria by
phagocytosis. MBP is present in amniotic fluid, and its con-
centration increases sharply from about 32 weeks of ges-
tation. MBP may play a role in the antibody-independent
recognition and clearance of pathogens in the amniotic cav-
ity toward term (Malhotra et al., 1994). It is interesting that
at 32 weeks the CH50 of amniotic fluids also increases and
exceeds significantly that of cord serum but is lower than
the CH50 of maternal sera.
2258  SECTION | I  Genitourinary Tract and Mammary Gland

CYTOKINES, CHEMOKINES, AND OTHER
MODULATORY AND GROWTH FACTORS
These small-molecular-weight peptides are usually pro-
duced by one or more cell types in various tissues and act
as messengers to induce various activities in nearby cells
or the producing cell itself. By sensitive immunoassay,
these factors can often be detected in fluids near the site
of production or can reach distant sites via the circula-
tion. Those mediators that have been detected in amni-
otic fluid are shown in Table 1. Unfortunately, the results
of such assays on amniotic fluid do not indicate which
cells are responsible for their production. These might
include cells of the fetus, fetal membranes, or placenta

TABLE 1  Cytokines, Chemokines, Growth Factors, and Prostaglandins in Amniotic Fluid

Gestational Age, Concentration, pg/mL

Weeks (Sample (Proportion Detectable,
Name Number) or Range) Alteration with References
IL-1β/IL-1RA 13–19 (4) 90.9 ± 26.8 – Srivastava et al. (1996)
IL-1RA 13–19 (11) 2835 ± 1775 – Srivastava et al. (1996)
IL-2 13–19 (13) 0.9 ± 3 (5/13) – Srivastava et al. (1996)
S IL-2R 13–19 (11) 609 ± 391 (8/11) – Srivastava et al. (1996)
IL-4 13–19 (14) 21 ± 47 (8/14) – Srivastava et al. (1996)
IL-6 13–19 (14) 3407 ± 236 – Srivastava et al. (1996)
Midtrimester to term 703 ± 401 No labor Santhanam et al. (1991)
2923 ± 5638 Labor
16,904 ± 9343 Labor plus infection
15–21 (22) 560 ± 1071 Weimann et al. (1995)
IL-8 13–19 (14) 2825 ± 690 Srivastava et al. (1996)
14–41 (80) <1800 Intraamniotic infection, Puchner et al. (1993)
>10,000 (9/12)
IL-6 and IL-8 Increase of IL-6 and Fetal chromosomal Vesce et al. (2002)
decrease of IL-8 abnormality
IL-10 13–19 (14) 24 ± 40 (8/14) Srivastava et al. (1996)
Midtrimester (66) <40 (<40–476) SGA > 40, 78 (<40–832) Heyborne et al. (1994)
Term (119) 20 Intraamniotic infection in Greig et al. (1995)
labor, 54 (0–759)
TNFα 13–19 (14) 183 ± 205 – Srivastava et al. (1996)
TNFα-active 14–20 (59) 4 Small for gestational Heyborne et al. (1992)
age, 10.5
INFα 13–19 (14) 11 ± 18 (4/14) – Srivastava et al. (1996)
GM-CSF 13–19 (8) 9 ± 14 (5/7) – Srivastava et al. (1996)
15–21 (22) <10 – Weimann et al. (1995)
G-CSF 15–21 (22) 1396 ± 2073 – Weimann et al. (1995)
HB-EGF Late pregnancy Rapid increase Not altered by infection Michalsky et al. (2002)
TGF-β1
Active 13–19 (11) 2.8 ± 2.9 – Srivastava et al. (1996)
Latent 13–19 (13) 167 ± 106 – Srivastava et al. (1996)

Continued
 Amniotic Fluid and the Fetal Mucosal Immune System Chapter | 115  2259

TABLE 1  Cytokines, Chemokines, Growth Factors, and Prostaglandins in Amniotic Fluid—cont’d

