Possible Developmental Early Effects of Endocrine Disrupters On Child Health

Endocrine disrupters and child health
Possible developmental
early effects of endocrine
disrupters on child health
Possible developmental
early effects of endocrine
disrupters on child health
WHO Library Cataloguing-in-Publication Data

Possible developmental early effects of endocrine disrupters on

child health.
1.Endocrine disruptors. 2.Disorders of sex development. 3.Sex

differentiation. 4.Environmental exposure. 5.Child. I.
World Health Organization.
ISBN 978 92 4 150376 1
(NLM classification: WK 102)
World Health Organization 2012

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Possible developmental early effects of endocrine disrupters on child health
This document was initially planned by an international working group

of experts convened by the World Health Organization and the United
Nations Environment Programme in a series of meetings (December
2009 in Geneva, June 2010 in Geneva, November 2010 in Stockholm) and
then developed by contributors from University of Turku (Finland) and
Rigshospitalet (Denmark).
Contributors to this document
Jorma Toppari, Departments of Physiology and Pediatrics, University of
Turku, Turku, Finland (leader of the writing team)
Annika Adamsson, Departments of Physiology and Pediatrics, University
of Turku, Turku, Finland
Malene Boas, University Department of Growth and Reproduction,
Rigshospitalet, Copenhagen, Denmark
Anders Juul, University Department of Growth and Reproduction,
Katharina M. Main, University Department of Growth and Reproduction,
Niels E. Skakkebaek, University Department of Growth and Reproduction,
Helena E. Virtanen, Departments of Physiology and Pediatrics, University
of Turku, Turku Finland
Reviewers
Heli Bathija, Department of Reproductive Health and Research, World
Health Organization, Geneva, Switzerland
Lizbeth Lpez Carrillo, National Institute of Public Health, Mexico
Secretariat
Nida Besbelli, Department of Public Health and Environment, World
Marie-Noel Brun Drisse, Department of Public Health and Environment,
World Health Organization, Geneva, Switzerland
iii
World Health Organization

Ruth A. Etzel, Department of Public Health and Environment, World
Agneta Sundn Bylhn, DTIE Chemicals Branch, United Nations
Environment Programme, Geneva, Switzerland
Simona Surdu, Department of Public Health and Environment, World
International working group of experts

contributing to the initial planning
l
Georg Becher, Norwegian Institute of Public Health, Oslo, Norway
ke Bergman, Stockholm University, Sweden
Poul Bjerregaard, University of Southern Denmark, Denmark
Riana Bornman, Pretoria Academic Hospital, South Africa
Ingvar Brandt, Uppsala University, Sweden

Jerry Heindel, National Institute of Environmental Health Sciences,
US
Taisen Iguchi, National Institutes of Natural Sciences, Okazaki, Japan
Susan Jobling Eastwood, Brunel University, United Kingdom
Karen Kidd, University of New Brunswick, Canada
Andreas Kortenkamp, University of London, United Kingdom
Derek Muir, Environment Canada, Canada
Roseline Ochieng, Aga Khan University Hospital, Kenya
Niels Erik Skakkebaek, University of Copenhagen, Denmark
Hans-Christian Stolzenberg, Federal Environment Agency, Germany
Jorma Toppari, University of Turku, Finland
Thomas Zoeller, University of Massachusetts, US
Tracey Woodruff, University of California San Francisco, US
iv
The development and publication of this document was funded by the

National Institute of Environmental Health Sciences through cooperative
agreement 1 U01 ES02617 to the World Health Organization and its
contents are solely the responsibility of the authors and do not necessarily
represent the official views of the NIEHS.
Contents
1. Introduction
a. Endocrine system
b. Endocrine regulation of development
i. gonadal hormones sex differentiation
ii. thyroid hormones significance in brain development
1
2
4
4
5
2. Endocrine disrupters (recognized on the basis of

experimental work in vitro and in vivo)
a. Sex hormone disrupters
b. Thyroid hormone disrupters
7
7
17
3. Early effects, child health problems putatively associated

with endocrine disruption
a. Cryptorchidism
i. Epidemiology
ii.
Mechanisms
iii. Endocrine disrupter association
b. Hypospadias
i. Epidemiology
ii.
Mechanisms
c. Timing of puberty
i. Epidemiology
ii.
Mechanisms
d. Thyroid effects
i. Epidemiology
ii.
Mechanisms
21
21
21
25
26
28
28
30
32
33
33
35
35
40
40
42
45
4. Data gaps and research needs
48
5. Summary
50
6. References
52
1. Introduction
In the 1960s, congenital malformations caused by drugs used during
pregnancy alerted the medical community to the fragility of the developing
fetus. The thalidomide tragedy changed the attitude to developmental
toxicology. Only a decade later, another sad story of pregnancy-related
medication started to unravel when an association between fetal exposure
to diethyl stilbestrol (DES) and vaginal clear cell adenocarcinoma in
teen-aged girls became evident. Later on, several other adverse effects
of DES were found both in boys and girls. These unfortunate human
experiments could have been avoided, if the drugs had been properly
tested and the results given proper attention. DES is a potent synthetic
estrogen that has been linked to cryptorchidism, hypospadias and reduced
sperm production after fetal and perinatal exposure in both the human
and the mouse. It may also increase the risk of testicular cancer. Data
from numerous reproductive and developmental toxicity tests that were
increasingly performed after the 1960s brought to light a large number of
chemicals that affected the endocrine system and showed adverse effects
in the reproductive organs. The rapid increase in the incidence of testicular
cancer and deteriorating semen quality plus the emerging problems in
reproduction of wild animals were linked to possible developmental
endocrine disruption, and the chemical compounds having this kind
of effects in experimental animals were called endocrine disrupters (or
disruptors). According to WHO, endocrine disrupting chemicals are
substances that alter one or more functions of the endocrine system
and consequently cause adverse health effects in an intact organism, or
its progeny, or (sub)populations (WHO, International Programme on
Chemical Safety). Estrogenic endocrine disrupters received much of the
early attention, but soon anti-androgenic and thyroid hormone disrupting
compounds came into the focus of endocrine research. Adverse effects
of endocrine disrupters on adipose tissue, the adrenal glands and the
endocrine pancreas have further widened this research area.
There is ample evidence of endocrine disruption in wildlife, and the
mechanisms of action of endocrine disrupters have been elucidated in
experimental animals, but there is limited knowledge of the association
of human disorders with exposure to endocrine disrupters. Accumulating
data suggest that many adult diseases have fetal origins, but the causes
have remained unexplained. Reproductive disorders, especially those
1

