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Introduction In the past, some believed that most problems with fertility, fetal damage, and congenital malformations were due to reproductive dysfunctions in the female. Recent years, however, have seen the ac- cumulation of a considerable body of knowl- edge regarding male-mediated effects on development and the effects of environmen- tal agents on male reproductive function (see Strobino et al., 1978; Soyka and Joffe, 1980; Wyrobek et al., 1 983a; and Schrag and Dixon, 1985 for reviews). The reproductive functions of the male mammal are to produce sperm, to attract receptive and fertile females, and to de- posit adequate numbers of genetically normal sperm in a manner and at a time suit- able for fertilization. Those functions involve numerous organ systems and complex brain functions. In human beings, social and psychologic factors are important for reproductive success. In this context, we attempt to develop markers for quantify- ing key aspects of human male reproductive processes and for detecting dysfunction associated with subfertility or abnor- malities resulting from exposure to xeno- biotic agents. The male's role in reproduction can be divided broadly into physiological and genetic functions. Any change in eith- er function can reduce the ability of sperm to fertilize an egg. Genetic defects in 39 male germ cells occurring during spermato- genesis or during their passage through the efferent ducts may persist and lead to infertility, early or late pregnancy loss, congenital malformations, perinatal problems, and heritable mutations (chro- mosomal or genie) that may cause disease later in life and be passed on to future generations. BIOLOGIC MARKERS OF MALE PHYSIOLOGIC DAMAGE Biologic markers of male reproductive physiology have at least four major appli- cations, as follows: Development and evaluation of safe and acceptable male contraception methods would be greatly facilitated by the exis- tence of reliable biologic markers of nor- mal male reproduction. About 15% of couples are infertile; in about 40% of infertile couples, the infertility is in the male (Mosher, 1980). Reliable biologic markers would help de- termine biochemical mechanisms for infer- tility and might help monitor treatment. Global industrialization has led to increased use of and dependence on chem- icals. There are no human or animal data on the reproductive effects of most chemi- cals. Reliable human biologic markers

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40 would permit direct measurements of repro- ductive effects in people exposed to xeno- biotic agents. Direct human studies would circumvent problems associated with extrapolation of results of toxicity studies from animals to humans. Comparable markers in animals and human beings would provide a quantitative means for extrapolating animal data to man and would allow investigations of phys- iologic and toxicologic mechanisms. The development and validation of mark- ers for human male reproductive health typically require a multidisciplinary approach that includes basic research in animal and human reproductive biology, engineering and statistical development of automated and quantitative procedures, clinical studies of human factors that affect variation and of the predictive value of individual markers, and epidemio- logic studies of populations exposed to xenobiotic agents. Fertility potential, which is a combined function of the male and female, is dif- ficult to assess in humans. Hence, the predictive value of abnormal ranges from biologic marker assessments requires knowledge of mechanisms. In the sections that follow, numerous ways of assessing normal and abnormal physiologic function and genetic variation in male germ cells and reproductive organs are discussed. In most instances, their utility as markers of exposure or markers of effect have not been assessed fully. The prevalence of humans exposed to en- vironmental, occupational, and therapeu- tic agents that are potential reproductive toxins argues strongly for the development of validated methods for measuring ger- minal and reproductive damage directly in people. Methods used in the evaluation of the reproductive health of human males are in three broad classes: personal his- tory, physical examination, and labora- tory analysis. The clinical application of personal history and physical examina- tion in fertility assessment is important in screening populations and evaluating laboratory analyses. Laboratory analyses include testicular biopsy, hormonal analyses, and semen analyses. ABLE REPRODUCTIVE TOXICOLOGY Markers differ in the numbers and kinds of assays available to measure them, in the degree of quantitation attainable so far, in the extent to which their under- lying mechanisms are understood, and in their feasibility for human studies. They also differ in sensitivity, specifi- city, and predictive value from assay