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Conclusions and Recommendations Maternal physiology is remolded during pregnancy to nurture a separate organism. The influence of xenobiotic agents must be considered for the conceptus and the mother; neither organism can be assessed separately, nor can knowledge of an agent in nonpregnant females be extrapolated to pregnant females. During the past decade, several tech- niques to assess fetal risk have become available, and increasing numbers of chem- icals have been recognized as having tera- togenic and mutagenic potential. Nonethe- less, no specific markers are available to indicate that exposure to a xenobiotic agent is directly associated with a cel- lular, subcellular, or pharmacodynamic event. Nor will such predictive markers be developed easily with existing methods. The greatest benefit of biologic markers is to establish risk of toxic response in mother or conceptus before the adverse response is expressed or becomes irrever- sible. No such biologic markers associated with pregnancy in animals or humans have been validated. Most descriptors of health status do not distinguish adverse health outcomes that resulted from environmental insult. Evaluation of potential biologic mark- ers is made difficult because of the fol- lowing factors: 253 Normative data are lacking for pro- posed markers. Multiple end points are possible for xenobiotic interaction with the mother or the conceptus. The effects of exposure are likely to depend on the stage of development of the fetus. Exacting validation is required for any putative markers of specific effects of xenobiotic exposure. RESEARCH STRATEGIES Developing biologic markers or new as- says is complicated. Insufficient data are available to formulate a strategy that could be universally applied for studying populations exposed to specific toxicants or linking assessments of various health end points to specific exposures. Normal frequencies of various health end points are unknown, as are other risk factors for the various health end points. However, some areas for immediate research initia- tives can be identified. Recent research emphasizing cellular and molecular aspects of mammalian devel- opmental and reproductive biology has identified an extensive directory of structural and functional markers. These could be used effectively to define a cause-and-effect relationship in which

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254 cell- or stage-specific events are pivotal to the differentiation of the embryo or particular endometrial cell type. Studies designed to determine the suc- cess rate of human pregnancy are needed. The developmental and biochemical aspects of fertilization, implantation, and growth of embryonic and extraembryonic tissues also must be determined. Embryonic signals around the time of implantation might be essential moderators of growth and development in the singular allogeneic environment of mammalian pregnancy, and absence of signals or presence of abnormal signals might be responsible for failed or abnormal pregnancies. Research has indicated that the tropho- blast produces and orchestrates the sig- nals that modulate maternal biochemical or immunologic acceptance or rejection of the blastocyst for implantation, but very little is known about this key tropho- blastic function. The nature of the sig- nals is not known, and this research should be expanded. New information in this basic aspect of cell-to-cell interaction should produce clinically relevant data to answer more general questions on the outcome of human pregnancy. Other evaluations of maternofetal func- tion can be obtained by pharmacokinetic studies, such as monitoring manganese in human placentas by magnetic resonance imaging. Those types of investigations offer an opportunity to study human placen- tal function with noninvasive tests for biologic markers, and the approach should be encouraged. Most toxicologic studies focus on popu- lations or animals exposed to large doses. Knowledge of events at clinically relevant serum or tissue concentrations and at spe- cific times in pregnancy is needed. Chronic exposure conditions in toxico- logic studies have different standards from acute exposure conditions during specific gestation intervals. Many mark- ers selected for consideration are not indicators of specific responses to par- ticular toxicants. The specificity of screening for a compound and its resulting insult has not been addressed as the capa- bility to detect the compound and its as- sociation with such other constituents TOXIC17YDU}RING PREGNANCY as DNA adducts in selected tissues and fluids has been. Biochemical, cellular, immunologic, and molecular assessments of tissues (e.g., chorionic villus sampling and ma- ternal blood sampling) should be empha- sized to develop assays for fetal assess- ments. A substantial contribution could be made to environmental health research by de- signing and carrying out studies to deter- mine the success rate of human pregnancy. The use of biologic markers of toxicity during pregnancy is intrinsic to such an investigation to establish quantitative and qualitative standards to be used in studies of pregnancy outcome. A few pre- dictive tests of high-risk pregnancies have been developed, but very few tests measure preimplantation events. More information is needed about the fertilized egg before implantation. At least three kinds of quantitative measurements allow determination of risk of pregnancy complications: measurements of alpha-fetoprotein, assessment of con- centrations of environmental toxicants in tissues, and classical physiologic determinations. Diagnostic and prognos- tic limits for the first two categories are being studied, and decisions must be made as to what physiologic tests to use. At least two qualitative determinations could be used in assessing possible preg- nancy complications: appearance of DNA adducts in extraembryonic fetal tissues and immunologic procedures that allow identification of high- risk couples, perhaps before they begin to produce fertilized eggs. Tests should be developed to classify high-risk couples according to the etiology of the problem; some spon- taneously aborting women could then be treated according to the pathophysiologic characteristics of the underlying defect. ACCOMPLISHING THE RESEARCH GOAL The following are recommendations are related to funding and the research agenda Encourage and support the continuing development of a data base identifying