of adult men, are strongly associated with congenital disorders such as
cryptorchidism and hypospadias. These disorders, together with testicular
cancer and impaired semen quality, form the testicular dysgenesis syndrome
(TDS) that by definition has a developmental origin. Epidemiological
studies on TDS components and other endocrine-related disorders
have often suffered from poor exposure assessment or inaccurate case
ascertainment particularly in registry-based studies. It is difficult to envisage
how epidemiological studies alone could either confirm or refute the role
of endocrine disrupters in common childhood (or adult) disorders. It is
becoming clear that we need to combine biological data on endocrine
signalling, chemical exposure data (including data on mixtures), genetics
and proper epidemiological methods by the means of systems biology to
advance the recognition of endocrine disrupters and the prevention of
adverse health effects.
The present document is a short summary of the current knowledge of the
effects of endocrine disrupters on child health. We focus on the congenital
disorders, cryptorchidism and hypospadias, which have a clear endocrine
connection, on thyroid hormone-related problems, and on puberty. Some
of the endocrine disrupters, such as polychlorinated biphenyls (PCBs)
also have adverse effects on neurocognitive development. However, that
is a topic of an entirely different large body of literature that is not related
to endocrine disruption and therefore not presented here. Even endocrine
disruption itself is a huge research area, and we have not been able to
include all studies here. We hope that this serves as an introduction to new
studies and aids in better understanding of the developmental effects of
endocrine disrupters on child health.
a. Endocrine system
The endocrine system regulates the metabolism and function of the body.
Endocrine glands secrete hormones that act on their target organs through
cognate receptors. The targets are in many cases also endocrine organs that
secrete hormones acting on the next level and also inhibiting the upper level
via negative feedback. We will focus only on the hormones that are essential
in the regulation of development of the brain and reproductive organs.
Sexual differentiation and reproductive functions are specifically under
hormonal control. Thyroid hormones are essential for brain development
and normal metabolism of the whole body. The regulatory system of both
reproductive hormones and thyroid hormones involves the hypothalamus
2
in the brain, the pituitary gland connected to the hypothalamus and

the peripheral thyroid gland and gonads. Hypothalamic gonadotropin
releasing hormone (GnRH) neurons stimulate pituitary gonadotropins to
secrete gonadotropins follicle stimulating hormone (FSH) and luteinizing
hormone (LH) that act on the gonads. FSH stimulates inhibin production
in the testis and ovary, which inhibits FSH production in the pituitary. LH
stimulates testosterone production, which serves an inhibitory function in
the upper level. Both gonadotropins influence estrogen secretion from the
ovary, and that has both inhibitory and, before ovulation, stimulatory effect
on GnRH neurons and the pituitary. This hypothalamo-pituitary-gonadal
(HPG) axis (Figures 1A and 1B) has also yet another regulatory network
Hypothalamus
Hypothalamus
GnRH
+
Pituitary
LH
Leydig cell
Testosterone
GnRH
Pituitary
FSH
LH
FSH
Testis
Sertoli cell
+
paracrine
factors
Ovary
Inhibin
Germ cell
Inhibin
Figure 1A. Schematic representation of the hypothalamopituitary-testis axis. GnRH, gonadotropin releasing hormone;
LH, luteinizing hormone; FSH, follicle stimulating hormone.
Progesterone
Estradiol
Figure 1B. Schematic representation of the hypothalamopituitary-ovary axis. GnRH, gonadotropin releasing hormone;
LH, luteinizing hormone; FSH, follicle stimulating hormone
in the brain controlling the GnRH neurons. In an analogous fashion,

thyreotropin releasing hormone (TRH) from the hypothalamus stimulates
pituitary thyrotropic cells to secrete thyroid stimulating hormone (TSH)
which in turn stimulates the thyroid gland to produce thyroxin (Figure 2).
Figure 2. Schematic representation of the

hypothalamo-pituitary-thyroid axis. TRH,
thyreotropin releasing hormone; TSH,
thyroid stimulating hormone; NIS, sodiumiodide symporter; T4, thyroxine; T3, triiodothyronine;

This inhibits TSH secretion to maintain a balance, called euthyroidism.
Two common diseases disturb this hypothalamo-pituitary-thyroid (HPT)
axis. In autoimmune hypothyroidism, the thyroid gland is affected by autoantibodies, which leads to low thyroxin levels and very high TSH levels.
In autoimmune hyperthyroidism (Graves disease) the thyroid gland is
stimulated by immunoglobulins that activate TSH receptors, which leads
to very high thyroxin levels and low TSH levels. Normal function of both
HPG and HPT axes is essential for normal development.
b. Endocrine regulation of development
i. Gonadal hormones sex differentiation
In the early embryo the two sexes are indistinguishable before the gonadal
sex is determined by a genetic programme involving SRY gene in the Y
chromosome. In the presence of SRY and several down stream genes the
gonad is directed to become a testis, whereas in the absence of SRY other
genes guide the gonad towards ovarian development. The fetal ovary
stays hormonally inactive, whereas fetal testis is producing large amounts
of hormones soon after testicular differentiation in gestational weeks
8-16. Somatic Sertoli cells in the testis produce anti-Mllerian hormone
(AMH) that induces involution of the paramesonephric ducts (also called
Mllerian ducts) that in the absence of AMH develop into the oviducts, the
uterus and the upper part of the vagina. Therefore male newborns do not
have these structures, whereas females do. Testicular Leydig cells produce
testosterone that stimulates fetal mesonephric ducts (also called Wolffian
ducts) to develop to epididymides, ejaculatory ducts and seminal vesicles.
These structures disappear in female fetuses, because the ovaries do not
secrete testosterone. Testosterone is further metabolized by 5-alphareductase enzyme to dihydrotestosterone (DHT) in the genital area. DHT
is needed for the development of the prostate and masculinization of the
external genitalia, i.e. development of scrota and the penis. If the DHT
is missing, fusion of the urethral folds can be insufficient resulting in
hypospadias and the penis may remain very small. In worst cases scrotal
fusion may also be deficient with the result that the 46,XY newborn looks
like a female. Leydig cells secrete also insulin like peptide 3 (INSL3) that
together with testosterone regulates testicular descent from the abdomen
to the scrotum.
Exposure of female fetuses to androgens leads to their masculinization,

whereas exposure of male fetuses to anti-androgens results in undermasculinization (feminization) (Welsh et al., 2008; Rey and Grinspon,
2011). Since the development of a male-type reproductive system is
dependent on multiple hormones, male fetuses are more susceptible to
endocrine disruption than females. Developmental disorders that appear
in newborn males include penile defects (hypospadias, micropenis) and
defects of testicular descent to the scrota (cryptorchidism). There is
strong evidence that testicular cancer, which appears several years later in
young adulthood, also has its origin in fetal life (Rajpert-De Meyts, 2006).
Furthermore, sperm production capacity may be largely determined during
early development (Sharpe et al., 2003). However, that can be measured
only after pubertal maturation. It is unknown whether the timing of
pubertal development is affected by fetal programming.
Although male fetuses appear more affected by endocrine disrupters,
female fetuses are also vulnerable. Androgen exposure can cause
masculinization when the doses are high, but lower doses have been
suggested to be associated with the development of the polycystic ovarian
syndrome later in adulthood (Pasquali et al., 2011). Breast development is
another sensitive target for endocrine disruption that may have serious
late-onset consequences (McLachlan, Simpson and Martin, 2006).
ii. Thyroid hormones significance in brain development
It is well established that thyroid hormones are of special importance in

the development of the brain. Numerous in vitro and animal studies have
shown that the absence of thyroid hormones reduces neuronal growth
and differentiation in the cerebral cortex, hippocampus, and cerebellum
(Nicholson and Altman, 1972; Auso et al., 2004; Lavado-Autric et al., 2003).
This is of special importance in fetal life, as development of the brain in
utero is dependent upon normal levels of thyroid hormones.
The fetal thyroid gland develops from the third gestational week and thyroid
follicles are formed and iodine concentration begins at approximately the
12th gestational week. However, the gland is not under feedback control by
TSH and fully functioning until approximately the 20th gestational week.
Thus, in the first trimester of gestation, before development and function
of the fetal thyroid gland, the fetus is dependent on transplacental supply
of maternal thyroxin (T4), and consequently on the ability of the maternal
5