to assay and from use to use. For example, as many as three applications of some of the markers (e.g., sperm concentration, motility, and structure) have been pro- posed: as markers of sperm production, as indicators of fertility status, and as indicators of exposure to a reproductive toxin. The validity of each marker depends on its specific application (e.g., see Chapter 7 for a discussion of sperm num- ber). A multistep process is required to vali- date all new markers of male reproductive health. Marker validation requires the description of measurement statistics of well-characterized groups and the un- derstanding of the biologic and technical factors that affect measurement variabil- ity. Validation also requires a critical and quantitative assessment of a marker's ability to discriminate, e.g., between men with normal sperm production and men with abnormal sperm production, fertile men and infertile men, and exposed men and unexposed men. However, the use of semen markers to discriminate the effects of environmental, therapeutic, and occupa- tional exposures does not necessarily require that a marker be associated with fertility status. In the latter applica- tions, distributional characteristics of semen values in exposed and unexposed cohorts can be compared with each other and with historical controls to identify exposed populations and to evaluate the effect of exposure. BIOLOGIC MARKERS OF GENETIC DAMAGE AND HERITABLE MUTATIONS IN HUMAN GERM CELLS Tests that measure the potential for mutagenicity in humans are important, because some populations are being exposed to drugs, as well as environmental

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INTRODUCTION and occupational chemicals, and the muta- genicity of those chemicals in laboratory animals, such as mice, is well established. Although induction of germinal mutations by mutagens is well documented in animals, there is no firm evidence that any agent has induced germinal mutations in people, and monitoring for increased mutations in humans has proved unsuccessful. From the animal literature, at least two broad types of induced genetic damage in exposed people-gene mutations and chromosomal alterations (in either chro- mosome structure or number)-can be ex- pected. As discussed in Chapter 9, current human methods might be inadequate to de- tect induced mutations among offspring using the sizes of exposed cohorts evalu- ated to date. Also, increasing evidence points to induced genetic damage in human male germ cells, especially for ionizing radiation. New approaches are under development to improve the detection of induced ger- minal mutations in people. These employ recent recombinant DNA and molecular techniques and use two sources of tissue for analyses: sperm of exposed men and somatic tissue from offspring of exposed individuals. These innovations promise the increased sensitivity needed to detect genetic defects in the germ cells of small cohorts of mutagenized people. Germinal mutations are rare events and, as described in Chapter 9, the devel- opment of human assays for measuring gene- tic damage in germ cells presents special validation challenges, including a pre- cise understanding of the spectrum of mutational damage detected, as well as an understanding of underlying mechanisms and assay responsiveness. Once developed, these detection assays would provide a means to identify human germinal mutagens and to manage human exposure so that the associated risk of inherited genetic de- fects and diseases could be reduced. Also, investigations of germinal mutations in laboratory animals, including mice (Chapter 8), is continuing to increase understanding of the relative sensitivity of germ cell stages, mutational mechanisms in germ cells, and the spectrum of genetic lesions induced by mutagen exposure. 41 Germinal exposures to mutagens clearly are not a male issue solely. In animals, male and female germ cells are known to be sensitive to germinal mutagens. Chapter 9, which discusses human germinal mutagens is included in this report because part of the progress in new technologies in- volves sperm-based assays. However, any new mutational assay that uses offspring tissue clearly would be applicable for studies of either or both human male and female exposures. IMPORTANCE OF ANIMAL STUDIES IN MARKER DEVELOPMENT Humans would be the species of choice for all investigations of human reproduc- tive health and of factors leading to in- fertility. However, human studies are constrained by patients' needs, human subjects' rights, and the difficulties of controlling genetic, environmental, and exposure factors. Most discoveries in human reproductive biology and the markers in use today were based on earlier investigations in ani- mals. Animal studies of basic biochemical mechanisms, cellular processes, and the effects of genetic and environmental fac- tors require continued support. Multi- generational studies in