Gestational Age, Concentration, pg/mL

Weeks (Sample (Proportion Detectable,
Name Number) or Range) Alteration with References
TGF-β1
Active 14–16 (12) 540 ± 60 – Lang and Searle (1994)
TGF-β2
Active 13–19 (14) 10 ± 11 – Srivastava et al. (1996)
Latent 13–19 (13) 1178 ± 469 – Srivastava et al. (1996)
TGF-β2
Active 14–16 (12) 2300 ± 400 – Lang and Searle (1994)
MIP1-α 13–19 (14) 241 ± 402 – Srivastava et al. (1996)
GRO-α Midtrimester (18) 1100 (400–5000) Intraamniotic infection Cohen et al. (1996)
Term (20) 1900 (1200–4200) 2700 (1400–12,700) Cohen et al. (1996)
Preterm (20) 2300 (500–10,000) Intraamniotic infection Cohen et al. (1996)
5000 (600–47,900) Cohen et al. (1996)
MMPase-9 Intraamniotic infection Harirah et al. (2002)
Stem cell factor 13–9 (14) 1047 ± 383 (13.6–541.9 ng/mL) Srivastava et al. (1996)
SLPI Second trimester (15) 106 ± 15 pg/mL Zhang et al. (2001)
Third trimester (12) 802 ± 138 pg/mL
Prostaglandin 444 ± 78 pg/mL Intraamniotic infection Hillier et al. (1993);
(2.238 ± 434 pg/mL)

SGA, small for gestational age; HB-EGF, heparin-binding EGF-like growth factor; MMPase-9, matrix methalloproteinase-9; SLPI, secretory leukocyte protease
inhibitor.