thyroid gland to increase the hormone production during pregnancy in
order to meet the needs of both fetus and mother.
Thyroid function is regulated by a finely tuned endocrinological
homeostasis maintaining relatively stable serum levels of thyroid
hormones. Thyroid hormone serum levels are monitored by a negative
feedback mechanism mediated by the effects of circulating thyroid
hormones at the hypothalamic and pituitary levels. In response to low
levels of thyroid hormones in the blood, the pituitary secretes thyroid
stimulating hormone (TSH), which stimulates the synthesis and release
of triiodothyronine (T3) and thyroxine (T4). In serum, these hormones
are transported to the tissues bound to transport proteins, among which
thyroxine binding globulin (TBG) is the most important thyroid hormone
transport protein in humans, whereas transthyretin (TTR) is the major
transport protein in many animals. T4 is converted to the active hormone
T3 in the liver or in local tissues by iodothyronine deiodinases. The highly
sensitive feedback regulation results in a remarkably stable concentration
of TSH in blood (except for known diurnal variations) and consequently
of circulating thyroid hormones in an individual.
Interference with thyroid homeostasis can take place on many different
levels of the HPT-axis and may result in alterations of thyroid hormones
available for the TH-receptors. In cases of markedly reduced hormone
production capacity in both maternal and fetal glands, e.g. in iodinedeficient countries, severe brain damage may occur. Similarly, normal
levels of thyroid hormones are important for postnatal neurological
development in early childhood. Consequently, children who are born
with congenital hypothyroidism and not treated with substitution therapy
from the neonatal period develop severe central nervous system damage.
Minor changes in the thyroid homeostasis may also affect neurological
development. Epidemiological studies have documented that even
a marginally low thyroxine level in a pregnant women may give rise to
reduction of cognitive functions of the offspring (Haddow et al., 1999;
Pop et al., 2003; Berbel et al., 2009). In this way, exposure to thyroiddisrupting chemicals may result in decreases of serum hormone levels
and consequently neurological damage.
Additionally, a normal thyroid function presupposes a successful
development of the thyroid gland itself and establishment of a wellfunctioning HPT-axis. Thyroid homeostasis may be disturbed by
6
hyperthyroidism or the presence of thyroid autoantibodies. However, it

is not yet clear whether some environmental chemicals may interfere with
thyroid function through these pathways.
2. Endocrine disrupters (recognized on the

basis of experimental work in vitro and in
vivo)
a. Sex hormone disrupters
The list of chemical compounds affecting the synthesis, transport,
metabolism and action of sex hormones is growing, and it is not possible
to include all studies in a review, since there are several hundreds of studies
of each of them. The US National Toxicology Program (NTP) and the
WHO International Programme on Chemical Safety (IPCS) among
others have published comprehensive reviews on individual chemicals.
Tables 1 and 2 provide short summaries of the main findings relevant to
reproductive development.
Hypospadias and cryptorchidism in experimental animals can be induced
by several endocrine disrupters that are either anti-androgenic or
estrogenic (Toppari, 2008). Examples of anti-androgens are the fungicides
vinclozolin and procymidone and DDE, the persistent congener of
estrogenic dichlorodiphenyltrichloroethane (DDT), that act as androgen
receptor antagonists (Gray et al., 2006), and phthalate esters, dibutyl
phthalate and diethyl hexyl phthalate that disturb androgen biosynthesis
(Mylchreest et al., 2002; Fisher et al., 2003). Some compounds disrupt
both receptor action and biosynthesis, e.g. linuron and prochloraz (Gray
et al., 2006). Dioxins act via aryl hydrocarbon receptors and interfere
with several nuclear receptors, causing genital malformations (Peterson,
Theobald and Kimmel, 1993). Penta-brominated diphenyl ethers are also
anti-androgenic (Stoker et al., 2005; Lilienthal et al., 2006), while some
polybrominated diphenyl ether metabolites can stimulate aromatase
activity in cells derived from human adrenocortical carcinoma (Song
et al., 2008), which also disturbs the androgen-estrogen balance. These
chemicals show additivity of the effects in low doses making the mixtures
harmful even when none of the individual compounds is present higher
than the no observed adverse effect level (NOAEL) (Kortenkamp and
7

TABLE 1 Effects of endocrine disrupters observed
in the human reproductive system
Contaminant
Sex
Male
Diethylstilbestrol
(DES)
Female
Observation
References
Increased risk of hypospadias
Brouwers et al., 2006; Klip et

al., 2002
Tendency towards smaller testes
Bibbo et al., 1977; Gill et al.,

1977, Ross et al., 1983,
Increased prevalence of
cryptorchidism
Palmer et al., 2009
Capsular induration of testis

1977
Severe sperm abnormalities

1977
Epididymal cysts

1977; Palmer et al., 2009
Infection/inflammation of testis
Palmer et al., 2009
Increased risk of breast cancer
Palmer et al., 2006
Vaginal adenosis
Bibbo et al., 1977; Sherman et

al., 1974
Oligomenorrhea
Bibbo et al., 1977
Increased risk of clear cell

adenocarcinoma of the vagina and
cervix
Herbst et al., 1971; Herbst et

al., 1979; Verloop et al., 2010
Increased frequency of preterm

delivery, first-trimester spontaneous
abortion, second-trimester pregnancy
loss and ectopic pregnancy
Kaufman et al., 2000
Associated with anogenital index
Swan et al., 2005
Positive correlation with increased

serum LH/testosterone ratio
Main et al., 2006a
Phthalate
esters (DBP,
DMP,BBP,DEHP,
DEP, DOP)
Male
Flame retardants
(Polybrominated
diphenyl ethers)
Male
Associated with cryptorchidism
Main et al., 2007
Phytoestrogens
Male
Associated with hypospadias
North et al., 2000
Dioxins
Female
Increased probability of female births
Mocarelli et al., 1996; Mocarelli

et al., 2000
Male
Higher percentage of oligospermia,

abnormal morphology and reduced
sperm capacity of binding and
penetration to hamster oocyte
Hsu et al., 2003
Polychlorinated
biphenyls (PCBs)

TABLE 2 Effects of endocrine disrupters observed
in the reproductive system of animals
Contaminant
Sex
Observation
Sterility
Epididymal cysts
Cryptorchidism
Reduction in testis weight
Male
Diethylstilbestrol
(DES)
Testicular lesions
Inflammatory disease of the
accessory sex glands
Reduction in the number of
spermatogonia with multinucleate
cells in lumina of testis
Nodular enlargements of the
seminal vesicles and/or prostate
Distension and overgrowth of the
rete testis
Distension and reduction in
epithelial height of the efferent
ducts
Underdevelopment of the
epididymal duct epithelium
Reduction in epithelial height in the
vas deferens
Convolution of the extraepididymal vas
Decreased testosterone levels
Female
Increased gonadotrophin levels