animals are the cornerstone of reproductive toxicity testing of chemicals (Zenick and Cleeg, 1989~. Animal end points include markers of gonadal, extragonadal, seminal, and hormonal pathophysiology and markers of offspring quantity and quality. Animal experiments permit control of such vari- ables as age and genotype (i.e., pharmaco- kinetics and metabolism), as well as exposure routes, dosages, and durations of exposure. Animal studies are not lim- ited to noninvasive markers, as are human studies. The effects of many chemicals have been evaluated in animals with varied study designs and exposure conditions, and animal data have been used to provide presumptive evidence of human reproduc- tive toxicity. However, animal data on a given chemical are typically incomplete, and it is difficult to come to a definite conclusion regarding reproductive ef- fects. In addition, there are uncertain- _ _

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42 ties in quantitative interspecies com- parisons, and large safety factors are involved in extrapolation of risk to humans. Animals and humans can differ markedly in their responses to chemical exposure. That is well illustrated by comparing the germinal effects of 1,2-dibromo-3-chloro- propane (DBCP) in animals and humans. Human exposure to DBCP, a highly effective nematocide, resulted in male infertility and germ-cell aplasia at doses that showed no other signs of organ or system toxicity (Whorton et al., 1977~. Some data suggest increased frequencies of spontaneous abortions among the wives of exposed work- ers, and there is indirect evidence that DBCP is a human germinal mutagen (Wyrobek et al., in press). However, the response among animals is highly species-depen- dent. At one extreme, mice are resistant to DBCP; essentially no induced germ- cell killing or germinal mutagenesis has been observed after varied exposures of different strains. Clearly, the negative germinal-mutagenicity data from the mouse are not relevant for human mutagenic risk assessment. In contrast, DBCP exposure of rats at similar and lower doses induced extensive germ-cell killing, subfertili- ty, and dominant lethality. Those results suggest that further efforts are needed to develop the rat and other mammals as models for assessing human germinal toxi- city and mutagenicity. Species differences underscore the need for improved strategies for extra- polating reproductive effects from ani- mals to humans. Ideally, animals with metabolism and biologic effects most simi- lar to humans' would provide the most reli- able data for extrapolation to humans. Detailed molecular comparisons of metab- olites, adduct formation, and molecular damage (e.g., DNA strand breakage) might provide a means for comparing responsive- ness quantitatively among mammals. For example, certain types, quantities, and kinetics of the formation and removal of DBCP metabolites, adducts, and other mole- cular damage in mouse, rat, and human, may be associated with induced germinal cell MAll,E REPRODUCTIVE TOXICOLOGY killing, infertility, and mutagenicity. As part of this research, improved tech- niques to detect adducts, metabolites, and molecular damage are required (the use of monoclonal antibodies, high-per- formance liquid chromatography, etc.~. Sensitive detection methods would benefit studies of both animals and humans and ultimately the assessment of human ger- minal risk. ORGANIZATION OF MALE REPRODUCTION SECTION This section evaluates markers that could be used to assess reproductive ef- fects of pathophysiologic changes and heritable genetic damage in males. The markers discussed are at varying stages of validation, ranging from markers that already are used to assess the effects of human exposure to reproductive toxins (e.g., sperm number) to markers that are only promising concepts and very early in their development. The section begins with a review of the clinical procedures for evaluating male infertility, includ- ng medical history, physical examina- tion, and semen analyses (Chapter 3~. This is followed by detailed evaluations of available methods and promising research related to markers of the structure and function of the testis, epididymis, acces- sory sex organs, and semen and sperm (Chapters 4-7~. Semen analysis is dis- cussed in several chapters because it is a noninvasive means of obtaining informa- tion regarding testicular, epididymal, and accessory organ function. Chapter 8 discusses the concept and status of gene- tic risk assessment. It is followed by a discussion of methods for detecting ger- minal and heritable mutations in human beings (Chapter 9~. Relevant research questions and promis- ing concepts that may lead to future im- proved markers of male reproductive and genetic toxicity are identified through- out the section and are summarized in Chap- ter 10. Detailed consideration of sexual behavior, sexual differentiation, and puberty is beyond the scope of this report.