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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 pigsand 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 experimentsparticularly in .

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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 OCR for page 223
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- tioni.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.

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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

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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- nentspossibly glycoproteinsto 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

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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- tionare 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

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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.,

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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

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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

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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

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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

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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) 5and 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)

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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-

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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 filamentsboth primary cytoskeletal proteinsin 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

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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

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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.

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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-

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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.