Decreased AR expression in testis,
epithelium of the rete testis, caput
and cauda epididymis and vas
deferens
Decrease in reproductive capacity
Impaired ovarian function
Increased uterus weight
Squamous metaplasia in the
oviducts, uterus and cervix
Increased the size of sexually
dimorphic nucleus of the preoptic
area
Cystic hyperplasia of the
endometrium and uterine
adenocarcinoma
Epidermoid tumors of the cervix
and vagina
Glandular elements and cellular
atypia in the vaginal epithelium
References
McLachlan, 1977
McLachlan, 1977
McLachlan, 1977
Fisher et al., 1999; Lewis et al., 2003;
McKinnell et al., 2001
McLachlan, 1977
McLachlan, 1977
McLachlan, 1977
McLachlan, 1977
Fisher et al., 1999; McKinnell et al.,
2001; Rivas et al., 2002
Fisher et al., 1999; McKinnell et al.,
2001; Rivas et al., 2002
McKinnell et al., 2001; Rivas et al.,
2002
McKinnell et al., 2001;
Rivas et al., 2002; Yamamoto et al.,
2003
Yamamoto et al., 2003
McLachlan, 1977
McLachlan, 1977
Lewis et al., 2003
McLachlan, 1977
Faber and Hughes, 1991; Lewis et
al., 2003
McLachlan, 1977
McLachlan, 1977
McLachlan, 1977
Diethylstilbestrol
(DES)
Female
Male
Tributyltin
Female
Male
Phtytoestrogens
(Genistein, Daidzein)
Female
Advanced development of primary

and secondary follicles in the ovary
Decreased pituitary responsiviness
to GnRH
Increased pubertal FSH levels
Increased anogenital distance
Reduced the number of Sertoli
cells and gonocytes in fetal testis
Reduced the number of germ cells
in fetal ovaries
Increased post-implantation loss
Impaired erectile function
Decreased plasma
testosterone levels
Increased testis weight
Reduction in epithelial height of
the efferent ducts
Increased pituitary response to
GnRH
Decreased pituitary responsiviness
to GnRH
area
Increased the weight of uterus
Decreased the weight of uterus
Decreased the weight of ovaries
Reduced serum estradiol levels
Reduced serum progesterone
levels
Irregular estrus cycle
Histopathological changes in the
ovaries and uterus
Induced permanent estrus
Decreased the age of vaginal
opening
Decreased testis weight
Alkyl phenol
ethoxylates (p-tertoctylphenol,
p-nonylphenol)
Male
Female
10
Decreased seminiferous tubule

diameter
Decreased epididymal weight
Decreased total cauda epididymal
sperm count
Reduction in epithelial height of
the efferent ducts
Post-implantation embryonic loss
Irregular estrus cycle

Faber and Hughes, 1991
Adeeko et al., 2003
Kishta et al., 2007
Kishta et al., 2007
Adeeko et al., 2003
Pan et al., 2008
Pan et al., 2008
Fisher et al., 1999
Fisher et al., 1999
Faber and Hughes, 1991; Lewis et
al., 2003
Lewis et al., 2003
Awoniyi et al., 1998
Awoniyi et al., 1998; Lewis et al.,
2003
Nagao et al., 2001
Nagao et al., 2001
Lewis et al., 2003
Lewis et al., 2003
Fisher et al., 1999
de Jager et al., 1999; Pocock et al.,
2002
de Jager et al., 1999; Pocock et al.,
2002
de Jager et al., 1999
de Jager et al., 1999
Fisher et al., 1999
Harazono and Ema, 2001
Katsuda et al., 2000; Pocock et al.,
2002
Alkyl phenol
ethoxylates (p-tertoctylphenol,
p-nonylphenol)
Female
Increased sexual motivation

towards a female teaser
Decreased the weight of ovaries
area
Decreased the age of vaginal
opening
Persistent estrus
Increased relative uterine weight
Decreased serum gonadotrophin
levels
Decreased serum progesterone
levels
Increased serum inhibin levels
Nipple retention
Phthalate esters
(DEHP, BBP, DINP,
DBP)
Pocock et al., 2002

Pocock et al., 2002
Herath et al., 2001
Katsuda et al., 2000
Barlow et al., 2004; Borch et al.,
2004; Gray et al., 1999b; Gray et
al., 2000; Mylchreest et al., 1999;
Mylchreest et al., 2000
Gray et al., 1999b; Gray et al., 2000;
Mylchreest et al., 1999; Mylchreest et
al., 2000; Parks et al., 2000
Reduced anogenital distance
Borch et al., 2004; Barlow et al.,

2004; Gray et al., 1999b; Gray et
Mylchreest et al., 2000; Parks et al.,
2000
Cryptorchidism
Gray et al., 1999b; Gray et al., 2000;

Mylchreest et al., 1999; Mylchreest
et al., 2000
Andrade et al., 2006; Barlow et al.,
2004; Gray et al, 1999b; Gray et
Barlow et al., 2004
Gray et al., 1999b; Gray et al., 2000
Barlow et al., 2004; Gray et al.,
1999b; Gray et al., 2000; Mylchreest
et al., 1999; Mylchreest et al., 2000
Gray et al., 2000; Parks et al., 2000
Gray et al., 2000; Mylchreest et al.,
2000
Gray et al., 2000
Barlow et al., 2004; Gray et al., 2000
Gray et al., 2000
Barlow et al., 2004; Gray et al.,
1999b; Mylchreest et al., 1999;
Barlow et al., 2004; Mylchreest et al.,
1999; Mylchreest et al., 2000; Parks
et al., 2000
Reduced accessory sex organ

weights
Male
Lesion of the rete testis
Hemorrhagic testis
Cleft phallus and hypospadias
Multinucleated gonocytes
Agenesis of the seminal vesicles
and coagulating glands
Agenesis of bulbourethal glands
Agenesis of ventral prostate
Agenesis of gubernacular cords
Agenesis of epididymis and vas
deferens
Histopathological changes of testis
11

Delayed preputial separation
Phthalate esters
(DEHP, BBP, DINP,
DBP)
Male
Female
Reduced fertility
Reduced fecundity
Reduced cauda epididymal sperm
numbers
Reduced daily sperm production
Reduced plasma and/or testicular
testosterone levels
Chlorinated pesticides
(DDE)
Female
Borch et al., 2004; Parks et al., 2000
Reduced serum inhibin B levels
Borch et al., 2004
Increase plasma LH levels
Borch et al., 2004
Uterine abnormalities
Reduced fertility
Gray et al., 1999b

Gray et al., 1999b
Gray et al., 1999b; Kelce et al., 1995;
You et al., 1998
Gray et al., 1999b
Hypospadias
weights
Abnormally small penis
Poorly organized testis
Decreased plasma testosterone
levels
Increased plasma estradiol levels
Abnormal ovarian morphology with
large number of polyovular follicles
and polynuclear oocytes
weights
Male
Delayed testis descent
Epididymal malformations
Altered sex behavior
Decreased sperm numbers
12
Andrade et al., 2006
Andrade et al., 2006

Dioxins
Gray et al., 1999b
Increased serum testosterone

levels
Nipple retention
Male
Gray et al., 1999b; Mylchreest et al.,

1999
Gray et al., 1999b
Gray et al., 1999b
Gray et al.,1999b; Kelce et al., 1995

Kelce et al., 1995; You et al., 1998
Kelce et al., 1995
Guillette et al., 1994
Gray et al., 1995; Mably et al., 1992a;
Mably et al., 1992b; Ohsako et al.,
2001; Simanainen et al., 2004
Gray et al., 1995; Mably et al., 1992b
Gray et al., 1995a
Gray et al., 1995; Mably et al., 1992a;
Ohsako et al., 2001; Simanainen et
al., 2004
Mably et al., 1992a
Gray et al., 1995; Simanainen et al.,
2004
Gray et al., 1995
Gray et al., 1995; Mably et al., 1992b;
Simanainen et al., 2004
Male
Dioxins
Female
Decerased daily sperm production

Dose-related tendencies to
decrease plasma testosterone
and DHT
Delayed puberty
Clef phallus
Vaginal thread
Reduced ovarian weight
Enhanced incidences of constant
estrus
Cystic endometrial hyperplasia
Decreased fertility rate
Reduced fecundity
weights
Polychlorinated
biphenyls (PCBs; PBC
77, 118, 126, 132,
169)
Male
Female
Hypospadias with cleft phallus
Dicarboximide
Fungicides
Male
(Vinclozolin,
Procymidone)