or cells from maternal tissues that transit across the fetal
tissues.
Interest in cytokines, chemokines, growth factors, and
prostaglandins in amniotic fluid has been stimulated by the
possibility that they are involved in the control of parturi-
tion. Enhanced production of cytokines and chemokines
can be induced by various stimuli, particularly infections.
Thus, increased mediator levels have been investigated as
a potential explanation for premature onset of labor due
to intraamniotic infections (Romero et al., 1989). More
recently, the potential effects of potent mediators on the
growth and welfare of the fetus and unborn infant have
begun to be evaluated (Heyborne et al., 1992, 1994). Some
of the amniotic fluid cytokines, their concentrations, and
alterations in intraamniotic infection and growth retardation
are shown in Table 1.
To date, there has been little discussion about the specific
effects of the various cytokines, chemokines, and growth
factors demonstrated in amniotic fluid on the development
of the mucosal immune system of the infant. However, one
might draw an analogy between amniotic fluid and maternal
milk, which also contains numerous cytokines, chemokines,
and growth factors.
The possibility that these milk constituents may alter
the development or immune function of the infant is dis-
cussed elsewhere in this book (see Chapter 72). The idea
that some of these factors are produced at the mucosal sur-
faces of the infant and provide important signals to other
fetal tissues or to the uterus or placenta should also be con-
sidered.
Cytokines
The function of cytokines in normal pregnancy and deliv-
ery is not fully established. The cytokines IL-1, IL-6, and
TNFα, as well as epidermal growth factor (EGF), have
been detected in human amniotic fluid before and dur-
ing labor (Table 1). Amniotic fluid mean values of IL-1β,
IL-6, TNFα, IFNγ, and EGF were approximately 4, 140,
4, and 3 times those of maternal serum values, respec-
tively. Thus, it seems likely that the fetus and/or placenta
contributes to the production of the cytokines in amniotic
2260  SECTION | I  Genitourinary Tract and Mammary Gland
fluid. This supposition is in agreement with Menon et al.
(2001), who reported that amniochorionic membranes are
a site of inflammatory cytokine production. Increases in the
concentration of cytokines in amniotic fluid may represent
enhanced macrophage activity for immunosurveillance of
the fetus. In addition, the elevation of amniotic fluid cyto-
kines may be a preparatory step in the initiation of labor,
since cytokines play an important role in the stimulation of
prostaglandin synthesis.
An increase of IL-6 (P = 0.034) and a decrease of IL-8
(P ≤ 0.0001) in amniotic fluid, as well as a decrease of IL-6
in the maternal plasma (P = 0.026), were associated with
pregnancies with fetal chromosomal abnormalities (Vesce
et al., 2002).
Heparin-binding EGF-like growth factor (HB-EGF) is
a member of the EGF family that has been implicated in
the healing of various organ injuries. HB-EGF production
is upregulated in response to injury to the kidney, liver,
brain, skin, and intestine. Recently HB-EGF was detected
in human amniotic fluid and breast milk. The HB-EGF in
amniotic fluid may play a role in the development of the
gastrointestinal tract in utero, whereas that in breast milk
may protect gut mucosa against postnatal injury (Michalsky
et al., 2002).
Amniotic fluid matrix metalloproteinase-9 and IL-6
are significantly elevated in women with intraamniotic
infection (Harirah et al., 2002), and the levels of matrix
metalloproteinase-9 are correlated with IL-6 (r = 0.813,
P < 0.001); both are positively correlated with amniotic
fluid leukocytes and inversely correlated with amniotic
fluid glucose (­Harirah et al., 2002). Therefore, amniotic
fluid matrix metalloproteinase-9 has been proposed as an
accurate biochemical marker of intraamniotic infection
with better sensitivity, specificity, and positive and negative
predictive values than IL-6 (Harirah et al., 2002). Elevated
matrix metalloproteinase-8 (>23 ng/mL) in amniotic fluid
is also a powerful predictor of spontaneous preterm deliv-
ery (<32 weeks), with an odds ratio of 68.4. Thus, amniotic
fluid studies can be used to improve the risk assessment
for preterm delivery in women who undergo midtrimester
amniocentesis for genetic indications (Yoon et al., 2001).
Secretory leukocyte protease inhibitor (SLPI), a potent
inhibitor of human leukocyte elastase, is a protein found
in various human fluids, including amniotic fluid, cervical
mucus, seminal plasma, and ascites. SLPI is produced by
amniotic membranes in response to cytokines (Zhang et al.,
2001). The SLPI in the amniotic fluid may contribute to
immune mechanisms during pregnancy (Zhang et al., 2001).
The binding of lipopolysaccharide (LPS) with LPS bind-
ing protein (LBP) and sCD14 activates macrophages at LPS
concentrations up to 1000 times lower than those required
with LPS alone. The presence of LBP and sCD14 in amni-
otic fluid might explain the high concentrations of cytokines
present in the amniotic fluid of culture-negative women. In
fact, the LBP and sCD14 concentrations in amniotic fluid of
women in preterm labor are linearly associated with amni-
otic fluid IL-6 concentrations (Gardella et al., 2001).
Adrenomedullin, a novel hypotensive peptide with pos-
sible immunological function, was discovered in human
pheochromocytoma (Kitamura et al., 1993). In humans,
adrenomedullin was detected in amniotic fluid and plasma
as well as in many tissues, such as adrenal medulla, aorta,
heart, lung, brain, and kidney (Macri et al., 1996). The
physiological function of increased adrenomedullin during
pregnancy is still not established, although it was suggested
that adrenomedullin plays an important role in the adapta-
tion of the vascular system to pregnancy needs and may
regulate placental vascular tone.
Plasma adrenomedullin concentrations increase with
advancing gestation and are highest in the second trimester
of pregnancy. Adrenomedullin concentrations in amniotic
fluid are significantly higher than in the maternal plasma
throughout gestation and also increase with advancing
gestation. There is a significant linear correlation between
amniotic fluid and maternal plasma adrenomedullin during
pregnancy. Adrenomedullin mRNA was detected not only
in the amniotic membranes and intermediate trophoblast
of third trimester pregnancy but also within the endothelial
layers of villous blood vessels (Kanenishi et al., 2001).
NONSPECIFIC ANTIMICROBIAL FACTORS
The inability of amniotic fluid to support the growth of dif-
