Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 223
an
Cell Biology:
Identiiiing Biologic Markers
Expressed During F,nriv Pr~,onnnov
This chapter discusses the biologic
processes that are important before and
around the time of implantation. Many
cellular and developmental stages at this
early time of pregnancy are critical to
the further development of the pregnancy.
In the section of this report on female
reproductive markers, one hormonal
change (hCG) is discussed (see Chapter
15~. In this section, many more potential
markers are discussed, including cellular
differentiation, diffusible cellular
products, and additional hormonal con-
centration changes. The markers discussed
here will lead to better understanding
of the biologic processes and possible
mechanisms of toxic action.
The greatest risk to successful eesta-
tion occurs around the time of implanta-
tion, when the maternal uterine environ-
ment and the embryo interact to establish
pregnancy. The chance of a couple of proven
fertility to conceive offspring in any
menstrual cycle is about 25% (Vessey et
al., 1976; Short, 1979), but it is diffi-
cult to determine the extent to which this
low success rate is due to errors or dys-
functions in ovulation, fertilization,
implantation, or later development. Live-
stock have a high incidence of early embry-
onic loss-up to 40% in pigs—and up to 50%
of human conceptions are estimated to un-
223
_ _ _ _ _O ~ J _ _ _ =~ ~ ~ _~ ~ _d
dergo early embryonic termination (Leri-
don, 1977; Short, 1979).
Records of human IVF/ET programs indi-
cate a 15-20% rate of completed pregnancy
(Webb and Glasser, 1984); however, the
fertilized embryos that are transferred
are selected for apparent viability.
IVF/ET failure is attributable to events
related to implantation (Edwards et al.,
1980; Webb and Glasser, 1984~. Implanta-
tion errors constitute one of the largest
causes of failure in reproductively com-
petent persons in IVF programs (Fig. 20-
1~. The high risk of implantation failure
might be compounded by xenobiotic agents
introduced into the intrauterine environ-
ment.
In the United States and other countries,
experimentation in humans is problematic
because of ethical and legal restrictions
(Andrews, 1984a,b). Knowledge of early
human development necessarily depends
more on comparative studies than does
knowledge of other human biology.
Although imperfect as analogies of human
reproduction, examples of many mammalian
reproductive systems are available for
study, and comparative analysis has pro-
vided insight into identification of com-
mon mechanisms characteristic of the pre-
implantation period (Amoruso, 1981~.
Animal experiments—particularly in
.
OCR for page 224
OCR for page 226
OCR for page 227
OCR for page 228
OCR for page 229
OCR for page 230
OCR for page 231
OCR for page 232
OCR for page 233
OCR for page 234
OCR for page 235
OCR for page 236
OCR for page 237
OCR for page 238
OCR for page 239
OCR for page 240
Representative terms from entire chapter:
cone cells
224
FIGARO 201 Relative contn~ution of differ-
ent states of very eartr gestation to outcome of
early IVF/ET. Summary of data on treatment
group resulted in first successful IVF/Er preg-
nanc~r (Edwards et al., 1980~. Of 79 women
monitored during menstrual cycles, 68 under-
went laparoscopy for attempted oo
MARKERS DURING EARLY PREGNANCY
Conditions in the female reproductive
tract necessary to maximize the opportuni-
ty for embryo implantation in the uterine
environment are well understood (Psychoy-
os, 1973; Glasser and Clark, 1975; Glasser
and McCormack, 1980, 1982~. However, the
understanding does not explain the specif-
ic effects of hormones, drugs, and toxic
agents on pregnancy. More incisive methods
of investigation-such as those developed
for cell biology, immunology, and molecu-
lar biology-must be found to define the
regulatory biology of blastocyst-endome-
trial interactions and to show how they
can be interrupted by xenobiotic agents.
The implantation process is initiated
when trophectoderm cells of the blastocyst
come into intimate contact with the recep-
tive uterine endometrium (Sherman and
Wudl, 1976; Glasser and McCormack, 1980,
1982~. Progressive phases of this process
are controlled by molecules exchanged
directly by cell-to-cell communication
(Enders et al., 1981 ~ and modulated by
molecular signals from stromal-epithelial
communication (Cunha et al., 1985~. These
molecules are expressed in response to
the same steroids that synchronize the
blastocyst and uterus from conception.
To interpret specific cell-to-cell
interaction during implantation events,
homogeneous populations of individual
cell types involved directly in implanta-
tion—i.e., endometrial epithelial and
stromal cells, blastocyst trophectoderm
and ectoplacental cone cells, and tropho-
blast giant cells-are isolated. The cell
populations can be cultured in vitro so
that biochemical mechanisms that regulate
their differentiation and interactions
can be studied (McCormack and Glasser,
1980; Glasser and McCormack, 1981; Scares
et al., 1985; Glasser and Julian, 1986;
Glasser et al., 1987b). Recent develop-
ments for studying trophoblast interac-
tions used three-dimensional culture
systems, in which trophoblast cells are
grown as free-floating spheroids (White
et al., 1988a). Such trophoblast spheroids
can be used to study interactions with
explants or monolayer cultures of other
tissues, e.g., endometrium (White et al.,
1988b).
To apply the concept of biologic markers
225
to the interaction of xenobiotic compounds
with the early mammalian developmental
processes, a research strategy that de-
pends on animal experimental models can
be formulated (Glasser, 1985~. Cellular
and biochemical methods can be used to
identify and validate critical structural
or functional markers of the regulatory
processes involved in the differentiation
of tissue during each step. Those markers
provide a basis for selecting the most
appropriate markers for use outside the
laboratory.
ASSESSING ENDOMETRIAL
SIGNALS
The role of the uterus is defined by
the responses of its epithelial and
stromal cells to a specific sequence of
ovarian hormones (Psychoyos, 1973; Glas-
ser and Clark, 1975; Glasser and McCormack,
1982~. Whether the uterine epithelial
cells respond directly to hormone instruc-
tions or indirectly to signals emanating
from hormone-regulated uterine stromal
cells is unknown (Cunha et al., 1985; Bigs-
by and Cunha, 1986~. In uterine epithelial
or stromal cells, xenobiotic agents might
interfere with the binding of a steroid
hormone to a target-cell receptor or might
affect steps in the biochemical responses
to hormonal regulation initiated by the
binding of hormones. These effects might
be independent or could be coupled, so a
response could be additive or synergistic.
Use of receptor analysis to identify
biologic markers of endometrial cell biol-
ogy is limited by the difficulty of gaining
access to markers and target cells.
Current data concerning effects of xeno-
biotic agents on the uterus describe re-
sponses of the whole uterus and do not re-
veal the extent to which each cell type
contributes to the net uterine response
or which cell type is at risk under differ-
ent environmental conditions. If informa-
tion of this nature were available,
efforts could focus on risk reduction.
Homogeneous populations of endometrial
cell types from the uterus can be isolated
(McCormack and Glasser, 1980; Glasser and
Julian, 1986) and their regulatory biology
and differentiation studied in vitro.
226
Experiments with primary cultures of uter-
ine epithelial cells must have confluent
and polarized monolayers to obtain biolog-
ically relevant data identifying and as-
sessing markers and their interaction with
xenobiotic agents. This research design
is useful, because the basal surface of
the epithelial cells is accessible for
experimentation and analyses, inasmuch
as the cells are cultured on semipermeable,
matrix-impregnated supports.