Increased epididymis weight
Increased anogenital distance
Delay in onset of spermatogenesis,
preputial separation and sex
accessory growth
Decreased sperm number and
total motile sperm count
Increased daily sperm production
Decreased serum testosterone
levels
Increased the number of abnormal
sperm
Altered sex behavior
Vaginal thread
Mild hypospadias
Delayed the timing of vaginal
opening

Cryptorchidism
Mably et al., 1992b

Mably et al., 1992a
Gray and Ostby, 1995
Faqi et al., 1998; Gray et al., 1999b;
Hsu et al., 2007; Kuriyama and
Chahoud, 2004
Gray et al., 1999b; Kuriyama and
Chahoud, 2004
Faqi et al., 1998
Faqi et al., 1998
Faqi et al., 1998
Kuriyama and Chahoud, 2004
Gray et al., 1999b
Gray et al., 1999b; Hsu et al., 2007;
Faqi et al., 1998
Faqi et al., 1998
Faqi et al., 1998
Gray et al., 1999b
Gray et al., 1999b
Faqi et al., 1998
Gray et al., 1994; Gray et al., 1999a;
Gray et al., 1999b; Hellwig et al.,
2000; Ostby et al., 1999
Cowin et al., 2010; Elzeinova et al.,
2008; Gray et al., 1994; Gray et al.,
1999a; Gray et al., 1999b; Hellwig et
al., 2000; Ostby et al., 1999
Elzeinova et al., 2008; Hellwig et al.,
2000
Gray et al., 1994; Hellwig et al., 2000;
Ostby et al., 1999
13

Increased the number of apoptotic
germ cells in testis
Nipple retention
weights
Glandular atrophy and chronic
inflammation of prostate
Reduced secretion and chronic
inflammation of seminal vesicles
Epididymal granulomas
Dicarboximide
Fungicides
(Vinclozolin,
Procymidone)
Herbicides (Linuron)
14
Male
Male
Chronic inflammation of epididymis

Agenesis of prostate
Spermatogenic granuloma
Decreased sperm number and
daily sperm production
Increased sperm head
abnormalities
Reduced elongated spermatid
content per testis
Low ejaculated sperm count
Abnormal morphology of
seminiferous tubules
Decreased fertility
Reduction of erections during the
ex copula penile reflex test
Increase in seminal emissions
during the ex copula penile reflex
tests
Decreased serum testosterone
levels
Nipple retention
weights
Reduced spermatid number
Decreased anogenital distance
Epispadias
Testicular and epididymal
malformations
Cowin et al., 2010

Hellwig et al., 2000; Ostby et al., 1999
Cowin et al., 2010; Elzeinova et al.,
2008; Gray et al., 1994; Gray et al.,
1999a; Gray et al., 1999b; Hellwig et
al., 2000; Ostby et al., 1999
Cowin et al., 2010; Gray et al., 1999b;
Hellwig et al., 2000; Ostby et al., 1999
Hellwig et al., 2000
Ostby et al., 1999
Gray et al., 1994
Elzeinova et al., 2008; Gray et al.,
1994; Gray et al., 1999a
Elzeinova et al., 2008
Cowin et al., 2010
Gray et al., 1999a
Elzeinova et al., 2008; Gray et al.,
1994
Gray et al., 1994
Colbert et al., 2005
Colbert et al., 2005
Gray et al., 1994
Gray et al., 1999b
Gray et al., 1999b
Gray et al., 1999b
Gray et al., 1999b
Gray et al., 1999b
Gray et al., 1999b
Gray et al., 1999b
Gray et al., 1999b
Male
Lead
Female

weights
Enlarged prostate weight
Reduced serum testosterone
levels
Decreased sperm counts
Reduced serum LH levels
Reduced volume of the sexually
dimorphic mucleus of the preoptic
area
Less masculine sex behavior
Irregular release pattern of
gonadotrophins
Delayed the timing of vaginal
opening and the day of first
diestrus
Prolonged and irregular periods
of diestrus
Disruption of estrus cycling
Suppressed serum levels of IGF-1,
LH and/or estradiol
Irregular release pattern of
gonadotrophins
Ronis et al., 1996

Ronis et al., 1996
McGivern et al., 1991
Ronis et al., 1996
Ronis et al., 1996
Dearth et al., 2002; Kimmel et al.,
1980; McGivern et al., 1991; Ronis
et al., 1996
McGivern et al., 1991;
Ronis et al., 1996
Dearth et al., 2002; Ronis et al., 1996
15
Male
Cadmium
Female
Male
Manganese
Female
16
Time- and dose-dependent

decrease in sperm motility
Partial or entire evacuation of the
Increased the diameter of
Reduced epithelial volume and
increased lumen of tubule in the
epididymis
Hyperemic testes with extensive
haemorrhaging, destruction of all
of the presperm spermatogenic
cells, and general necrosis and
shrinkage of the seminiferous
tubules
Decrease in sperm output
Reduced size of the testis
Reduced number of differentiating
germ cells in 16.5-day embryos
Spermatozoa had poor ability to
capacitate in vitro and showed a
low fertilizing capability
Perturbed estrus cycles
Reduced number of differentiating
germ cells and the size the ovary
in 16.5-day embryos
Tendency towards delayed timing
of vaginal opening
Earlier onset of vaginal opening
Increased the epithelial area
and the number of terminal end
buds in the mammary glands and
decreased the number of alveolar
buds
Increased serum gonadotrophin
levels
Increased serum testosterone
levels
Increased daily sperm production
and efficiency of spermatogenesis
Increased serum gonadotrophin
levels
Increased serum estradiol levels
Earlier onset of vaginal opening
Benoff et al., 2008

Toman et al., 2002
Toman et al., 2002
Toman et al., 2002
Foote, 1999
Foote, 1999
Tam and Liu, 1985
Tam and Liu, 1985
Tam and Liu, 1985
Ishitobi and Watanabe, 2005
Tam and Liu, 1985
Ishitobi and Watanabe, 2005
Johnson et al., 2003
Johnson et al., 2003
Lee et al., 2006

Lee et al., 2006
Lee et al., 2006
Pine et al., 2005
Pine et al., 2005
Pine et al., 2005
Faust, 2010). Thus, when animals are exposed to the chemicals at levels
that never cause hypospadias, they can together elicit hypospadias in 100%
of offspring (Jacobsen et al., 2010; Rider et al., 2010).
b. Thyroid hormone disrupters
Numerous chemicals have been shown to interfere with thyroid function
in experimental studies. Several groups of chemicals, e.g. dioxin-like
compounds and certain flame retardants, have a high degree of structural
similarity with the thyroid hormones T3 and T4, thus competing with the
hormones for the TH-receptor and transport proteins.
PCBs and dioxins
Polychlorinated biphenyls (PCBs), dioxins (PCDDs) and furans (PCDFs)
are widespread, persistent and toxic environmental pollutants from
industrial production or production of herbicides. They comprise a group
of highly persistent lipophilic chemicals that can be detected in samples
from human and wildlife populations, although banned for decades in
most countries. Many of these compounds, especially the hydroxylated
metabolites, which are also biologically active, have a high degree of
structural resemblance to thyroxine (T4).
The negative effect of PCB-exposure on peripheral thyroid hormone
levels is well documented by studies in laboratory animals. Thus, PCBs
and dioxins decrease the levels of circulating thyroid hormones in rats,
especially T4 (Gauger et al., 2004; van der Plas et al., 2001; Hallgren et al.,
2001; Hallgren and Darnerud, 2002; Martin and Klaassen, 2010; Viluksela
et al., 2004; Nishimura et al., 2002). Similarly, monkeys exposed to PCBs
showed dose-dependent reductions of thyroid hormone levels (van den
Berg, Zurcher and Brouwer., 1988). Mixtures of dioxin-like compound
also decrease levels of T4 in an additive manner (Crofton et al., 2005).
There is substantial evidence that perinatal exposure to PCBs and their
hydroxylated metabolites decreases thyroid hormones in the offspring.
This has been shown for exposure to PCBs in rats (Crofton et al., 2000;
Meerts et al., 2002; Donahue, Dougherty and Meserve, 2004; Meerts et
al., 2004; Zoeller et al., 2000), in sled dogs (Kirkegaard et al., 2010), and
exposure to dioxins in rats (Nishimura et al., 2003; Seo et al., 1995). Mouse
studies have demonstrated accumulation of hydroxylated metabolites in
the fetal compartment (Darnerud et al., 1996).
17