ferent bacteria was noted a long time ago. It was not clear
whether this was due to an antimicrobial activity or to a
deficiency of one or more nutrients. Amniotic fluid sup-
plemented by bacterial growth media was shown to allow
growth of bacteria. However, when the constituents of the
growth media were added individually, only phosphate was
required (Larsen et al., 1974). Subsequently, Schlievert
et al. (1976) found that phosphate inhibits an antimicrobial
property of amniotic fluid that includes a hexapeptide and
zinc. The activity of the hexapeptide was inhibited by phos-
phate. Antimicrobial activity against Escherichia coli was
totally lost after addition of phosphate, suggesting that the
hexapeptide was a major antimicrobial agent against this
bacterium.
A number of the antimicrobial substances found in
amniotic fluid are also present in other mucosal secretions,
including milk (see also Chapter 15). Several of these com-
ponents are presumably produced by epithelial cells of the
fetus or by amniotic epithelial cells. We therefore examined
the relationship among the concentrations of fSC, lactofer-
rin, and lysozyme (Cleveland et al., 1991a). fSC is pro-
duced by secretory epithelial cells and serves as a marker
for sites of polymeric Ig transport (see also Chapter 20). As
in the case of fSC, lactoferrin and lysozyme concentrations
increase in the amniotic fluid with increasing gestational
 Amniotic Fluid and the Fetal Mucosal Immune System Chapter | 115  2261
age. We also found that the concentrations of both lactofer-
rin and lysozyme are correlated with fSC concentrations in
amniotic fluid and that the concentrations of lactoferrin and
lysozyme are correlated with each other (Cleveland et al.,
1991a). This suggests that both these factors are produced
by the same cells or that the cells that produce them are
under similar regulatory influences.
Defensins are nonspecific antimicrobial factors that
may be important in the early defense against infection (see
also Chapter 16). Defensins are neutrophil peptides in the
azurophilic granules released from activated neutrophils
­(Faurschou et al., 2002). Defensins can eliminate microor-
ganisms by forming pores in their plasma membrane (Bals,
2000). Amniotic fluid defensin levels measured by enzyme-
linked immunoabsorbent assay were fourfold to 24-fold
higher in patients with intrauterine infection than in normal
preterm and term controls. An amniotic fluid defensin level of
400–2500 ng/mL identified over 80% of patients with a pos-
itive amniotic fluid culture. Amniotic fluid defensin levels
also identify accurately patients with subclinical intrauter-
ine infection, as defined by placental histologic findings and
positive amniotic fluid culture. It was also suggested that
the defensin level may contribute to the increased risk of
spastic cerebral palsy and other fetal morbidity in newborns
from patients with intrauterine infection. It is interesting
to note that labor alone was not associated with elevation
of the defensin level. Therefore, it was also suggested that
amniotic fluid defensin levels could be another marker of
subclinical intrauterine infection (Heine et al., 1998; Balu
et al., 2002).
HUMORAL IMMUNITY
Immunoglobulins and Immunoglobulin
Transporters
During pregnancy, the maternal immune response is gen-
erally modulated toward humoral immunity (Stirrat, 1994;
Jensen et al., 1995). Activated lymphocytes during murine
pregnancy produce cytokines that preferentially stimulate
antibody production versus T cell cytotoxic responses. This
effect is most prominent within the uterine decidua but also
affects systemic immunity. The discussion below consid-
ers the maternal and fetal adaptive immune responses, with
emphasis on humoral immune factors and their receptors in
amniotic fluid.
The sources of the immunoglobulins in the chorionic
and amniotic fluids are known in some detail, but the selec-
tivity and mechanisms of their transport are not yet clear.
As described previously, Jauniaux et al. (1995) found that
the gradient between maternal serum and chorionic fluid
was greater for IgA than for IgG and that neither IgM nor
C3 were detected in this fluid. The absence of detectable
amounts of the larger IgM molecules in this fluid might be
due to its limited concentration in the interstitium of the
decidua or to lack of transport across the chorionic mem-
brane. C3 has a size and serum concentration in the same
range as that of monomeric IgA. However, the inability of
Jauniaux et al. (1995) to detect similar amounts of C3 as IgA
in chorionic fluid could have been due to insensitivity of the
C3 assay used or selective transport of IgA. Specific trans-
porters for IgG and IgA are known to be produced by the
fetus. One of them, the polymeric immunoglobulin receptor
(pIgR), has been identified by immunofluorescence in the
fetal membranes (Tourville et al., 1969). The neonatal Fcγ
receptor (FcRn) is expressed in the syncytiotrophoblasts of
the placenta (Leach et al., 1996), but to our knowledge its
presence in the chorioamniotic membranes has not been
reported. However, Bright and Ockleford (1995) did not
find evidence of endogenous IgG staining within either the
cytotrophoblast or the viable amniotic epithelial cells of the
chorioamniotic membrane. IgG was detected throughout
the interstitium of the decidua, chorion, and amnion and in
dying amniotic epithelial cells. They concluded that, at least
late in gestation, the entry of IgG into the amniotic cavity is
by passive diffusion.
We have quantified albumin and each of the major Ig
classes in amniotic fluids collected by amniocentesis for
genetic analysis and determination of fetal maturity (Hilton,
1993). Figure 3 demonstrates the albumin concentration in
amniotic fluid as a function of gestational age. The progres-
sive fall during gestation is similar to that described by oth-
ers for total protein concentration. In contrast to the pattern
for albumin, the concentrations of each class of Ig in amni-
otic fluid did not seem to vary significantly with gestational
age, as shown in Figure 4. When concentrations of amniotic
fluid proteins are considered in relation to the concentrations
4000
y = 3324.6 – 49.562x R2 = 0.363
3000
Albumin, µg/ml
2000
1000
0
10 20 30 40 50
Gestational age, weeks
FIGURE 3  Albumin concentration in amniotic fluid versus gesta-
tional age. The concentration of albumin was measured by nephelometry
in 31 amniotic fluid samples. Gestational age was calculated from the last
menstrual period. Reprinted with permission from Hilton (1993).
2262  SECTION | I  Genitourinary Tract and Mammary Gland