Tables 20-1 and 20-2 list candidate mark-
ers to assess the status of uterine epi-
thelial or uterine stomal cells. These
markers are not practical for medical moni-
toring because they are not readily acces-
sible. Nevertheless, they are useful
to identify critical targets, times,
and processes.
TOXICI7~YDURING PREGNANCY
Uterine Secretions
Early morphologic studies suggested
that uterine secretions might be biochemi-
cal correlates of receptivity develop-
ment. The quality and quantity of secre-
tions that accumulate in the uterus
during various phases of the reproductive
cycle have been studied in several species,
to identify phase-specific or hormone-
specific secretory products that might
be markers of particular facets of implan-
tation. Few attempts have been made to
examine critically which cell type is
the source of each secretory product,
whether proteins that are secreted are
synthesized de nova, or whether the appear-
ance of such possible markers represents
selectively stimulated protein synthesis.
Analyses of human endometrial washings
have not revealed large concentrations
TABLE 20 1 Putative Biologic Markers to Assess Status of Uterine Epithelial Cells
Cellular or
Developmental
Stage
Biologic Marker
Comments
Proliferation
Postmitosis
Cell number; mitotic
index; labeling index
Short-term biosynthetic
and metabolic index;
profiles of apical versus
basal cell surface and
secretory proteins/glyco-
proteins
Long-term biosynthetic and
metabolic index; differen-
tial response to steroid
hormones; differential
trafficking of apical versus
basal cell surfaces and
secretory proteins/glyco-
proteins
Preimplantation Timing of final stages of
differentiation; protein/
glycoprotein profiles of
apical versus basal surface
secretions; timing of rising
titers of progesterone and
estrogen; differential
receptor response
Implantation Biochemical index of
terminal differentiation;
different changes in ster-
oid hormone receptors;
specific early pregnancy
factors
These markers assess mitogenic response of uterine
epithelial cells
These markers evaluate physiochemical and biologic responses
to regulatory factors (hormones, growth factors); can be used
to evaluate analogues, congeners (phytoestrogens, catechol
E); can use other ceil and tissue models
These markers assess hormonaLly regulated differentiation
during transition of hostile to neutral uterus; changes blocked
by castration; because of embryonic diapause in some animals,
these markers can describe ability to reactivate blasto~rtes
These markers describe transition from neutral to
sensitized or receptive uterus
These markers describe uterine epithelial receptivity to
blastopyst, attachment, uterine influence on the blastopyst, and
initiation of stromal cell differentiation; growth factors not
well studied
MARKERS DURING EARLY PREGNANCY
TABLE 2~2 Putative Biologic Markers to Assess Status of Utenne Stromal Cells
Cellular or
Developmental
Stage Biologic Marker Comments
Proliferation
Postm~tosis
Changes in number of
pytoplasm~c and nuclear
endoplasnuc reticulum
Rate of cell division;
continued changes In
number of endoplasm~c
reticulum
Pre~mplantation Number of endoplasm~c
reticulum and frequency
of stromal mitosis
Number of endoplasm~c
reticulum; mammal rate
of stromal cell division
Implantation
of specific proteins. Most studies have
found that uterine secretions consist
mainly of common serum proteins (Wolf and
Mastroianni, 1975; Roberts et al., 1976;
Hirsh et al., 1977~; however, transudation
of serum proteins appears to be selective,
but variable throughout the endometrial
cycle (Beier and Beier-Hellwig, 1973~.
Electrophoretic analysis and more recent
radiolabeling studies have revealed spe-
cific proteins not found in serum. These
range from low-molecular-weight compo-
nents—possibly glycoproteins—to pro-
teins of 60-67 kilodaltons (Wolf and
Mastroianni, 1975; Tzartos and Surani,
1979; Sylvan et al., 1981~; several appear
to be specific to the secretory phase of
the endometrium during the menstrual
cycle.
In uterine washings, various enzymes
have been found at concentrations above
those in serum; for example, glycosidase
(Hansen et al., 1985), antitrypsin (Rob-
erts et al., 1976; Casslen and Ohlsson,
1981), and fibrolytic activity (Werb et
al., 1980) in human uterine fluid vary
throughout the menstrual cycle. Those
enzymes might be involved in the implanta-
tion process (Tzartos and Surani,1979~.
Maathuis and Aitken (1978) have shown
that proteins are secreted throughout the
proliferative and secretory phases of the
cycle, and their concentrations are lower
after ovulation. That is in accord with
227
Not well studied
These markers describe responsiveness of cells to steroids; not
sensitive to dec~duogen~c stimuli; uterine stromal growth
factors not well studied
These markers assess uterine sensitivity
These markers describe stromal component of receptive
uterus
the finding of lower fluid volume in the
secretory phase (Clemetson et al., 1973~.
The finding does not preclude secretion
of specific proteins into the uterus during
this phase. Associated with lower fluid
volume is increased potassium ion concen-
tration, which is particularly high
around the time of implantation. Other
nonprotein components also change
throughout the cycle. The concentration
of fructose increases around the midsecre-
tory phase, but glucose concentration
changes little throughout the cycle (Doug-
las et al., 1970; Maathuis and Aitken,
1978~.
In rats and mice, high estrogen concen-
trations elicit intrauterine secretions,
particularly of proteins; high concentra-
tions of progesterone reverse this effect
(Armstrong, 1968; Surani, 1975; Aitken,
1977; Pratt, 1977; Fishel, 1979~. Unique
uterine secretory proteins have been re-
ported-one protein appeared 18-20 hours
after estrogen injection (Surani, 1975~.
That interval corresponds with the pulse
of ovarian estrogen that is released by
normal rats late on day 3 and during early
phases of implantation on day 4. The ap-
pearance of unique proteins could be coin-
cidental, and the relationship of induced
proteins to a specific embryonic or endome-
trial function is unproved. Intrauterine
proteins might be serum transudates, meta-
bolic products, or degradation products
228
that are unrelated to the specific process
being studied.
Because they affect the uterine environ-
ment, uterine secretions probably help
to regulate the blastocyst awaiting im-
plantation. Such regulation might be:
· Direct, i.e., secretions might be
information proteins that signal the
blastocyst or adhesive proteins that in-
crease cell-to-cell communication.
· Indirect, i.e., secretory proteins
might serve as nutrients or as modulators
of pH or isotonicity of the uterine envi-
ronment.
· Passive, in that secreted proteins
contribute to endometrial cell mainte-
nance or the pharmacodynamics of the myo-
metrium.
The opportunity for proteins to influ-
ence implantation success exists for ap-
proximately 72 hours in humans (Hertig
and Rock, 1945; Hodgson and Pauerstein,
1976; Croxatto et al., 1978), but only 18-
24 hours for species with short pre-
implantation periods (Glasser and
McCormack, Webb and Glasser, 1984~.
Studies of uterine secretions have
been unrewarding in demonstrating a regu-
latory role for some proteins or in sug-
gesting a cause-and-effect relationship
that might make the marker useful to detect
specific effects. In part, the difficulty
arises from heterogeneity of the uterine
secretions, which prevents us from distin-
guishing between the degrees to which ovi-
ductally and transepithelially trans-
ported stromal secretions contribute to
the secretory profile.