Negative correlations between serum levels of PCBs or other
organochlorine pollutants and thyroid hormones are reported among
wildlife, including polar bears (Skaare et al., 2001), seals (Chiba et al., 2001;
Sormo et al., 2005), and nestling eagles (Cesh et al., 2010).
In conclusion, experimental and wildlife observations point towards
subtle, but significant, effects of exposure to dioxin-like chemicals and
PCBs on mammalian thyroid function.
Flame retardants
The industrial use of flame retardants is abundant and this group
of chemicals is found in a wide range of products such as electronic
equipment, plastics, paints and synthetic textiles. This group of chemicals
includes different compounds such as tetrabromobisphenol A (TBBPA),
polybrominated diphenyl ethers (PBDEs) and polybrominated biphenyls
(PBBs), of which TBBPA and PBDEs show an even closer structural
relationship to T4 than PCBs.
Numerous, but not all (Van den Steen et al., 2010), studies in rats have
demonstrated that PBDEs and commercial mixtures of flame retardants
decrease the levels of circulating thyroid hormones (Fowles et al., 1994;
Zhou et al., 2001; Stoker et al., 2004; Hallgren et al., 2001; Lee et al., 2010).
Perinatal maternal exposure of rats to different mixtures and congeners
of PBDEs similarly reduced thyroid hormones in the fetuses (Zhou et al.,
2002; Kodavanti et al., 2010; Kim et al., 2009), and this has been confirmed
in other species including kestrels (Fernie et al., 2005) and minks (Zhang
et al., 2009). Recently, several studies have demonstrated that even low
doses of maternal PBDE exposure, comparable to levels of human
environmental exposure, may similarly disrupt thyroid homeostasis in rat
pups (Kuriyama et al., 2007) or lambs (Abdelouahab et al., 2009).
Pesticides
Innumerable different chemicals are used as pesticides and are
part of potentially widespread human exposure. Many animal and
toxicological studies suggest that multiple pesticides may have thyroiddisrupting properties. Both persistent organochlorine pesticides and
non-persistent pesticides such as organophosphates, carbamates and
pyrethroids, may interfere with thyroid function. The persistent chemicals
dichlorodiphenyltrichloroethane (DDT) (and the metabolite DDE),
18
hexachlorobenzene (HCB), and nonylphenol (NP; a surface active

substance used in pesticide aerosols) are among the most studied with
regard to thyroid-disrupting effects. Although use of these chemicals
has long been banned in many countries, they are still present in the
environment due to their long environmental half-lives and continuous
use in some countries.
Exposures to DDT (Scollon, Carr and Cobb, 2004), HCB (Rozman et
al., 1986; van Raaij et al., 1993a; van Raaij et al., 1993b; Foster et al., 1993;
Alvarez et al., 2005), and different mixtures of pesticides (den Besten et
al., 1993; Rawlings, Cook and Waldbillig, 1998) decrease serum levels of
thyroid hormones in rats. Similarly NP decreases the level of T4 in studies
of salmon (McCormick et al., 2005) and lambs (Beard et al., 1999).
Perfluorinated chemicals
The use of perfluorinated chemicals (PFC) in industrial and consumer
products is increasing due to their surface protection properties, which are
exploited in products such as stain- and oil-resistant coatings, but also in
floor polishes and insecticide formulations. The group comprises several
chemicals, e.g. perfluorooctanoic acid (PFOA) as well as perfluorooctane
sulfonate (PFOS), which is also the metabolic end product of other PFCs.
PFCs are extremely persistent in the environment.
Exposure to PFOS and PFOA decreased levels of T4 after both short-term
(Martin and Klaassen, 2007; Chang et al., 2007) and long-term exposure
(Yu, Liu and Jin, 2009). A study of monkeys showed reduction of T3 after
exposure to PFOS (Seacat et al., 2003).
Perinatal exposure to PFOS also reduced serum levels of T4, both in
pregnant dams (Thibodeaux et al., 2003) and in the offspring (Lau et al.,
2003; Luebker et al., 2005). Cross-over studies of rats exposed in utero or/
and in lactation document that both prenatal and postnatal exposure to
PFOS may reduce thyroid hormone levels in the offspring (Yu et al., 2009).
Phthalates
Phthalates are widely used as plastic emollients and additives in various
industrial and consumer products, and exposure to phthalates is inevitable.
For certain groups, such as hospitalized neonates and premature babies,
exposure may be massive. In these patients, changes in thyroid hormone
levels as a result of exposure to phthalates may be transient, but could
19

nonetheless have permanent effects on the development of the central
nervous system, if changes occur in a developmentally critical phase.
Studies of the thyroid-disrupting effects of phthalates and their monoester
metabolites are scarce. In rats, di-n-butyl phthalate (DBP) decreased
T3 and T4 in rats in a dose-dependent manner (OConnor, Frame and
Ladics, 2002), and several studies have shown histopathological changes
in the thyroid after exposure to phthalates (Howarth et al., 2001; Poon et
al., 1997). In vitro studies indicated antagonistic properties of DBP and
DEHP (Sugiyama et al., 2005; Shen et al., 2009).
Bisphenol A
Bisphenol A (BPA, 4,4-isopropylidenediphenol) is widely used to
manufacture numerous plastic products including food can linings
and clear plastic bottles and several population studies have reported
a high degree of human exposure (Calafat et al., 2008; Ye et al., 2008).
Young children can be particularly exposed via baby bottles and plastic
baby products. Several countries have banned BPA from baby products
following the precautionary principle.
Despite the current debate on reproductive effects of BPA, only a few
animal studies of thyroid-disrupting effects of BPA exist. BPA fed
to pregnant rats was associated with a significant increase of T4 in the
pups, compatible with thyroid resistance syndrome (Zoeller et al., 2005).
However, other studies have found no or contrasting effects on thyroid
hormone levels (Nieminen et al., 2002a; Nieminen et al., 2002b; Xu et al.,
2007) after exposure to BPA.
Ultraviolet filters
Several ultraviolet (UV) filters used in sunscreens are suspected to have
thyroid-disrupting properties. 4-methylbenzylidene-camphor (4-MBC)
and octyl-methoxycinnamate (OMC), and benzophenone 2 (BP2)
decreased serum levels of thyroid hormones in rats (Seidlova-Wuttke et
al., 2006; Klammer et al., 2007; Jarry et al., 2004; Schmutzler et al., 2007).
20
3. Early effects, child health problems

putatively associated with endocrine disruption
a. Cryptorchidism
i. Epidemiology
Congenital cryptorchidism is defined as a condition in which one or