D 600 100
y = 2.2063e-2 x 10 (61783e-2x) Rz = 0.658

500

10
400
IgG, Pg/mL

fSC, µg/mL
300

1
200

100
10 20 30 40 50
.1
Gestational age, weeks 10 20 30 40 50
Gestational age, weeks
FIGURE 5  fSC concentration in amniotic fluid versus gestational
40
E age. fSC in amniotic fluid was quantified by ELISA. fSC concentration
was significantly correlated with gestational age.

30
of these same proteins in the maternal serum, albumin and
IgG were of the same magnitude (approximately 1/10–1/30
IgA, Pg/mL

20
of maternal serum concentration). However, the ratios of
amniotic IgA and IgM to maternal serum concentrations
were much lower (1/100 and 1/1000, respectively). The lat-
10 ter finding is consistent with later reports on chorionic fluid
(Jauniaux et al., 1995).
Cederqvist et al. (1978) have stated that the level of IgA
0 in amniotic fluid increases until midgestation and decreases
10 20 30 40 50 thereafter. Because there were so few samples available
Gestational age, weeks between weeks 20 and 30 of gestation, such a pattern is dif-
ficult to discern from their data or our own (Figure 4). Given
the decreasing concentration of albumin during gestation
F 10 and the data of Gitlin et al. (1972) that all radiolabeled pro-
teins are cleared from the amniotic fluid at a similar rate,
it seems likely that there is more than one source of IgA in
the amniotic fluid. The extent to which nonspecific diffu-
1 sion and receptor-mediated transport of maternal IgA and
IgM, Pg/mL