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CONCLUSIONS AND RECOMMENDATIONS the factors that affect the various stages of pregnancy. This data base would iden- tify stage- and cell-specific biologic markers that optimize detection of cause- and-effect relationships, as well as con- stitutive factors that predispose to the success or failure of any pregnancy in a neutral environment. No informative risk assessments can be made now Nile of the processes that characterize the pre-implantation and pert-implantation periods of mammalian development. Stimulate and support studies that focus on the response to exposures to xeno- biotic agents during specific stages of gestation. The research would develop the data to characterize markers of expo- sure. Coupling these data with those de- veloped according to the recommendation above would provide a basis for risk as- sessment. Encourage active and aggressive par- ticipation of established developmental and reproductive biologists in cellular and molecular toxicology. Establish postdoctoral fellowship programs that encourage new Ph.D.s and M.D.s in toxicology to train in genetic, immunologic, and biochemical aspects of cellular and molecular developmental and reproductive biology. Support a biennial forum to encourage interaction between active contributors to the various disciplines relevant to reproductive toxicology. Because of the breadth of biologic disciplines that could be brought together in such a forum, a re- volving task force is recommended. With such a task force, a core group of scholars representing the various fields would be charged to make recommendations regarding research and to encourage research through position papers, forums, and demonstra- tion projects funded by small amounts of research money. The core group should be augmented with a rotating group of scien- tists from the same and additional biologic disciplines. 255 BRINGING THE ASSAY OUT OF THE LABORATORY AND INTO THE PUBLIC HEALTH DOMAIN In addition to the need for laboratory exploration of promising ideas, resources must be allocated to determine adequately the utility and validity of these ideas in human populations. Some markers will be useful only in carefully defined popula- tions. Such limitations need to be de- fined. Even when markers have been determined to be potentially sensitive and specific for use in the public health sector to es- tabl~sh exposure and disease potential, information collected from large, care- fully documented studies is lacking, ex- cept for a few cases, such as alpha-feto- protein. Because most exposed populations are likely to be small, a central clear- inghouse for information from isolated studies of specific exposures will be need- ed to validate markers. The Food and Drug Administration and the Environmental Protection Agency have offices to collect case studies; however, an office is needed to compile and perhaps fund studies of exposed populations. Measurement of indexes of exposure is complicated, not only because laboratory procedures are insufficiently refined, but also because obtaining samples is com- plex. Consideration must be given to the fact that therapeutic options are available for adverse conditions confirmed in the fetal period. Options might include fetal surgery (e.g., bone-marrow transplanta- tion and drainage of fluid in hydrocephalic fetuses) and choice of modes of delivery (e.~. cesarean section versus vaginal delivery for spine bifida). However, risk is associated with the use of any of these. ASSESSMENTS OF THE STATUS OF SPECIFIC MARKERS RELATED TO PREGNANCY The following recommendations concern- ing potentially useful biologic markers of exposure and effect during pregnancy represent current knowledge in clinical and basic sciences of reproduction and

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256 TOXICITY DURING PREGI~NCY TABLE 23~1 Biologic Markers Associated with Pregnancy and Possible Reproductive Hamrds Marker Immediately Usable in Large Scale Human Studies Usable in Studies of Selected Individuals Promising for Human Studies; Needs More Animal Studies Development Needed Exposure MarkersConcentrations ofTomcants or Metabolites in: Matemal Blood + Urine + Feces + Hair + Nails Tissue biopsy Endometnum Cervical mucus Uterine washings Fat Conception products Placenta Conception products Chorionic villus sample Placenta at delivery Amnion at delivery Chorion at delivery Umbilical blood Embty - Fetus Conception products Tissue Blood Hair at deliver Nails at delivery Urine at delivery Feces Amniotic fluid Amniotic cells Foreskin + + Effect Markers Matemal Clinical history General health + Reproductive history + Menstrual history + Steroid-LH-FSH concentrations Uterine prolactin Immune status hLA types TLX antigens Histology Vaginal epithelium End am et riu m Cervical mucus Globulin and other protein types Uterine blood flow + + + + + + + + + + + + + + + +