Uterine Epithelial Cells
Hormone-regulated expression of spe-
cialized uterine epithelial cell func-
tions-recognition, adhesion, and secre-
tion—are related to differentiation of
epithelial cell structure and functional
polarity. Polarity depends on establish-
ment of cross- linked interepithelial
tight junctions and results in distinct
apical and basal surface membrane domains.
Development of cross-linked tight junc-
tions coincides with active remodeling
TOXICI1~YDURING PREGNANCY
of the apical surface. Both development
and remodeling are stimulated by proges-
terone and occur in viva immediately before
implantation. Putative biologic markers
of these processes are listed in Table
20-1.
Experimentally polarized uterine epi-
thelial cells are necessary to analyze
hormonal mechanisms that regulate spe-
cialized epithelial cell functions, and
enough information has been collected
to support an in vitro model of polarized
epithelial cells. Proliferation, growth,
and differentiation of polarity occur in
epithelial cells isolated from immature
rat uteri and cultured on matrix-impreg-
nated filters in the presence of estrogen,
progesterone, or both (Carson et al., 1988;
Glasser et al., 1988; Glasser and Julian,
1989~. The model approximates the in viva
situation by providing access to the epi-
thelial cell through its basal surface.
Supplemental regulatory factors stimulate
polarized cells and validate the experi-
mental model for use in studying expression
of epithelial cell-specialized functions.
Stromal proteins, which can modify
the hormonal response, have access to
the cell through its basal surface. Pro-
files of glycoconjugates and proteins
associated with epithelial cells in
apical and basal surface secretions or
in the apical surface membrane are analyzed
during hormone-regulated proliferation,
growth, and differentiation of filter-
cultured epithelial cells. Differential
changes in the apical surface membrane
and its secretions are detected by differ-
ences in the protein/glycoprotein pro-
files and their distributions (Carson et
al., 1988; Glasser et al., 1988; Glasser
end Julian, 1989~.
Uterine Stromal Cells
Studies of uterine stromal cells have
produced a variety of biologic markers
that can be used to monitor responses to
regulatory agents and to identify proc-
esses that limit the responses. (Table
20-2~.
It has been suggested that uterine epi-
thelial cell response to estrogen (and
perhaps progesterone) does not involve
AL9RKERS DURING EARLY PREGNANCY
the steroid hormone receptors endogenous
to those cells in fetal neonatal uteri
(Cunha et al., 1985; Bigsby and Cunha,
1986~. Rather, the response is indirect
and is stimulated by interaction between
steroids and hormone receptors in the un-
derlying stromal cells. The extent to
which these principles apply to cells in
sexually mature animals remains to be in-
vestigated, but evidence points to modula-
tion of uterine epithelial cells by stromal
cells; furthermore, stromal cell decidu-
alization might require signal transduc-
tion via epithelial cells (Lejeune and
Leroy, 1980~. Decidualization is a unique
structural (and presumably functional)
transformation of fibroblastlike cells
of the uterine stroma to a distinctive
tissue that is later discharged. The new
tissue has giant, polygonal, multinucle-
ate, endoreduplicative cells with abun-
dant thin cytoplasmic filaments. These
cells are rich in glycogen and lipids;
specific alterations in nucleic acid
(Glasser, 1975) and protein synthesis
(Glasser, 1972; Glasser and Clark, 1975;
Bell, 1979) are believed to be associated
with the process. The decidual cell re-
sponse can be induced in uteri, by
blastocysts or by a variety of artificial
stimuli in laboratory animals (Glasser,
1972~. Decidualization is a progesterone-
dependent process (Glasser and Clark,
1975), and its response to the blastocyst
signals that the uterus has matured.
Research has provided many interesting
clues and directions for potential mark-
ers, but has not yielded markers that are
practical for medical monitoring.
Further research should focus particular-
ly on two processes sensitive to xenobiotic
agents: stromal-epithelial cell communi-
cation and hormone-regulated differentia-
tion of stromal cells and remodeling of
their extracellular matrices.
Recombination of epithelial and mesen-
chymal cells from various tissues has been
instrumental in showing that stromal cells
give rise to directive and permissive fac-
tors that influence epithelial cell dif-
ferentiation. Studying recombinations
of uterine epithelial cells cultured on
a matrix-impregnated filter with the stro-
mal cells or their conditioned media ap-
229
plied to the basal side of the filter prom-
ises to yield more detailed and specific
data and permit analysis of functional
differentiation. Accessibility to the
basal secretory compartment also will
permit research on the influence of xeno-
biotic agents on secretions from epitheli-
al cells or on the effect of secretions on
the morphologic and functional differen-
tiation of uterine stromal cells. Studies
probably will not yield useful markers
in the immediate future, but they will
determine whether cell-to-cell communica-
tion might be subject to toxic effects.
The role of decidual tissue remains
to be defined, although several functions
have been ascribed to it (Glasser and Mc-
Cormack, 1980~. Decidualization probably
is a conservative mechanism in which troph-
oblast invasion of the endometrium is con-
trolled and limited during establishment
of the hemochorial placenta (Bryce and
Teacher, 1908~. Regardless of function,
Decidualization reflects a change in the
synchronized endometrial substrate that
follows blastocyst attachment, and it
might be sensitive to xenobiotic agents.
Studies of in vivo and in vitro rat decid-
ualization (Glasser and Julian, 1986;
Glasser et al., 1987b) have demonstrated
that non-coordinate expression the inter-
mediate filament subunit, desmin, and
v~ment~n is a correlate of hormone-
regulated stromal cell differentiation.
Desmin is marginally detectable in undif-
ferentiated stroma and accumulates at a
£reater rate than cell protein. Vimentin
expression is a marker of decidual cell
growth; vimentin increases in proportion
to decidual cell protein. Ninety-six hours
after decidualization is initiated, the
concentration of decidual cell desmin
is equal to or greater than that of
vimentin.
Inductive accumulation of the extra-
cellular matrix proteins laminin and
entactin, which are absent from undiffer-
entiated stroma, is also a marker of de-
cidualization (Wewer et al., 1985; Glasser
et al., 1987b). Other indexes include de-
creased production and reorganization
of fibronectin (Grinnell et al., 1982;
Glasser et al., 1 987b), expression of a
decidual luteotropin (Markoff et al.,
230
1983; Maslar et al., 1986), and appearance
of heparin sulfate proteoglycan and chon-
droitin sulfate proteoglycan (Wewer et
al., 1985~. These markers suggest that
the surfaces and later the extracellular
matrix of epithelial and stromal cells
are remodeled in response to the hormonal
interactions that regulate uterine recep-
tivity to the blastocyst. The changes
alter cell-to-cell interactions and ac-
commodate the specialized attachment and
invasive functions of the differentiat-
ing trophoblast. Interference with the
remodeling of the stromal extracellular
matrix into a basal, laminlike structure
might have far-reaching consequences,
not only for the programmed advance of
trophoblast through stroma, but also for
mobilized migration of B and T lymphocytes
into the uterus as elements of implantation
and establishment of the hemochorial
placenta.
Those alterations are ordered by a pre-
cise program of hormone-specific synthe-
sis of informational proteins (Glasser
and Clark, 1975; Glasser and McCormack,
1980~. One of the instructions might be
provided by a luteotropinlike peptide
hormone synthesized by decidual cells
(Markoff et al., 1983; Maslar et al.,
1986), thereby involving decidual cells
in the regulation of endometrial response
to the trophoblast. If that endocrine
capability also occurs in humans, the pre-
implantation e ndome trial s tro ma mig ht
directly affect the intricate modulation
of the preparatory processes required for
implantation.