the fetal mucosa contribute to amniotic IgA concentration

may vary with gestational age. This issue will be considered
further in the context of the structure of the amniotic IgA.
.1
fSC, the cleaved extracellular portion of the pIgR
expressed in secretory epithelium, has also been quanti-
fied in a set of amniotic fluid samples (Cleveland et al.,
1991a,b; Hilton, 1993). Figure 5 shows the relationship
.01
10 20 30 40 50 between fSC concentration and gestational age (Hilton,
Gestational age, weeks 1993). There is a strong positive correlation between fSC
FIGURE 4  Ig concentration in amniotic fluid versus gestational age. and gestational age from about 15 or 20–40 weeks of gesta-
(a) The concentration of IgG was measured by nephelometry. (b) The con- tion. As discussed earlier, the anatomical site of production
centration of IgA was measured by ELISA. (c) The concentration of IgM of most of the fSC in amniotic fluid is not known. In most
was determined by ELISA. The number of samples was 31 for each of the biological solutions, some of the SC is found complexed
Ig classes. There was no significant correlation between the concentration
with its ligands, polymeric immunoglobulins (IgA and
of any Ig class and gestational age. Reprinted with permission from Hilton
(1993). IgM), which have been transported across these epithelial
cells. After proteolytic cleavage of the pIgR from the api-
cal surface of the epithelial cell, the complexes (Ig-SC) are
 Amniotic Fluid and the Fetal Mucosal Immune System Chapter | 115  2263
called secretory immunoglobulins (e.g., S-IgA). Approxi-
mately 80% of the amniotic fluids that we have examined
contained small amounts of S-IgA of typical size (390 kDa),
as indicated by Western blots stained with anti-α chain and
anti-SC antibodies (Cleveland et al., 1991b). This finding
suggests that this portion of the amniotic IgA is derived
from epithelial cell transport of dimeric IgA molecules. No
S-IgA molecules of this size were detectable in unconcen-
trated urine collected from the first postnatal void. How-
ever, high-­molecular-weight bands consistent with 390-kDa
S-IgA were recovered from lysates of amniotic membranes
and from the supernatants of short-term cultures of washed
amniotic membranes (Cleveland et al., 1991b). This sug-
gests that, at least near the end of gestation, some—presum-
ably maternal—IgA is actively transported into the amniotic
fluid by amniotic epithelial cells. This maternal IgA may
be produced locally in the decidua, since Cederqvist
et al. (1978) reported that up to one-half of the IgA in amni-
otic fluid from 11 to 40 weeks of gestation was of the IgA2
isotype. Normally, only about 10% of serum IgA is IgA2,
whereas IgA produced at mucosal sites contains a larger
proportion of IgA2, depending on the site.
Western blotting also demonstrated the presence in all
amniotic fluids examined of bands in the 200-kDa range,
which stained with polyclonal and monoclonal antibodies
against α chain and SC (Cleveland et al., 1991a). Similar-
sized bands were also seen in the first postnatal urine sam-
ples and in lysates and supernatants from cultured amniotic
membranes. A preliminary characterization of these unusual
IgA molecules indicated that they contain full-length α and
light chains, suggesting that they are formed from the 390-
kDa molecules by dissociation of interchain disulfide bonds.
Subsequent studies (Hilton, 1993) examined various meth-
ods for isolating and analyzing the small molecular form
of IgA from amniotic fluid. After examination by several
different techniques, it seemed clear that these molecules
did not contain substantial amounts of the J chain present
in typical 390-kDa S-IgA. It was also clear that not all the
IgA molecules of this size contained SC. When the approxi-
mately equal amounts of 390- and 200-kDa forms were
compared by several analytical techniques, SC could be
detected in only the 390-kDa forms. However, the amount
and purity of the 200-kDa forms available for analysis were
not adequate to allow detection of SC if it was present on
only a subset of these molecules. It is likely that the prelimi-
nary observations of Cleveland et al. (1991b) were based on
a population of 200-kDa IgA molecules that were enriched
in their SC content by isolation with the use of monoclo-
nal anti-S-IgA affinity methods. Despite the presence of
this IgA form in the urine of newborns, the low concentra-
tion of urinary IgA at this time (<0.2 μg/mL) (­Gitlin et al.,
1972) and the rapid clearance rate of amniotic fluid make it
unlikely that fetal urine is a major source of even the small
amount of IgA found in late-gestation amniotic fluid.
The ability of Igs in the amniotic fluid to protect the
developing fetus from infection is difficult to ascertain.
Jauniaux et al. (1995) detected specific antibodies against
Toxoplasma, cytomegalovirus, and rubella virus in cho-
rionic fluids as early as 9 weeks of gestation. These anti-
bodies were present only when the maternal serum had
antibodies to the same agents. The ratios of maternal serum/
chorionic fluid titers varied from 5:1 to 32:1, consistent
with their mean ratio of 28:1 for total IgG concentration.
Mellander et al. (1986) measured antibodies in amniotic
fluid and found that a minority of samples contained IgA
antibodies against E. coli surface antigen and poliovirus.
IgG and IgA antibodies to Candida have been detected
in the majority of amniotic fluids as early as 15–18 weeks
of gestation by two different groups (Auger et al., 1980;
Mathai et al., 1991). Interestingly, in contrast to Candida
antibodies in serum, where IgG predominates, IgA antibod-
ies predominated in the amniotic fluid. This suggested that
these antibodies were not derived from maternal serum. It
is interesting to speculate that these antibodies might have
been derived from maternal secretions (cervical or vaginal)