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CONCL USIONS AND RECOMMENDS TIONS 257 Immediately Usable in Large~cale Human Studies Usable in Studies of Selected Individuals Promising for Human Studies; Needs More Arsenal Studies Development Needed Placental Placental hormone concentrations: hCG hPL EPF cACrH cTSH PAPP-A Interferon Interleukin I Estradiol Estriol Progesterone Receptor number and affinity for: Bet a-a drenergic Diazepam Glucocorticoids Epidermal growth factor Opiates Somatomedin Testosterone I(DD Binding site number for: IgG-Fc Low-density lipoproteins Retinol binding protein Transcobalamin II Transferrin Concentrations of associated proteins SP1 Alpha-fetoprotein Alkaline phosphatase Transcobalamin I/II/III DNA adduct frequency Metabolic rates of: Nutrients Xenobiotics Transport efficiency of: Nutrient Xenobiotic KaIyotyping Morphometr; Histology of placer." tissues Embryo, Fetus, and Neonate Physical examination + at delivery Apgar or Brazelton scales Presence of dysmorphology + + + + + + + + + + + + + + + + + + +

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258 TOXIC17YDURING PREGNANCY Immediately Usable Promising for Human In Large Scale Usable In Studies of Studies; Needs More Animal Studies Marker Human Studies Selected Individuals Development Needed Death Carcinogenesis Growth measures + In utero + + Growth measures + Death + Presence of + dysmorphology Plasma growth factors IGF + EGF + NGF + Physiology Cardiac output + (ultrasound or Doppler) Assessment of inborn + metabolism errors or other genetic disorders Reproductive MIF + Fetal breathing rate + Techniques That Might Yield New Markers Analytic techniques for + xenobiotics Amniocentesis + Chononicvillus sampling + Doppler or biophysical + monitoring Kalyotyping Fluorescent-activated cell sorting of fetal blood samples Fetoscopy Magnetic resonance imaging Conception products Measurements of tissu~pecif~c antigens CEA AFT ACHE + + + + development that can be applied to toxi- cologic studies during pregnancy. The techniques of basic research that might be useful to assess risk associated with toxic exposure in the conceptus and mother are considered, as well as the clinical application of the techniques. Markers are divided into two major cate- gories: markers of exposure and markers of effect (Table 23-1~. Table 23-1 also lists laboratory techniques that might yield new markers. These markers have potential utility only during certain periods of the pregnancy. Subdivisions of particular periods during pregnancy reflect changing conditions, sensitivi- ties, and responses. Three periods during which relevant exposures or events could occur are considered: before and around implantation, during organogenesis, and during fetal development until just after birth. Table 23-2 lists some biolog- ic markers by the periods during pregnancy for which the marker is informative. The first period comprises any time before conception until the anticipated menstru- al period in that cycle (14 days after con- ception). Organogenesis constitutes the

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CONCLUSIONS AND RECOMMENDATIONS interval from the anticipated menstrual period until 8 weeks of embryonic age. The fetal-peripartum period extends from 8 weeks of embryonic age until 24 hours after birth. Markers of Effect Before and Around Implantation Success of a pregnancy is established before and around implantation as a conse- quence of maternal immunologic status and synchronized interaction of the uterine endometrium and the developing embryo. The greatest risk to successful completion of pregnancy occurs during the interval encompassing conception, embryo attach- ment to the uterine epithelium, and embryo advancement into the differentiating stroma. Although failed pregnancies re- sult from numerous mechanisms, includ- ing physical defects in the uterus or fal- lopian tube, endocrine abnormalities, and immunologic alterations, the risk before and around implantation might in- crease if xenobiotic agents are introduced into the intrauterine environment. Frag- mentary data indicate adverse influences of toxic agents, but most investigations have not been designed stringently and their results have often been analyzed retrospectively. The lack of biologic markers might be attributed to the paucity of stage-specific indicators of develop- mental and reproductive events that could be used in prospective studies of xenobiot- ic exposure. Epithelial cell markers are profiles of proteins and glycoconjugates that char- acterize the distinct apical and basola- teral cell surfaces and their secretory compartments. Analyses of these profiles in animals provide probes to monitor pro- gressive development from a neutral to an ova-receptive uterus, determine the role of the individual steroid hormones in regulating changes associated with epithelial cells, and monitor alterations in secretion rates. Also, biochemical and immunocytochemical matrix proteins- laminin, entactin, and fibronectin-and expression of decidual luteotropin enable the sensitivity of stromal cells to xeno- biotic exposure to be determined. 259 Biologic indicators of endometrial status are more promising than practical; the endometrium is not readily accessible for monitoring. Extending the protocols developed to analyze endometrial cell differentiation to the cervix and vagina would identify biologic markers that are more readily accessible (and thus depend less on animal models) and more readily extrapolated to studies of health and en- vironment. Differentiation of rodent trophectoderm to trophoblast giant cells has proved to be a productive experimental model for identifying potential markers of the sta- tus of the developing embryo. Trophoblast antigens and placental hormones (hCG and hPLinhumansandPL-1 andPL-2inrodents) appear to be the most promising biologic markers. Little useful information is available about the biochemical or physiologic regu- lation of placental lactogen synthesis and secretion by trophoblast giant cells. Recombinant-DNA technology, using specif- ic molecular probes, is needed to identify the mechanism of progesterone regulation. Around the time of implantation, the only proven indicator of implantation in humans is hCG. However, the presence of hCG reflects the presence of differenti- ated trophectoderm and the blastocyst, and hCG cannot be detected before blasto- cyst implantation. hCG is not a marker of a specific toxicant or response to a toxi- cant; therefore, it is not effective in identifying risk. However, hCG does re- flect the status of successful or failed pregnancy. The effectiveness of hCG as a marker of early postimplantation devel- opment would be increased if the regulatory relationships between the undifferenti- ated (cytotrophoblast) and differentiated (syncytiotrophoblast) trophoblast cells and their intermediate stages were better understood. Indicators of preimplantation status, such as early pregnancy factor (EPF) or pregnancy-associated proteins (some pur- ported to originate in the trophoblast), have been proposed as biologic markers. Until these proposed indicators are es- tablished as markers and their usefulness is proved, there is no basis for attempting