Evidence of an endocrine function of
decidual tissue is the demonstration
that human prolactin (hPRL) is a separate
hormone discrete from growth hormone and
that amniotic fluid contains extremely
high concentrations of immunoreactive
hPRL. Specific functions of hPRL in the
female include regulation of postpartum
lactation in mammary glands, reproductive
cycle regulation, pregnancy maintenance,
and embryonic growth and development.
hPRL in amniotic fluid is involved in fetal
osmoregulation. Suppression of maternal
pituitary hPRL during pregnancy does not
affect amniotic fluid concentrations.
It has been determined that the source of
TOXIC17~YDURING PREG~4NCY
amniotic fluid hPRL is decidualized en-
dometrium of pregnancy.
Proliferative human endometrium cul-
tured in the presence of progesterone-
with or without estrogen-has been report-
ed to produce immunoreactive hPRL (Daly
et al., 1983a). Immunoreactive hPRL is
an in vitro culture product of decidua from
day 23 of the menstrual cycle through
term (Daly et al., 1983b). The amount of
hPRL produced is a function of the extent
of decidualization and is progesterone-
dependent. Production of hPRL by prolifer-
ative endometrium after 6 days in culture
with progesterone in the absence of a
blastocyst suggests that hPRL synthesis
and secretion could be produced in vivo
by early luteal endometrium, including
predecidual cells. If hPRL synthesis and
secretion could be produced in vivo, matur-
ation of the uterus to a receptive environ-
ment might occur earlier in the human than
anticipated and provide an endocrine basis
tor the success of early cleavage-stage
embryos after IVF/ET. The specific decidu-
al hormone in the circulation of women
presumed to be pregnant then could be as-
sayed, and a practical marker of endome-
trial differentiation around the time of
implantation would be identified. Experi-
mental data suggest that synthesis and
secretion of this hormone occurs before
the earliest time reported for hCG expres-
sion (Saxena et al., 1974~.
Detection of markers of changes around
the time of implantation may be possible,
in light of recent studies of endometrial
cell types and their interaction with the
blastocyst (Copp, 1979~. Interpretation
of risk-assessment data developed in the
laboratory must take into account func-
tional polarity and epithelial-stromal
cell communication, as well as possible
toxic effects on matrix remodeling as an
expression of hormonal regulation of these
cells.
Investigations of Cervix and Vagina
Although studies of uterine epithelial
and stromal cells have not yielded a prac-
tical biologic marker, investigations
should be extended to the cervi~c and va-
gina. Vaginal and cervical changes
AtARKERS DERRING EARLY PREGNANCY
reflect uterine changes, and the vagina
and cervix are accessible; correlative
studies of epithelial and stromal cells
from these areas might identify markers
useful for studies of human reproductive
toxicology. Specific hormone-regulated
changes in cervical glycoproteins (Chil-
ton et al., 1981) and in the expression of
cytokeratin patterns by differentiating
vaginal epithelial cells (Kronenberg and
Clark, 1985) offer strong support for such
correlative studies.
TROPHOBLAST BIOLOGIC
MARKERS
Prospective studies of human tropho-
blast development or studies seeking
trophoblast signals of early human preg-
nancy (0 to 3 weeks) are so constrained
by social and ethical restrictions (An-
drews 1984a,b) that they are not practical.
Use of nonhuman trophoblasts prevents the
study of hCG as a pert-implantation marker,
but laboratory and domestic animals offer
many other advantages for critical experi-
mental analysis. The following discussion
outlines current understanding and sug-
gests new approaches that might yield
valid and accessible markers of tropho-
blast response to xenobiotic agents (Table
20-3).
Trophectoderm
The first cells to differentiate in
the mammalian embryo are the trophectoderm
FIGURE 2~2 Schematic diagram of rat con-
ception during midterm pregnancy. Dashed lines
show regional limits of dissection to harvest ~ndi-
vidual groups of trophoblast giant cells. Diagram
based on information presented by Davies and
Glasser, 1968. Source: Glasser et al., 1987a.
231
cells. This differentiation occurs at
embryo compaction during the cleavage
stage, at which time the blastomeres assume
an inside-outside orientation (Johnson
et al., 1981~. Although trophoblast cells
do not contribute to embryo formation,
they become an integral part of the placen-
ta(ShermanandWudl, 1976~. Trophectoderm
and its differentiated derivatives-the
trophoblast giant cells (TGCs)-are in-
volved intimately and structurally in most
of the placental functions that are criti-
cal to viviparity (Billington, 1985~.
Thus, compaction and later the process
of blastocoelation are critical points
at which adverse effects of toxic agents
could influence development.
The mammalian embryo comprises an inner
cell mass-the presumptive embryo-and
a blastocoele; both are surrounded by
trophoblast cells. The trophectoderm
cells covering the inner cell mass are
termed ~polar," and those surrounding the
blastocoele are called "mural" (Fig. 20-
2~. Preimplantation and early postim-
plantation trophectoderm cells are pro-
liferative and diploid.
When trophoblast cells lose contact
with the inner cell mass or attach to the
uterine epithelium, they lose their abili-
ty to divide. The trophoblast cells cease
division and become giant cells, which
have more DNA than other cells (Ilgren,
1983~. In the human, the increase in DNA
is accomplished mainly by cell fusion (the
cytotrophoblast becomes the syncytio-
trophoblast). In the mouse and the rat,
~~\ Decidua basalis
Polar Trophoblast If\
1,
Labyrinth
Reichert's
membrane
Mural Trophoblast \~V- /
\ /
~ ~ Decidua capsularis
232
TOXICI7YDURING PREGNANCY
TABLE 2~3 Putative Biologic Markers to Assess Status of Trophoblast
Cellular or
Developmental
Stage Biologic Marker Comments
Syngamy Number of ceils with
n + n versus 2n chro-
mosomes
Totipotency Number of viable
embryos; 37-kilodalton
one-cell embryo marker
Compaction Allocation of cells
inside versus outside;
number of tight junc-
tions and desmosomes;
efficiency of ion
channels
Blastocoelation Macromolecular
Early blastocyst
This marker might assess sensitivity of nuclear target
2n nucleus might be different target from an n + n
nucleus; can evaluate nuclear determinant of sensitivity, such as
maternal versus paternal genome; can use microinjection to
introduce toxins to cytoplasm or nucleus
These markers assess deletion of affected pronucleus,
sensitivity of cleavage stages, expression of genome
(paternal, maternal), and influence of xenobiotic on progression
These markers assess effect on determination and
differentiation of cell lineage between inner cell mass
and trophectoderm
synthesis; expression of
paternal genome markers;
efficiency of proton pumps
and ion channels
Histology of inner
cell mass
Trophectoderm: con-
centrations of cytokera-
tins; changes in lectin-
binding specificity;
changes in profiles of
surface and secretory
proteins and glycoproteins;
chorionic gonadotropin
concentrations
These markers assess transepithelial and paraepithelial
transport; methods are improving rapidly
Not well-studied markers of cell lineage; several models
have been described (embryonic carcinoma cell model and
embryonic stem cell model)
