**3.2 Epidemiologic approaches**

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exert their effects, *b*) identify populations at risk, and *c*) evaluate reproductive and

The determination of sperm concentration, morphology, and motility remains the primary clinical assay for male infertility (WHO, 1999). The WHO guidelines on semen quality provide reference semen parameters as follows: sperm concentration >20 million/ml, 50% motility, and at least 50% normal morphology (WHO, 1999). *Oligospermia* is the term for semen with <20 million/ml sperm in ejaculate, and *asthenospermia* for semen with sperm <50% motility or <25% rapid progressive motility, *teratospermia* for reduced percent sperm with normal morphology, and *azoospermia* for the absence of sperm in the ejaculate (Braude & Rowell, 2003). It is unknown whether endocrine disrupter exposure represents a subset of the environmental risk factors for male infertility, as there are few studies that examine exposure and human semen quality. Moreover, toxicants can affect the male reproductive system at one of several sites or at multiple sites. These sites include the testes, the accessory

sex glands, and the central nervous system, including the neuroendocrine system.

The usefulness of experimental animal models is usually perceived as limited to hazard identification using the test protocols specified by regulatory agencies. However, animal models can also provide valuable support to reproductive risk assessment on many other fronts. If the focus is on human exposure, animal studies can be designed to confirm reproductive toxicity when initial observations in exposed humans are suggestive of an adverse effect. Furthermore, such observations can be extended across a wide range of exposures in animals, using any route of exposure and any specified dose versus time scenario. For example, when human exposure is likely to be acute or intermittent, animal models are ideal for defining critical exposure windows based on developmental stage or for revealing the pathogenesis of an effect at various times after exposure through recovery. This is particularly important with respect to male reproductive effects because alterations in semen quality or fertility may not become evident until sometime after the exposure, particularly if an early stage of spermatogenesis is targeted. A rodent model is most commonly used for the study of reproductive and developmental toxicity (Claudio et al., 1999). To use toxicology data derived from animal studies to advantage in risk assessment, it is critical to identify and understand species-specific differences in physiology and metabolism that may affect the response to the toxicant in question. Rodent models have, for example, been used to determine the relationship between sperm end points and function (fertility) (Chapin et al., 1997). Determining that a substance is toxic to the male reproductive system is only the first step: The next step is to examine its mechanisms of toxicity. Mechanistic information allows for predictions about the potential toxicity of individual compounds or complex mixtures in humans, for better understanding of the windows of vulnerability in the development of the male reproductive system, and for developments of possible preventive or curative measures. Acute short-term exposure models combined with serial exposure models give a complete picture of the range of effects (Claudio et al., 1999). Exposing animals over a long period of time allows for the detection of transgenerational effects from chemicals, such as male-mediated developmental effects. If developmental

developmental hazards to improve public health.

**3.1 The contribution of experimental models** 

**3. Methods of assessing male reproductive capacity** 

Epidemiologic methods for assessing the impact of hazardous substances on male reproductive health include *a*) questionnaires to determine reproductive history and sexual function, *b*) reproductive hormone profiles, and *c*) semen analysis. The choice of appropriate methodologies to study the effects of reproductive toxicants is predicated on the investigators' understanding of several factors: the nature of the exposed population; the source, the levels, and the known routes of exposure; the organ systems in which a toxicant exerts its actions; the hypothesized mechanisms of a toxicant's actions; and the techniques available to assess the effects of toxicants in the relevant organ systems (Wyrobeck et al., 1997). **Table 2** outlines the methods currently available for assessing the principal targets of male reproductive toxicants in humans--the testes, the accessory sex glands, the neuroendocrine system, and sexual function. Researchers and clinicians interested in male reproductive health and fertility are using increasingly sophisticated methods adapted from the fields of assisted reproductive technology and reproductive toxicology, including assays of sperm function, genetic integrity, and biomarkers of DNA damage. For population-based studies involving occupational groups or communities with environmental exposures, issues related to the cost, validity, precision, and utility of these methods must be carefully considered.