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260 TOXICITY DURING PREGNANCY TABLE 23-2 Biologic Markers Associated with Pregnancy and Possible Reproductive Hazards, by Period of Gestation l Up to 14 Days After Conception Useful: hCG Promising: Potential: Organogenesis Useful: Promising: Clinical history Steroid concentrations Blood or urine concentrations EPF TLX HLA types Uterine prolactin concentration Uterine secretions of hormones Uterine secretions of xenobiotics Sonography for size and location hCG Clinical history Hormone concentrations Sonographic measurements: Growth Size Movement Magnetic resonance imaging: Image dysmorphology Identify energy sources using p32 Assess xenobiotic or nutrient concentrations using C~3 Potential: Chorionic villus sampling: Karyotyping Enzyme activity levels and isotypes Localization-metabolism of xenobiotics DNA-adducts Flow-activated sorting During Fetal Growth Period Until Birth Useful: Sonography to assess: Growth Fetal breathing CNS function Dysmorphology Doppler blood flow measurement of: Uterine blood flow Umbilical blood flow Fetal heart rate Biophysical monitoring: Heart rate Amniocentesis to perform: Promising: Karyotyping Xenobiotic analysis Toxicity screen of embryo culture Alpha-fetoprotein Maternal serum-cord to measure concentrations of: CEA Alpha-fetoprotein HPL Xenobiotics Fetal cord sampling Fetos~copy Flow-activated sorting

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CONCL USIONS AND RECOMMENDS TIONS to use them in assessing risk associated with toxic exposures during mammalian development. The field of reproductive immunology has provided some potentially useful indicators for evaluating repeated preg- nancy losses. This documentation and categorization can reveal cases in which therapeutic intervention, such as immuno- therapy, would be useful. Toxicology assesses the effects of toxi- cants and environmental pollutants on pregnancy outcome. However, some couples are at high risk for reproductive failure. Inclusion of high-risk couples in large epidemiologic studies of the effects of environmental factors on reproductive performance clouds the issue of pathophys- iologic effects on normal patterns of re- production, because such at-risk couples are not rare. At-risk couples should be identified for any population to be inves- tigated. The study of trophoblast antibodies is essential because trophoblast tissues form the operational interfaces between maternal and extraembryonic cells in al- logeneic relationships of human pregnan- cy. None of the currently studied placen- tal proteins has proved valuable in this regard, but not all placental proteins have been studied. AA3 should be studied, because it appears to be central to the allogeneic relationship of maternal en- dothelia with extraembryonic cytotropho- blasts. Furthermore, inasmuch as placental perfusion with oxygen, nutrients, and antibodies depends on maternal blood flow through spiral arteries, it is important to develop techniques to measure spiral arterial function. Ultrasonography has provided a means to determine the effec- tiveness of spiral arterial function, but quantitative biochemical measures of placental bed perfusion would be more in- formative. Organ ogen es is Organogenesis is the period of greatest embryonic vulnerability to insult and permanent structural and functional al- teration. Such alterations are difficult 261 to assess, because the conceptus is in- accessible; no invasive procedures are performed earlier than week 8 of gestation, for fear of interrupting the pregnancy. Thus, morphologic and biochemical assess- ments of the embryo are nonexistent. How- ever, advances in ultrasonography allow early visualization of the embryonic sac and the conceptus, and the placenta and its attachment to the uterus can be exam- ined. Biochemical assessments of trophoblast hormone production can be made by detection of hCG and hPL, associated proteins of undefined function (SP1, PAPP-A, and PP11/12), and steroids. Yet these only indicate general viability of the embryo and do not provide information concerning development. Markers of embryonic devel- opment, such as fetal red blood cells, can be assessed by applying fluorescent tag- ging techniques to maternal blood sam- ples. Such techniques also might provide direct information concerning the devel- opment of specific organ systems (e.g., Mullerian inhibiting substance concentra- tion) and the relative contributions of the embryonic genome and maternal genome in producing the effects of an insult. Immunologic tests for anti-RHO, lupus, and lymphocytotoxic antibody are useful in assessing viability of a pregnancy. Biophysical monitoring can be used to de- termine uterine vascular responses to maternal toxicity. Magnetic resonance imaging is being used increasingly to as- sess structure and to determine substance localization within fetal tissues. As molecular probes specific for events in organ development are developed, markers of toxicity of specific toxicants might become evident. The Fetal and Neonatal Period The fetal and neonatal periods of devel- opment are the most productive for assess- ing effects of xenobiotic agents and the pharmacokinetics of such agents, because the conceptus is sufficiently large and accessible during these periods. Tests performed during the fetal period might only document events that occurred before and around the time of implantation and