These markers describe increasing complexity and
cleavage stages of trophectoderm differentiation
Concentration of early Role of these factors still unclear; existence is not
pregnancy factors confirmed in all species
Late blastocyst Loss of zone pellucida This marker describes time of hatch and changes in
hatching biochemistry of zone pellucida
Recognition Expression of trophec- These markers describe immunologic response to embryo;
toderm recognition can identify implantation defects
antigens
Attachment Presence of specific lectin These markers assess implantation and postrecognition
receptors and oligonucleo- attachment of trophectoderm and uterine epithelium;
tide acceptors; changes in changes suggest reorganization of trophectoderm cell
surface and secretory surface
proteins, and glycocon-
jugates; cytokeratin
expression
Cessation of Decrease in mitotic This marker identifies effect of mitosis-inh~iting factors
mitosis labeling index in mural in initiating differentiation
trophectoderm followed by
polar trophectoderm
Binucleation Proportion of cells
with 2n chromosomes;
increase in nuclear
and cytoplasmic areas
These markers assess endocycles and role of DNA in
sensitivity
AL 4RKERS DURING EARLY PREGNANCY
233
Cellular or
Developmental
Stage Biologic Marker Comments
Endoreduplica- Proportion of ceils with
lion >4nchromosomes;shift
in activity of DNA
potrmerases; patterns
of DNA
DNA fragments on southern
blots or RNA fragments on
northern blots
Concentration of
specific proteins cytokeratins (40, 51, 55,
44 and 46 kilodaltons),
actin, and tubulin
Depression of
Secretion of tin
sue remodeling
enzymes
Synthesis and
secretion of
steroid
hormones
Synthesis and
polypeptide
hormones
Concentrations of early
pregnancy factors
Concentration of 37-
ldlodalton mitogen
Concentrations of
growth factors (insulin
growth factor-I, -II,
platelet-derived
growth factor)
Loss of adhesive
properties, matrix pro-
teins and receptors;
migration and invasion
of cell types; activity
of specific enzymes,
such as plasminogen
activator
Concentrations of
progesterone, estrogen,
and testosterone;
density of steroid
receptors
Humans: concentrations
of hCG a and B
subunits and hPL
Concentrations of
various pregnancy-
associated factors
Rodents: concentrations
of placental lactogen~
PL-1 and PL-2
These markers assess sensitivity of trophoblast giant cells.
(For instance, do changes in chromosome number alter
sensitivity? Is entire genome being replicated? Are
genes expressed differentially?)
These are cell lineage markers for trophoblast giant cells;
simple epithelial cell type not found in inner cell mass;
markers also assess role of cytoskeleton in differentiation;
changes in actin and tubulin not well studied, but do not
appear to be specific responses
These markers assess establishment of trophoblast-uterine
relationship; the role of these factors is not well defied
This marker assesses fetal growth
Role of these factors in organogenesis and fetal growth
unknown
These markers assess sensitivity of differentiation
to regulation by Intracellular environment; specific
enzymes not well studied; role of enzymes might be
secondary
These markers can be used to study factors that initiate
up and down regulation of steroids and explore possible
autocrine and paracrine regulation; Reimplantation
synthesis and secretion have been validated only in pig,
cow, and sheep; postimplantation validated in many species; role
not well understood
These markers assess differentiation secretion of human
cytotrophoblast to syn~tiotrophoblast
These measurements have been described, but roles are
not adequately defined
These markers assess differentiation of trophoblast giant
cells; PL'1 and PL'2 are under different regulatory
mechanisms
the DNA increase occurs in the nucleus via
endomitotic and endoreduplicative mechan-
isms (Nagl, 1978; Ilgren, 1983~.
Blastocyst attachment is an initial
step in implantation, starting differen-
tiation processes that manifest them-
selves in the establishment of a definitive
placenta. Highly regulated structural
and functional differentiation is found
during the interval of blastocyst trophec-
toderm attachment to a receptive uterine
epithelium and TGC apposition with ele-
ments of the maternal vascular system
(Sherman and Wudl, 1976; Glasser and Mc-
Cormack 1980, 1982~. These modifications
in trophoblast function support viability
and the ordered patterns of embryonic
growth and development. Progression of
234
the trophoblast through the remodeled
substrate of uterine decidual cells is
ensured by increased secretion of proges-
terone by either the corpus luteum or the
trophoblast cells.
Steroidogenesis
Studies of rat blastocysts and their
trophoblastic outgrowths cultured in
vitro (Beier and Beier-Hellwig, 1973)
have yielded information on the patterns
of secretion of various hormones. Rat
blastocysts secrete progesterone at in-
creased rates (0.1 to 0.5 pa/ml per
blastocyst) during the initial phases of
hatching-equivalent gestation day (EGD)
5—and outgrowth (EGD 6~; the rate in-
creases to 6 or 7 pa/ml per blastocyst on
EGD 8-13 and then falls to a lower, but
still high, rate of 4.5 pa/ml per blasto-
cyst after EGD 14 (Fig. 20-3~. Estradiol
and testosterone patterns of the rat
blastocyst and trophoblast outgrowths
can be demonstrated, but they are so er-
ratic as to be unreliable as markers of
trophoblast differentiation.
Progesterone production by rat
blastocyst or trophoblast outgrowths
occurs during gestation (EGD 6 to 8) when
maternal plasma progesterone already has
increased to nearly 60 ng/ml (Glasser and
McCormack, 1980~. Maternalplasmaproges-
FIGURE 2~3 Steroid production by rat blastm
cyst outgrowths. Day 4 blasto~rsts (sp + =
day 0) were recovered from uterus and cultured
In groups of 1~15 In 3 ml of NCIC-135 plus 10%
fetal calf serum In 35-mm plastic dishes. Medi-
um was changed daily. Hormones assayed with
specific radio~mmunoassay of spent medium.
Equivalent gestation day = age of blastoc~st In
culture equivalent to age that it would have been
if left In utero. Source: Glasser et al., 1987a.
TOXICI7'YDURING PREGNANCY
terone plateaus at 90-120 ng/ml between
EGD 10 and 12.
In the presence of this great pool of
maternal plasma progesterone, no role
has been identified for progesterone,
estradiol, or testosterone contributed
by the trophoblast cell. Trophoblast pro-
gesterone might have a paracrine role
in ontogeny of decidual cell differentia-
tion or an endocrine role in trophoblast
regulation of its estradiol receptor (Mc-
Cormack and Glasser, 1978) or in rat pla-
cental lactogen synthesis (Scares et al.,
1985~. If trophoblast steroidogenesis
does have a regulatory role, how progester-
one affects development, and the relative
sensitivity of trophoblast steroidogene-
sis to toxic insult should be compared with
the sensitivity of steroid hormone produc-
tion by the corpus luteum.
Trophoblast Giant Cells and Placental
Hormones
Differentiation of the fetal placenta
is essential to embryonic development in
mammals. In rodents, TGCs are an integral
component of the fetal placenta. In viva
and in vitro primary and secondary TGCs
are derived from mural and polar blasto-
cyst trophectoderm, respectively. Cells
of the ectoplacental cone are derived from
polar trophectoderm (Ilgren, 1983) and
-
llJ
is
o
en
In
o 8
v)
o
D -
o
O _
-
4
of
~0
Us
V
o
cc 1
11
.
11
' C— 4
Us) 1 2
18
10
In
o 08
UO
o
D 06
04
o 0.2
_ . ~
DAYS IN CULTURE.