The testis, the site of sperm cell production and the target organ for genetic damage, is most often studied. To establish the extent of toxicity to the testis, researchers can measure the size of the testis, obtain a semen sample, or take a testicular biopsy. Standard semen analyses (including semen volume, sperm concentration, total sperm count, motility, and morphology) have been the primary research tools for studying the effects of toxicants on the male reproductive system. Epidemiologic studies have successfully utilized semen quality as a marker of fertility (Fisch et al., 1997). The uncertainties associated with traditional semen measures have led to the recent development of assays of sperm function and genetic integrity; these assays may prove more sensitive and more specific reflections of toxicant-induced effects (e.g., aneuploidy or reduced sperm motility) in individuals (Martin et al., 1997). However, The accessory sex glands, which include the epididymis, prostate, and seminal vesicle, may also be targets of toxicants (Schrader, 1997). Ethylene dibromide is one substance that affects the accessory sex glands after occupational exposure. Alterations in sperm viability, as measured by eosin stain exclusion or by hypo-osmotic swelling or alterations in sperm motility variables, suggest a problem with the accessory sex glands. Biochemical analysis of seminal plasma provides insights into glandular function by

Environmental Toxicants Induced

factors (Schrader, 1997).

**3.3 Biomarkers of genetic damage** 

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secreted primarily by the gonads and that act on the pituitary to increase (activin) or decrease (inhibin and follistatin) FSH synthesis and secretion. Within the gonads, these peptides regulate steroid hormone synthesis and may also directly affect spermatogenesis. Ongoing studies are investigating the utility of serum inhibin-B level as an important marker of Sertoli cell function and *in utero* developmental toxicity (Jensen et al., 1997). Other indicators of central nervous system toxicity are reported alterations in sexual function, including libido, erection, and ejaculation. There is not much literature on occupational exposures causing sexual dysfunction in men (Schrader, 1997); however, there are suggestions that lead, carbon disulfide, stilbene, and cadmium can affect sexual function. These outcomes are difficult to measure because of the absence of objective measures and because sexual dysfunction can be attributed to and affected by psychologic or physiologic

Biomarkers of chromosomal and genetic damage are increasingly used in the search to understand abnormal reproductive health outcomes, in part because of the possibility that there may be identifiable genetic polymorphisms which make an individual more susceptible to the adverse reproductive effects from exogenous substances. These assays provide promising and sensitive approaches for investigating germinal and potentially heritable effects of exposures to agents and for confirming epidemiologic observations on smaller numbers of individuals. Efficient technology for examining chromosomal abnormalities in sperm has only been developed recently. Chromosomal abnormalities are primarily of two types: numerical and structural. Both kinds can be attributed in some cases to paternal factors. Karyotype studies have shown that although oocytes demonstrate a higher frequency of numerical chromosomal abnormalities, human sperm demonstrate a higher frequency of structural abnormalities with less frequent numerical abnormalities (Moosani et al., 1995). In assessing sperm exposure to toxicants, it is therefore imperative to assess DNA structural integrity and not just chromosomal count. Aneuploidy is a chromosomal abnormality that causes pregnancy loss, perinatal death, congenital defects, and mental retardation. Aneuploidy, a disorder of chromosome count, is observed in approximately 1 in 300 newborns. It is speculated that of all species, humans experience the highest frequency of aneuploidy at conception, with estimates ranging from 20 to 50% (Moosani et al., 1995). Spontaneous abortions occur in at least 10-15% of all clinically recognized pregnancies. Of these, 35% contain chromosomal aneuploidy. Despite such a high frequency, there is little information about what causes this abnormality in humans. Paternal origins of aneuploidy and other genetic abnormalities can be analyzed by studying chromosome complements in human sperm. Two types of analyses provide data on chromosomal abnormalities in human sperm: sperm karyotype analysis and fluorescence *in situ* hybridization (FISH) (Moosani et al., 1995). Each technique has advantages and disadvantages. Sperm karyotyping is performed after sperm have fused with hamster oocytes. It provides precise information on numerical and gross structural abnormalities of all chromosomes from a given spermatozoon. However, only a limited number of sperm can be evaluated in each assay, and only those sperm that fertilize the oocytes are analyzable. Furthermore, this assay is technically difficult, labor intensive, expensive, and requires the use of animals. Also, it is better suited for clinical than for field studies because it must be performed on fresh semen. FISH, on the other hand, relies on the use of chromosomespecific probes to detect extra chromosomes (aneuploidy) or chromosome breaks or