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262 during organogenesis, but increasingly sophisticated technology should make earlier assessments possible. Biophysical monitoring provides infor- mation concerning heart rate and blood flow velocity in maternal, fetal, and pla- cental vessels. Ultrasonography measures structure, fetal weight, growth (bipari- etal diameter, femoral length, cardiac function, placental size, and maturity), and function (chest wall movements and body movements). These assessments are used to develop normative data, but no markers have been associated with specific environmental exposures. Fetal blood samples and tissue biopsies can be obtained with fetoscopy, and amnio- centesis provides amniotic fluid and cells for evaluation. The combination of chori- onic villus sampling and molecular probes for specific genetic disorders can be used to assess the capability of the conceptus to respond to environmental insult. Func- tional alterations can be demonstrated by inducing specific enzymes, such as AHH, or by interactions with specific cellular constituents, such as DNA adducts. Prog- ress is expected to come from combining magnetic resonance imaging with molecular probes. Major basic protein concentrations are markers of the onset of labor. Preterm labor-labor before 37 weeks of gestation- increases the risk of infant morbidity and mortality. The mechanisms causing preterm delivery and the effects of toxi- cants on the likelihood of preterm delivery need to be assessed. MARKERS OF EXPOSURE Biologic markers of exposure represent the detection and assessment of a specific chemical or its metabolites in an organism and demonstrate that exposure to a sub- _ stance occurred. Unfortunately, this G concentrations. information, especially on a virtually TOXICI7YDURING PREGNANCY inaccessible conceptus, usually is un- available. Measuring the amount of a chem- ical in air, water, or food to which the pregnant female is exposed (environmental monitoring) is one means to determine ex- posure. Other methods include analyses of readily accessible maternal body prod- ucts, such as blood, urine, feces, and hair. DNA-adduct evaluation is used as an in- dicator of exposure to a compound that has a specific pattern or fingerprint on 2- D gel electrophoresis and has been depen- dent on monitoring specific fluids, such as blood and urine. However, with the ad- vent of magnetic resonance imaging, spe- cific substances can be monitoredes- pecially paramagnetic ions-in specific organs without compromising pregnancy. Laboratory assessment of internal dose in fetal or embryonic compartments re- quires invasive techniques to obtain tissues or fluids. If tests of human fluids could be refined so that the effects of nutrition could be distinguished from the effects of xeno- biotics, in vitro tests could be used to screen populations for persons suscep- tible to adverse effects of exposure. For example, rat embryo culture and early mouse blastocysts have been used to screen serum from high-risk populations of women who repeatedly abort spontaneously. The rat embryo culture has been used to screen patients exposed to tobacco smoke, drugs, and anticonvulsants. Nonmammalian sys- tems have been used to screen amniotic fluids. These in vitro systems have been proposed to test whether xenobiotics are producing toxic changes in the serum of patients exposed to them. Changes might be the production of reactive metabolites of the xenobiotic compound, nutritional alterations, or the products of maternal changes such as changes in immunoglobulin