~ :~ 4't
· 4 +2 +4
EOUIV. GESTATION DAYS: (6) (8)
RAT BLASTOCYST (day 4)
In vitro culture
24 hr. steroid production/blastocyst
1'-''-
+8 +10 +1 2
(12) (14) (16)
MARKERS DURING EARLY PREGNANCY
are precursors for additional secondary
TGCs. TGCs cease to divide as a primary
step in their differentiation from troph-
ectoderm to ectoplacental cone cells:
15-25% of the trophectoderm cells become
binucleate or multinucleate. Morpholog-
ically, TGCs are nondividing, polytene
giant cells. The c number (number of copies
of haploid DNA) in TGCs increases from
about 2-4 to as much as 1,024, as a result
of endoreduplication (Barlow and Sherman,
1972~. In human trophoblast cells, no more
than 40% become endoreduplicative
(Friedman and Skehan, 1979~. Functional-
ly, TGCs express secretory proteolytic
enzymes (proteases, collagenases, and
elastases), steroid hormones (progester-
one, testosterone, and estrogen), and,
at midgestation, one or more peptide hor-
mones (placental lactogens). Rodent
trophoblast does not produce a hormone
similar to hCG.
Placental peptide hormones display
characteristics similar to hPRL and
growth hormone (GH). For this reason,
they have been termed placental lactogens
( PLs ) o r c ho rionic so matomammo trop ins
(Talamantes et al., 1980; Josimovich,
1983~. PLs are the dominant trophic hor-
mones affecting fetal development during
the latter half of pregnancy (Brinsmead
et al., 1981). They can regulate fetal
tissues development directly. For exam-
ple, ovine PL stimulates amino acid trans-
port and ornithine decarboxylase activity
in fetal tissues (Hurley et al., 1980;
Freemark and Handwerger, 1982) and indi-
rectly alters maternal protein, carbohy-
drate, and lipid metabolism (Kaplan and
Grumback, 1981).
The biologic and biochemical charac-
teristics of PLs vary among species, but
all are secretory products of placental
giant cells. In the human, PLs are produced
by the syncytiotrophoblast (Watkins,
1978~; in sheep, by the trophoblast binu-
cleate cells (Martal et al., 1977; Watkins
and Reddy, 1980; Wooding, 1981~; and in
rats, by the TGCs (McCormack and Glasser,
1980; Soares et al., 1985~. These PL secre-
tory cells differentiate from readily
identifiable precursor cell populations-
human cytotrophoblasts (Enders, 1965),
sheep uninucleate cells (Wooding, 1981),
235
and mouse trophectoderm and ectoplacental
cone cells (Rossant and Tamura-Lis,
1981; Ilgren, 1983~. TGC differentiation
has not been studied rigorously; the most
thorough investigations have been done
in rodents, primarily mice. The
transformation of mouse ectoplacental
cone cells to differentiated TGCs has been
analyzed (Rossant and Ofer, 1977; Johnson
and Rossant, 1981; Rossant and Tamura-
Lis,1981~.
Rats and mice produce two types of PLs
that can be distinguished biochemically,
immunologically, and temporally by their
appearance during pregnancy (Kelly et al.,
1975; Robertson et al., 1982; Soares et
al., 1985~. The early form (PL-1) is pres-
ent during midpregnancy (days 9- 11 in the
mouse; days 10-12 in the rat), and the late
form (PL-2) predominates during the latter
half of pregnancy. PL- 1 has a higher mole-
cular weight and is more acidic than PL-2
(Soares et al., 1985~. Both are active in
radioreceptor assays and bioassays for
lactogenic hormones (Soares et al., 1985~;
however, neither is active in a growth
hormone radioreceptor assay. Serum PL-2
has been measured in the mouse throughout
pregnancy and around parturition (Soares
et al., 1982; Soares and Talamantes, 1984~.
Ovaries have an inhibitory influence on
serum PL-2 concentration; the fetus has
a trophic influence (Soares and Talaman-
tes, 1985~. Distinct genetic differences
in serum profiles of PL-2 have been report-
ed (Soares et al., 1982~. PLs are products
of the trophoblast, rather than of other
components of the placenta (Glasser and
McCormack, 1981; Soares et al., 1985; Glas-
ser and Julian, 1986), and the change from
PL-1 to PL-2 that occurs in vivo has not
been reproduced in vitro.
Differences in hormone oroduction
~ ^^ . ~ ~ ~
.
might be et~rects of local environment.
For example, polar TGCs are in contact
with maternal decidua basalis and the mes-
enchymally induced trophospongiosum of
the chorioallantoic placenta; mural
TGCs are between the decidua capsularis
and the parietal endoderm (Davies and Glas-
ser, 1968~. Production might be mediated
by signal differences from the different
environments or by their interpretation
by TGCs. That suggests that residual ef-
236
facts of xenobiotic agents in undifferen-
tiated uterine stromal cells might influ-
ence trophoblast endocrine function when
these cells decidualize. The absence of
qualitative regional effects confirms
that all TGCs are similar; functional dif-
ferentiation of an individual TGC results
in sequential expression of PL-1 and PL-2
by the same cell. Thus, the response of
the single trophoblast target to toxic
exposure is influenced by the developmen-
tal stage at which the trophoblast cell
is at risk.
Expression of PL- 1 during organogenesis
might be coincidental but PL- 1 and organo-
genesis could be more closely related-
possibly through a reciprocal relation-
ship with insulin growth factors (Adams
et al., 1983~. Thus, early exposure to a
xenobiotic agent might produce more seri-
ous consequences than if exposure occurred
as TGC differentiation and organogenesis
were terminating.
Trophoblast Giant Cell Cytoskeleton
Morphologic and functional differentia-
tion have been linked to alterations in
the expression of cytoskeletal proteins;
therefore, microtubules and intermediate
filaments—both primary cytoskeletal
proteins—in endocrine-competent TGCs
(Glasser, 1986) have been analyzed to iden-
tify markers.
Tubulin and polymerized microtubules
have been identified in TGCs, but the mi-
crotubule organizing center has not been
described. TGCs from trophectoderm are
unlike most other cells. A decentralized
system for microtubule renucleation oc-
curs at multiple sites throughout the cyto-
plasm, and microtubules may be sensitive
and ubiquitous targets for xenobiotic
agents. Disruption in microtubule assem-
bly might have extensive effects on cell
function.
In rodents, trophectoderm cells are
the first embryonic cells to exhibit inter-
mediate filament proteins (Glasser,
1986~. Mouse preimplantation blastocyst
trophectoderm cells displayed two inter-
mediate filament proteins (54 and 46 kilo-
daltons), identified as cytokeratins.
In contrast, midgestation TGCs displayed
TOXICI7~YDURING PREGN~4NCY
not only major components of 54- and 46-
kilodalton intermediate filament pro-
teins, but 52-, 45-, 43-, and 40-kilodalton
species. Of these additional keratins,
the 52- and 40-kilodalton species were
most prominent (Glasser and Julian, 1986~.
No specific function has been assigned
to any of the proteins of the various cyto-
keratin gene families, but these addition-
al landmarks might be important in mon-
itoring functional and morphologic
differentiation of specialized cells
(e.g., ectoplacental cone cells and other
stem or precursor cell populations) (Vene-
tianer et al., 1983~.