both testes are not located at the bottom of the scrotum at the time of
birth. Figure 3 describes the clinical classification of testicular position in
cryptorchidism (non-palpable testis excluded).
Inguinal
Suprascrotal
High scrotal
Normal
Figure 3. Clinical classification of testicular position in cryptorchidism (non-palpable testis excluded).
Testes descend to the scrota normally during the last trimester of

pregnancy. Preterm boys are often bilaterally cryptorchid, because they
have not yet reached the age at which the testes descend, and their testes
usually descend spontaneously before the due date. However, the incidence
of cryptorchidism at the expected time of delivery is still higher in this
group than in full-term babies. Therefore the incidence rates are usually
given separately for full-term and preterm infants, and the weight of < 2.5
kg is often used as a proxy for being preterm. In addition to maturity of
the baby, the exact position of the testis at examination is an important
determinant in the ascertainment of cryptorchidism. This can be assessed
reliably onlyFigure
in prospective
clinical studies, whereas registry- and interview3
based epidemiological studies tend to misclassify cases as normal.
Registries are unreliable sources of data for cryptorchidism (Toppari et al.,
2001). Interestingly, the reported prevalence of cryptorchidism can vary
21

from 1 to 9% in the same population, depending on the data source (1%
orchidopexy rate, 2% hospital discharge registry, 4% mothers interview,
9% clinical examination at birth; Boisen et al., 2004; Strandberg-Larsen et
al., 2009).
Scorer (1964) used the distance of the testis from the pubic bone as a
criterion to classify the testis as descended or undescended. The position
of the undescended testis can be abdominal, inguinal, suprascrotal or high
scrotal. Non-palpable testes are either absent or abdominal or sometimes
deep in the inguinal canal or they may be ectopic, which means that they
are outside their normal route of descent, e.g. above the pubic bone or
in the thigh. Normal testes locate at the bottom of the scrotum, whereas
retractile testes move freely up and down, but can be manipulated to the
bottom at least for some time. The high scrotal testes may locate in the
upper part of the scrotum or they may also be manipulated down, but
return immediately back to their higher position (Boisen et al., 2004).
Clear definitions of cryptorchidism have been used in several prospective
clinical studies, which makes them comparable with other studies using
similar definitions (Table 3). Table 3 demonstrates that there are large
regional differences and adverse trends. The incidence of cryptorchidism
at birth is much lower in Finland than in Denmark, and an increasing rate
can be seen in the United Kingdom and Denmark.
The majority of cryptorchid testes (up to 75%) descend spontaneously
during the first three months of life (Boisen et al., 2004) when the
hypothalamo-pituitary-testicular axis is very active (Andersson et al.,
1998). After that, the testes may reascend and also new cases of (acquired)
cryptorchidism appear (Hack et al., 2003a; Wohlfahrt-Veje et al., 2009).
Congenital and acquired cases are mixed in all epidemiological studies that
use the hospital discharge registries and interviews as data sources. The
cause of both congenital and acquired cryptorchidism remains elusive in
most cases, but it is most likely that the aetiology is different for these
conditions, which further complicates all association studies that do
not assess them as distinct outcomes. Entrapment of the testis into the
inguinal scar after previous operation (Eardley, Saw and Whitaker, 1994),
improper elongation of the spermatic cord during childhood (Clarnette
and Hutson, 1997) or spasticity of the cremaster muscle e.g. in patients
with cerebral palsy (Smith et al., 1989) have been proposed to cause
acquired cryptorchidism. Previous retractility of the testes has also been
reported in some cases (Lamah et al., 2001). In the Danish cohort study,
22

Table 3. Rate of congenital cryptorchidism in prospective clinical
studies using clearly defined criteria of cryptorchidism
Country
Reference
U.S., Rochester
Minnesota, St. Marys
Hospital
Denmark,
Copenhagen,
Rigshospitalet
U.K., West London,
Hillingdon Hospital
India, Kanpur, Dufferin
Hospital and U.I.S.E
Maternity Hospital
Taiwan, Provincial TaoYuan Hospital
(Harris and
Steinberg, 1954)
Korea, 38 hospitals
U.K., Oxford, John
Radcliffe Hospital
Number of
subjects
n=4474
Diagnosis based on
(Buemann et al.,
1961)
n=2701)
position (testis cannot

be manipulated into the
scrotum)*
position
(Scorer, 1964)
n=3612
distance measurement
(Mital and Garg,

1972)
n=2850
distance measurement
(Hsieh and Huang,

1985)
n=1208
(Choi et al., 1989)

(Group, 1992)
n=7990
n=7400
position (presence or
absence of testes in
the scrotum)*
position
position
distance
measurement
U.S., New York, Mount

Sinai Hospital
Malaysia, Kuala
Lumpur, University
Hospital
Italy, Pisa, S. Chiara
Hospital and Division
of Neonatology at the
University of Pisa
Denmark,
Copenhagen,
Rigshospitalet
Finland, Turku, Turku
University Hospital
Lithuania, Panavys
City Hospital
UK, Cambridge Baby
Growth Study
Rate of cryptorchidism
at birth
1.3% (BW>2500g), 1.5%
of all boys
1.8% (BW>2500g), 4.1%
of all boys
2.7% (BW>2500g), 4.2%
of all boys
1.6% of full-term single
born boys
4.1% in preterm boys,
1.4% in mature boys
0.7% of all boys
3.8% (BW>2500g),
4.9% of all boys
(excluding boys with
severe congenital
malformations)
4.1% (BW>2500g),
5.0% of all boys
(excluding boys with
severe congenital
malformations)
2.2% (BW>2500g), 3.7%
of all boys
2.4% (BW>2500g), 4.8%
of all boys
(Berkowitz et al.,
1993)
(Thong et al., 1998)
n=6935
n=1002
distance
measurement
position
(Ghirri et al., 2002)
n=10730
position
3.5% (BW>2500g), 6.9%

of all boys
(Boisen et al., 2004)
n=1046
position
8.4% (BW>2500g), 9.0%

of all boys
n=1455
position
(Preiksa et al.,
2005)
(Acerini et al., 2009)
n=1204
position
n=742
position
2.1% (BW>2500g), 2.4%

of all boys
4.6% (BW>2500g), 5.7%
of all boys
5% (BW>2500g), 5.9% of
all boys
*Does not seem to include high scrotal testis as cryptorchid testis
23

