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OCR for page 39
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
OCR for page 41
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.
Representative terms from entire chapter:
biologic markers