In vitro analysis of morphologic and
functional differentiation of blastocyst
outgrowths and isolated TGCs have been
effective in identifying valid biologic
markers of processes essential to estab-
lishment of the fetal placenta (Copp,
1979~. Other markers have not been dis-
counted, but these experiments have deter-
mined PLs to be unique among the candi-
dates. Identification of differentiated
TGC as the only cellular source of these
hormones is important. Further studies
to assess the risk of a presumably pregnant
female can now focus on the development
of the trophoblast and its expressed secre-
tions.
Ectoplacental Cone Cells
Ectoplacental cone cells arise from
polar trophectoderm and are diploid and
proliferative. Because of their numbers,
endoreduplication, and differentiation
in culture, they present a model that might
resolve some questions posed by studies
of other trophoblast cell populations.
Mouse ectoplacental cone cells trans-
planted ectopically or cultured transform
into giant cells (Rossant and Ofer, 1977;
Johnson and Rossant, 1981; Rossant and
Tamura-Lis, 1981~. TGCs of rodents endo-
reduplicate their nuclear DNA; they are
polyploid and synthesize proteins that
are characteristic of TGCs, ectoplacental
cone cells (Johnson and Rossant, 1981~.
The presence of the inner cell mass adja-
cent to ectoplacental cone cells is be-
lieved to maintain proliferation and in-
hibit transformation to TGCs (Rossant and
Af'4RKERS DURING EARLY PREGNANCY
Ofer, 1977). As gestation progresses,
some ectoplacental cells are pushed farth-
er from the inner cell mass or its deriva-
tives and transform into TGCs. Thus, ec-
toplacental cells provide a reservoir of
precursor cells that contribute to tropho-
blast growth and expansion during the sec-
ond half of pregnancy.
When ectoplacental cells lose contact
with the inner cell mass and its deriva-
tives (Rossant and Ofer, 1977; Wooding,
1982), they lose their ability to divide,
and they become giant cells with nuclear
DNA contents greater than 4 c. Very little
information has been developed since the
adventofrecombinantDNA methods regard-
ing the organization of the TGC genome or
gene expression of genomic DNA, which can
increase from 2 to 1,024 c. Extensive en-
doreduplication during the course of nor-
mal differentiation makes trophectoderm
cells, ectoplacental cone cells, and TGCs
unique mammalian cells- and thereby unique
models to study gene expression. Studies
of the locations of repetitive-DNA se-
quences and changes of protein expression
during endoreduplication in TGCs offer
opportunities to learn about the cellular
and molecular base of differentiation.
Regulation of Trophoblast Hormone
Synthesis
For the process of giant cell differen-
tiation, the relative advantage of nuclear
DNA endoreduplication compared with regu-
lation of early postimplantation interac-
tions is unknown. PL expression might
correlate with replication of the entire
trophoblast genome, rather than with am-
plification (Barlow and Sherman. 1972-
Sherman et al., 1972~.
itiate PL- 1 synthesis in the enlarging
genome, signals that direct the sequence
rearrangements obligatory for the transi-
tion of PL-1 to PL-2, and what initiates
PL-2 synthesis are unclear. In analysis
of human trophoblast, those questions have
to do with regulatory foci coincident to
cytotrophoblast differentiation. Xeno-
biotic agents might interrupt or redirect
normal development of the trophoblast.
, _,
Signals that in-
237
EXTRAPOLATION TO HUMAN
TROPHOBLASTS
Experiments with in vitro models of
trophoblast cells have identified the
importance of their postmitotic differen-
tiation and their derivatives in the con-
trol of normal mammalian development.
Markers of structural and functional
events critical to differentiation also
have been reported.
Although rodent trophoblast experi-
ments have enlarged understanding of some
aspects of postimplantation biology, they
have not yielded markers that reliably
signal the status of the trophoblast dur-
ing the high-risk periods before and
around implantation in mice, rats, or hu-
mans. The residence time of the free blas-
tocyst of these mammals in utero is rather
short. In contrast, some livestock-such
as sheep, pigs, and cows-have long uterine
residence time of the free blastocyst
(more than 12 days), and the blastocysts
synthesize and secrete gonadal signals
that mark the immediate Reimplantation
period (Heap et al., 1981~. Generation
of steroid implantation signals by blas-
tocysts with short residence in utero
(Dickmann and Dey, 1974) has not been con-
firmed (Heap et al., 1981~.
Human Chorionic Gonadotropin
Human chorionic gonadotropin is a prac-
tical marker of human trophoblast cell
development. The major recognized action
of hCG is its role in regulation of steroid-
ogenesis in the corpus luteum. Thus, hCG
serves a pivotal function in pregnancy
maintenance. hCG also has been implicated
in regulation of steroidogenesis in the
fetal testis and the fetal adrenal gland
(Stock et al., '971~. Its role in the regu-
lation of placental steroidogenesis is
controversial. In situ hybridization has
shown that the x-subunit mRNA of hCG is
found in cytotrophoblasts, syncytiotro-
phoblasts, and the intermediate forms be-
tween those two definitive cell types
(Pijnenborg et al., 1985~. However, the
,B-subunit mRNA can be found only in the
intermediate form and syncytiotropho-
blast (Dreskin et al., 1970; de Ikonicoff
238
and Cedard, 1973; Hoshina et al., 1985).
Those findings argue strongly that tropho-
blast differentiation must be in progress
before the §-subunit becomes available
for dimerization with the c-subunit and
before the rapid secretion of the intact
hCG molecule.
The widespread use of the radioimmuno-
assay (RIA) for hCG derives from ready
availability of specific antibodies at
reasonable cost, ease of using the assay,
extensive background on interpretation
of plasma titers, and absence of more spe-
cific markers. (See Chapter 15 for discus-
sion of the development of these assays
and the problems with using them in epide-
miologic studies.) Use of RIA for hCG has
received general acceptance for determin-
ing whether pregnancy has begun and is
based on the rationale that a rise in plasma
hCG reflects the presence of a functional
trophoblast and verifies the existence
of pregnancy (Canfield et al., 1984~.
TOXICITY DURING PREGNANCY
Decrease in hCG titers signals interrup-
tion of pregnancy, but does not identify
cause or tissue site of initial damage.
The earliest measurement of complete
hCG depends on differentiation of enough
intermediate and terminal syncytiotroph-
oblast cells to produce hCG that can be
detected with the assay. This occurs 3-
4 days after the blastocyst has become
implanted in the uterine endometrium.
Therefore, hCG is not an effective marker
of events associated with transition from
morula to blastocyst, of entry into the
uterus, and of various events before and
around implantation that occur during days
5-10 (Fig. 20-4~. Saxena and colleagues
( 1974) reported that a luteotropic
hCG-like factor is produced by the human
blastocyst and is detectable immediately
before implantation (day 6), but this has
never been confirmed. Aggressive prospec-
tive research with animal or experimental
models is required to identify trophoblast
(~=ertilization(~)?EPF, PAP, Decidual Luteotropin By) hPL detected
hCG detected (unconfirmed)
(a) Implantation (3 Anticipated Time of Menses
(3 hCG detected (confirmed)
Implantation Periods
~ .....
, Pre- , , Post-
......
~ ~-
.....