and transported into the amniotic fluid.
Blanco et al. (1983) found that amniotic fluid from 46
patients soon after the clinical onset of bacterial intraamni-
otic infection had slightly higher IgG concentrations than
those from 46 matched controls. However, they did not
determine whether there was a selective increase in spe-
cific antibodies against the infecting organisms. Thus, the
increased IgG noted may have been due to increased trans-
port of total IgG into the amniotic fluid. However, another
group has reported that bacteria from amniotic fluid of
women with chorioamnionitis were frequently coated with
antibodies (Moller et al., 1997).
INCREASED ACTIVITY OF THE IMMUNE
SYSTEM IN AFRICAN AMERICANS
Although there are currently no known markers for identi-
fying differences in immunity and inflammation in amni-
otic fluid in African Americans and Caucasians, these could
be important since the incidence of preterm labor is much
higher among African Americans. This may be correlated
with the higher serum Ig levels, particularly IgG, in African
Americans. This difference presumably reflects increased
B-cell activation (Tollerud et al., 1995). Serum IgG was
directly related to human leukocyte antigen (HLA)-DR
and the level of soluble IL-2 receptors (sIL-2R) and was
inversely related to the proportion of CD4 cells (Tollerud
et al., 1995). African Americans exhibit increased expres-
sion of the costimulatory molecules CD80 (B7-1) and CD86
(B7-2) (Hutchings et al., 1999). Costimulatory interactions
have been shown to be important for the production of the
proinflammatory cytokines TNFα and IL-1β (May et al.,
2000). Individuals with mutations at the TNF promoter
2264  SECTION | I  Genitourinary Tract and Mammary Gland
TNF(-238A) and TNF(-376A/-238A) consistently have
lower plasma levels of IL-10 than TNF. In children with
severe malaria, TNF promoter variants influence the bal-
ance of IL-10 to TNF in the plasma, which in turn affects
the outcome of clinical complications (May et al., 2000).
Altogether, African Americans appear to have inherently
higher levels of selected components of humoral immunity
and, therefore, an increased potential for complement and
anti-inflammatory responses in plasma. We know of no
studies comparing humoral immune factors in the amniotic
fluid of African Americans with other groups.
PATHOLOGICAL CONDITIONS
INVOLVING AMNIOTIC FLUID
Chorioamnionitis
Chorioamnionitis (amniotic fluid infection) is usually asso-
ciated with fever, purulent amniotic fluid (with bacterial
contamination) and tachycardia (maternal or fetal), and/or
increased maternal peripheral leukocyte counts. The fact that
bacterial cultures of amniotic fluid were positive for 75% of
women whose newborns weighed less than 1500 g provides
evidence that amniotic fluid infection is a major risk factor
for preterm delivery (labor) (Hitchcock et al., 1999).
Bacterial vaginosis is a risk factor for amniotic fluid
infection. The presence of several vaginal organisms in the
second trimester of pregnancy has been linked to the devel-
opment of amniotic fluid infection. Women vaginally colo-
nized by Gardnerella vaginalis, Bacteroides species, and
Mycoplasma hominis have more than a twofold increased
risk for amniotic fluid infection, compared with women not
colonized by any of these organisms (Krohn et al., 1991).
High vaginal IL-8 concentration, high neutrophil count,
abnormal vaginal gram stain, absence of hydrogen perox-
ide-producing Lactobacillus, and anaerobic vaginal flora
are strongly associated with amniotic fluid infection among
women in preterm labor (Hitti et al., 2001).
Most pathogens, including E. coli, develop unique viru-
lence mechanisms that allow them to colonize and invade
the urogenital tract (Goluszko et al., 2001; Das et al. 2005;
Wroblewska-Seniuk et al. 2005; Rice et al. 2006; Nowicki
et al. 2010; Sledzinska et al. 2010). Bacterial adhesins inter-
act with tissue receptors (Samet et al. 2013a; Samet et al.
2013b; Banadakoppa et al. 2014; Rana et al. 2014a; Rana
et al. 2014b), allowing ascending infection that triggers
TNF, IL-6, IL-8, and NO responses (Hedlund et al., 2001;
Frendeus et al., 2001; Fang et al. 2004). Lack of bacterial
attachment or prevention of colonization blocks signaling
and the host cytokine response. Because only certain genet-
ically related pathogens (not all bacteria) cause infection,
the identification of genetic and virulence factors could pre-
dict an association with preterm delivery (Hart et al., 2001).
For example, we found that vaginal colonization with
E. coli bearing class III P adhesin—but not the related class
I or II adhesins—was associated with a twofold increase in
the risk of preterm delivery. Clinical symptoms of amni-
otic fluid infection at term occur 1.5 times more often in
patients with bacterial vaginosis than in those without (Sil-
ver et al., 1989). A recent clinical study provides evidence
that treatment of bacterial vaginosis, or even asymptomatic
abnormal vaginal colonization, with oral clindamycin early
in the second trimester significantly reduces the incidence
of preterm deliveries (Ugwumadu et al., 2003).
Amniotic fluid infection is strongly associated with
the presence of a cytokine response in the amniotic fluid
(Romero et al., 1989; Hillier et al., 1993). Cytokine pro-
duction by macrophages is an immunological response
to pathogens that invade the amniotic fluid. The cytokine
concentration in the amniotic fluid of infected patients can
be even higher than the concentration of cytokines in the
cerebrospinal fluid of children during meningitis or in the
peritoneal fluid of patients during peritonitis (Mustafa et al.,
1989; Zeni et al., 1993; Hillier et al., 1993; Romero et al.,
1993; Greig, 1993). Prostaglandin E2 has also been associ-
ated with amniotic fluid infection. Although it is not clear
whether the macrophages that produce cytokines are of