0
......
a__ :::::: ~ I I I
-.: ~.:~_ T I I I
~~; ~~-_ ~ I T I I I
/ Jim - - -_ ~ 1 I I I I I
'/ ? Decimal Hormone
:::: GUT _ _ _ _ _ ~ 1 I I I I
~ ~ I ~ ~ ~ ~ 1 t , . _ _ _ _ _ _ , l
0 1 2 3 4 5 6 7 8 9 10 12 14 16 18
Days after Mid-Cycle LH Surge
FIGURE 2 - Some major events occurring in utero that define pert-implantation period in human. Note absence
of confirmed makers available. Sensitive radioimmunoassays of hCG that use specific monoclonal antibodies do not
detect hCG until day 10, at least 3 days after implantation and initiation of trophoblast differentiation. EPF and PAP
are trophoblast products that might be used as markers of risk status of embryo. Decidual luteotropic hormone might
senre as marker of endometrial condition. Source: Glasser et al., 1987a.
MERGERS DURING EARLY PREGNANCY
signals that might be expressed during
the critical high-risk periods of days
5-7and5-10.
Placental Lactogens
Although PLs are not hormones of the
period around implantation, they are im-
portant to fetal well-being. PLs are prod-
ucts of transcription and translation
of the five-gene human placental lactogen
(hPL) family. Translation and transcrip-
tion take place in the syncytiotropho-
blast, but not cytotrophoblast (McWil-
liams and Boime, 1980; Boime et al., 1982;
Hoshinaet all, 1982, 1985~.
Biologic actions of hPL are interpreted
in terms of its homology to growth hormone
and prolactin. Effects of hPLs have been
demonstrated in several nonprimates
(Friesen, 1966), but its role in humans
has not been resolved (Josimovich, 1966~.
Whether hPL directly affects fetal lipid
and carbohydrate metabolism in response
to transient fluctuations in nutrient
availability or has a chronic role in modi-
fying the set point and response time of
various systems of intermediary metabol-
· e
m 1S unc ear.
PLs might have indirect effects and
be mediated by reciprocal action with in-
sulin growth factor (IGF-I) (Pijnenborg
et al., 1985~. Some data have demonstrated
that hPL is not required to maintain human
pregnancy (Parks et al., 1985~.
An injury to trophoblast cells that pre-
vents normal expression of hCG would inter-
fere with early events involved in estab-
lishing the placenta and also interfere
with hPL expression. However, the sensi-
tivities of hPL and hCG genes and their
mRNAs to particular xenobiotic agents
could be different, and hPL and hCG gene
products should be monitored. Effects
on hPL would be detected only between
weeks 2 and 3 of gestation, when hPL is
scheduled to be secreted.
Pregnancy-Associated Factors
Owing to the lack of an hCG homologue
in nonprimate mammals and the inability
to detect very early status, further search
is under way for macromolecules that are
239
present in females during early gestation,
but absent in the nonpregnant females.
A directory of more than 20 factors specif-
ic or unique to pregnancy has been devel-
oped (Bohn, 1985~. New proteins detected
with immunologic methods in placental
extracts or in sera of pregnant women con-
tinue to be reported (see Chapter 19 and
Bell, 1983; Ellendorff and Koch, 1985~;
these proteins range in molecular weight
from 25,000 to 2.1 million and are mainly
glycosylated proteins. Detectable
amounts of factors thought to be pregnancy-
specific have been immunologically as-
sayed in oviduct secretions and in seminal
plasma.
The usefulness of these pregnancy-as-
sociated factors as markers is restricted
by important problems (Chard, 1985~. In
many cases, a secretion has neither cell-
nor tissue-specific origin. The presumed
uniqueness of a putative marker might be
related to design of an appropriate assay
and to physiologic relevance of that assay.
Much of the debate regarding specificity
of the early pregnancy factors rests on
the functional significance of rosette
inhibition assay (Beverley, 1985~. Ana-
lyzing and identifying the principal role
of single-pregnancy factors has led to
the suggestion that they are better under-
stood if considered as categories of preg-
nancy-associated proteins, rather than
as single, nominally specific factors
(Table 20-4) (Chard, 1985~.
Some proteins contribute to trophoblast
maintenance and function (perhaps through
their ability to bind steroids), some have
protease inhibitory functions, and others
have been implicated in the immunobiology
of pregnancy. The time of synthesis onset
and the identification or relevance of
a suggested specific function are not
clear. One protein, pregnancy protein
14 (PP-14, an ~x1 microglobulin), has been
shown to be in high concentrations in sem-
inal plasma and increases rapidly in the
plasma of pregnant females. For these
reasons, it has been suggested that PP-14
functions at implantation and has some
role in establishing the hemochorial pla-
centa. No protein, even hCG, can be
related unequivocally to specific events
associated with fertilization, concep-
240
TOXICITY DURING PREGNANCY
TABLE 2 - Categonzation of Pregnan~y-Associated Factors
Source Functions Control
Category 1
hCG, hPL, Trophoblast
specific protein (syncytim
(SP-1), trophoblast)
Bl-glycm
protein
Category2
Pregame protein 5,
PAPP-A
Category 3
Binding proteins,
PZP
Pregnant y
protein 12
Pregnancy
protein 14
Regulation of
growth and
differentiation
(autocrine? para-
crine? endocrine?)
Trophoblast
(emit
trophoblast)
Endometnum;
decidua; maternal
Liver
Local immune
and coagulation
reactions
Binds small molecules
molecules
Number of synthetic units;
changes in blood flow, ~port,
delivegr, steroids; growth factors
Not known
Estrogen and progesterone;
mar represent maternal red
pponse to pregnancy
Does not include endometnal
surface and secretory proteins
and ~ycoconjugates
tion, or the period before or around im-
plantation (Billington, 1985~.
The questions of source and function
of these proteins might be resolved in part
through the study of differentiation of
the human trophoblast cell in culture.
A variety of methods, including recombi-
nant-DNA technology, could be used to
analyze the transition from cytotropho-
blast to syncytiotrophoblast. The tech-
nology also would be excellent for inves-
tigating the consequences of introducing
xenobiotic agents at different steps in
trophoblast differentiation. Friedman
and Skehan (1979) described a directory
of morphologic and functional properties
that characterized the transition of cyto-
trophoblastlike (CTL) cells of the BeWo
choriocarcinoma cell line to syncytio-
trophoblastlike (STL) cells. Cytolog-
ically, CTL and STL cells were identical
with their counterparts in utero.
BeWo CTL cells constitute 96-99% of
the cell types of the stored cell line.
Cultured in the presence of subthreshold
concentrations of methotrexate, the CTL
cells differentiate. At the end of a 96-
hour culture, more than 90% of the cells
assume STL structure and function. When
methotrexate is removed, the STL cells
become CTL-like. Although methotrexate
suppresses DNA synthesis, the DNA content
per cell increases by 66% coincidentally
with expression of increased hCG.
Very little work has been done with
this model to study either cell invasive-
ness or hCG synthesis, nor have the ;nter-
mediate forms been analyzed. A substantial
data base has resulted from studies of
molecular biology of gene regulation of
hCG (Boime et al., 1982), the endocrine
physiology of response to cyclic AMP and
the molecular basis for second-messenger
response (Hilf and Merz, 1985), and the
cell biology of cytoskeletal changes
(Friedman and Skehan, 1979; Glasser,
1986~. Such data would make it possible
to expand the utility of hCG as a marker
of risk associated with exposure to ~ceno-
biotics in early pregnancy.