maternal or fetal origin, it is known that cytokine levels in
amniotic fluid are correlated with levels of prostaglandins.
Granulocyte colony stimulating factor (G-CSF) can
be detected in amniotic fluid. Amniotic fluid G-CSF con-
centrations are similar in preterm and term labor and are
not significantly influenced by labor, although intraamni-
otic infection is associated with increased concentrations
of G-CSF in amniotic fluid, neonatal urine, and maternal
serum (Calhoun et al., 2001).
The S100 proteins are a family of low-molecular-weight
(10–14 kDa) calcium-binding multifunctional proteins with
α and β subunits. They are present in the cytosol, intracel-
lularly and extracellularly, and via binding to a target pro-
tein may trigger processes activated by the calcium signal
transduction pathways important in cellular differentiation,
progression, and inflammation. Some pathological condi-
tions during pregnancy related to hypoxic stress can affect
the elevation of S100B concentration in the amnion. There
have been several studies investigating the correlations
among the S100B concentrations in the amniotic fluid, cord
blood, and maternal serum during pregnancy and pathologi-
cal conditions related to fetuses have been suggested in pre-
term delivery (Gazzolo et al., 2000), intrauterine fetal death
(Florio et al., 2004), pregnancy-related hypertensive disor-
ders (Schmidt et al., 2004), intrauteral growth restriction
(IUGR) cases, and preeclampsia (Tskitishvili et al., 2006).
The amniotic fluid S100B protein concentration of the
preeclampsia and normotensive IUGR cases was signifi-
cantly higher than that of the control. This study also shows
that amnion could be a source responsible for the increased
concentration of S100B in amniotic fluid.
 Amniotic Fluid and the Fetal Mucosal Immune System Chapter | 115  2265
Fetal Growth Restriction
EGF is present in large amounts in the amniotic membranes
and fetal urine. EGF is a 53-amino acid polypeptide mito-
gen that stimulates tissue growth (Laborda et al., 2000).
Receptors or binding sites for EGF have been identified in
placenta and fetal membranes. Amniotic fluid EGF levels
increase rapidly in late pregnancy but are not altered by
chorioamnionitis or by term or preterm labor. Although
intrauterine growth restriction is associated with lower EGF
levels in amniotic fluid, EGF is not altered in maternal or
fetal plasma. EGF increases near term and decreases in
pregnancies complicated by intrauterine growth restriction
(Varner et al., 1996).
EGF may also influence the level of parathyroid hor-
mone-related protein (PTHrP). PTHrP may regulate pla-
cental calcium flux, fetal tissue development, and uterine
contractions. Investigations have shown that EGF (10 μg/L)
increased PTHrP secretion by over 100% and that PTHrP
secretion by EGF was both dose and time dependent; in
contrast, estradiol, progesterone, and human chorionic
gonadotropin had no effect on PTHrP secretion (Dvir et al.
1995). Intrauterine PTHrP concentrations are reduced in
association with growth restriction in the spontaneously
hypertensive rat (SHR) in comparison with those of its nor-
motensive control, the Wistar Kyoto rat, implicating PTHrP
as a pivotal fetal growth factor (Wlodek et al., 2001). The
SHR fetus is growth-restricted and has suppressed amniotic
fluid PTHrP, which is largely determined by the fetus or
gestational tissues and is independent of maternal hyper-
tension or maternal PTHrP (Wlodek et al., 2001). The low
SHR amniotic fluid PTHrP may play a role in the growth
restriction of the SHR fetus (Wlodek et al., 2001).
Circulating S100B protein is increased in intrauterine
growth-retarded fetuses (Gazzolo et al., 2002). Insulin-like
growth factor binding protein-1 (IGFBP-1) is present in
higher concentrations in amniotic fluid than in serum, cer-
vical mucus, urine, or seminal plasma (Darj and ­Lyrenas,
1998). Therefore, detection of IGFBP-1 by the dipstick
test (Darj and Lyrenas, 1998) with monoclonal antibodies
to IGFBP-1 could be useful in the diagnosis of premature
rupture of membranes.
ANTIRETROVIRAL DRUGS AND AMNIOTIC
FLUID
The timing of mother-to-child transmission of HIV-1 and
implications for prevention has been studied (Kourtis et al.,
2006). Lamivudine is an antiretroviral drug that has been
widely used during pregnancy, and has been reported to
reduce significantly HIV-1, including zidovudine-resistant
strains, as well as hepatitis B virus in patients with chronic
hepatitis B infection. Therefore, lamivudine placental trans-
fer and amniotic fluid concentrations were investigated in
HIV-infected pregnant women. The lamivudine concentra-
tion in the amniotic fluid of pregnant patients who received
antiretroviral therapy was higher than that in maternal and
fetal plasma. It was postulated that lamivudine is able to
cross the placenta by simple diffusion and is concentrated in
the amniotic fluid (Mandelbrot et al., 2001). The pharmaco-
kinetics and safety of nelfinavir when used in combination
with zidovudine and lamivudine in HIV-infected pregnant
women have been studied (Bryson et al., 2008).
Valacyclovir has been recommended for prevention of
and therapy for herpesvirus infection and 10 years ago was
a new candidate for treatment of symptomatic herpes infec-
tion during pregnancy (Kleymann, 2003).
Studies have shown that steady-state nevirapine plasma
concentrations are influenced by pregnancy (Nellen et al.,
2008). Maternal-fetal transfer and amniotic fluid accumula-
tion of nucleoside analog reverse transcriptase inhibitors in
HIV-infected pregnant women provide evidence that amni-
otic fluid concentrations of nucleoside reverse transcrip-
tase inhibitors, such as lamivudine (3TC) and zidovudine
(ZDV), were up to14-fold higher than blood plasma drug
concentrations (Chappuy et al., 2004).
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