0.8% and 1.4% (accumulated rate) of boys had acquired cryptorchidism
(ascending testis) at the age of 18 and 36 months, and 0.6% and 0.8%
of boys, respectively, had recurrent cryptorchidism (spontaneous descent
at 3 months and reascent thereafter) (Wohlfahrt-Veje et al., 2009). In the
Cambridge cohort study, the prevalence of acquired cryptorchidism was
7.0% at 2 years of age (Acerini et al., 2009). In the Netherlands, prevalence
rates of up to 2.2% for acquired cryptorchidism between 6 to 13 years
of age were reported (Hack et al., 2007a). The Dutch have suggested a
wait-and-see policy in the treatment and follow-up of these cases because
>75% have spontaneous descent at puberty (Hack et al., 2003b; Hack et al.,
2007b). In the Nordic countries early orchidopexy is recommended to all
cryptorchid boys, because the possible adverse effects that delay may cause
are unknown (Ritzen et al., 2007). Semen quality is better in men with early
orchidopexy than in those with a later operation (Virtanen et al., 2007;
Canavese et al., 2009) and postpubertal orchidopexy may be associated with
a higher risk of testicular cancer than prepubertal operation (Pettersson
et al., 2007; Walsh et al., 2007), although a large Danish cohort based on a
national hospital discharge registry and cancer registry did not corroborate
any effect of the age at treatment of cryptorchidism on the risk of testicular
cancer (Myrup, Schnack and Wohlfahrt, 2007). The finding that the testis
cancer risk was higher in the men that were operated on after puberty
than before it (Pettersson et al., 2007) may reflect the fact that this group
included only those who did not have spontaneous descent of acquired
cryptorchid testes in puberty, whereas the prepubertally-operated group
included a large group of boys who would have had spontaneous descent
in puberty (Hack et al., 2003b; Hack et al., 2007b). The differences between
these groups may reflect the underlying pathology and explain the small
difference in the risk observed in the study by Petterson et al. (Pettersson
et al., 2007). The absence of putative spermatogenic stem cells, type A
spermatogonia, was linked to poor spermatogenic prognosis independent
of timing of surgery (Hadziselimovic and Herzog, 2001; Hadziselimovic
et al., 2007). However, the distinction of different types of spermatogonia
only on a morphological basis is difficult and immunohistochemical
analysis may differ from conventional histologic assessment (Wikstrm et
al., 2004; Wikstrm et al., 2007). Testicular biopsies are not recommended,
unless there is a specific reason such as suspicion of malignancy (Ritzen
et al., 2007).
Cryptorchidism is a well characterized risk factor for testicular cancer,
and men with a history of cryptorchidism have a 4 to 6-fold higher risk
24
of testicular cancer than men without cryptorchidism (Dieckmann and

Pichlmeier, 2004; Schnack et al., 2010b). However, most of the men with
a history of cryptorchidism never develop testicular cancer, and only
about ten percent of men with testicular cancer have been cryptorchid.
Furthermore, orchidopexy does not abolish the cancer risk. Thus,
although cryptorchidism is a risk factor for testicular cancer, it does not
seem to cause it. These two disorders most likely share aetiological factors.
Against this background it is not surprising that a high incidence of
cryptorchidism is accompanied by a high rate of testicular cancer, which is
apparent e.g. in Denmark and Finland, which have high and low incidence
rates, respectively (Boisen et al., 2004) (Jacobsen et al., 2006). This implies
that any causal relationship of cryptorchidism with environmental effects
can be considered a putative risk factor for testicular cancer.
Semen quality and fertility are also related to cryptorchidism (Lee and
Coughlin, 2001; Virtanen et al., 2007), and epidemiological findings reflect
also this connection. For example men in Finland and Denmark also differ
from each other in semen quality. Danish men have lower sperm counts
than do Finns (Jrgensen et al., 2001; Jrgensen et al., 2002). Features that
might predict such a difference can appear in early childhood, as seen in
the Finnish-Danish cohort study of cryptorchidism, in which the testes
were measured by ultrasound and reproductive hormones were analyzed
at the age of three months (Boisen et al., 2004; Main et al., 2006b). Danish
boys had smaller testes than Finnish boys and testicular growth was slower
in Denmark than in Finland (Main et al., 2006b). Similarly, inhibin B levels
were lower in Danish boys than in Finnish boys and correlated closely to
the testis volumes (Main et al., 2006b). All these findings together suggest
that cryptorchidism is also linked to semen quality.
Hypospadias is a disorder of penile development that is common, but
the incidence is still only approximately 1/10th of the cryptorchidism rate
(Toppari et al., 2001). Hypospadias is also linked to cryptorchidism and they
occur together more often than expected by chance (Schnack et al., 2010a).
The prevalence of hypospadias varies between Denmark and Finland
in a similar pattern as testicular cancer and cryptorchidism (Virtanen et
al., 2001; Boisen et al., 2005). All these disorders and sperm production
capacity of the testis are critically linked to androgen action and related
hormonal regulation during development (Sharpe and Skakkebaek, 2008).
One or more of the disorders may arise from maldevelopment of the
testis, called testicular dysgenesis syndrome (TDS) (Skakkebaek, Rajpert25

De Meyts and Main, 2001). It is useful, therefore, to consider all these
problems together in epidemiological and experimental studies.
ii. Mechanisms
Testes differentiate in the fetal gonadal ridge during early gestation

(embryonic weeks 6-7) and become hormonally active soon after
differentiation. The interstitial Leydig cells in the testis secrete testosterone
and insulin-like peptide 3 (INSL3) that regulate testicular descent. INSL3
stimulates outgrowth of the gubernaculum that is attached to the testis
and epididymis and anchors the gonad to the bottom of the pelvis close
to the inner opening of the inguinal canal. When the fetus grows rapidly,
the testes become separated from the kidneys and other organs that move
upwards along the growing body. During late gestation the testes rapidly
move through the inguinal canals to the scrota. This transinguinal descent
is dependent on normal androgen action. In androgen insensitive persons
and those with defects in androgen production, the gonads remain either
in the bottom of abdomen or in the inguinal canals. The same is true in
androgen deficient and androgen insensitive rats and mice. Thus, it is easy
to hypothesize that anything that will perturb INSL3 and/or testosterone
production or action can cause cryptorchidism.
Mutations in androgen receptor gene, steroidogenic enzymes needed for
androgen production, or hypothalamo-pituitary regulators needed for
testicular stimulation are all well characterized reasons for cryptorchidism,
but occur very rarely (Virtanen et al., 2007; Barthold, 2008). Chemicals that
inhibit androgen production or action (anti-androgens) can directly disturb
testicular descent, which has a robust experimental evidence. Mutations in
INSL3 and its receptor RXFP2 have been reported in heterozygous form
in cryptorchid boys (Ferlin et al., 2003; Foresta et al., 2008). However, these
may be polymorphisms rather than mutations, because they were found
as frequently in normal population as in cryptorchid subjects (El Houate
et al., 2008; Nuti et al., 2008). No mutations either in INSL3 or in RXFP2
were found in Finnish patients (Koskimies et al., 2000; Roh et al., 2003).
However, down-regulation of these genes might contribute to maldescent
of the testes. Estrogens can down-regulate Insl3 expression in mice, which
may explain why estrogens can cause cryptorchidism (Emmen et al., 2000;
Nef, Shipman and Parada, 2000). Lower cord blood levels of INSL3 were
found in boys with cryptorchidism persisting at 3 months compared to a
group of control boys, suggesting that perturbed INSL3 production may
have contributed to the disorder (Bay et al., 2007).
26
There are several other genes that have been linked to cryptorchidism
in experimental animals with knock-out techniques e.g., Hoxa10, Hoxa11
(Hsieh-Li et al., 1995; Rijli et al., 1995; Satokata, Benson and Maas, 1995;
Overbeek et al., 2001; Daftary and Taylor, 2006), but there is little evidence
for their role in humans. Cryptorchidism can also be found as a part of
several syndromes, many of which have an identified genetic reason
(Virtanen et al., 2007). However, a great majority of cryptorchidism occurs
as a single disorder. Genome-wide association analyses and transcriptome
analyses may bring new candidate genes, such as FGFR1 and downstream
signaling molecules SOS1 and RAF1 (Hadziselimovic et al., 2010) that
need to be tested in larger populations. A recent study did not find any
mutations in FGFR1 and heterozygous GnRHR mutations were found
in similar frequency as in a group of controls (Laitinen et al., submitted).
The genes may also be the targets of adverse environmental effects as
exemplified by estrogen-INSL3 interaction.
Risk factors for cryptorchidism that have been reported in several studies
include low birth weight, being small for gestational age, prematurity and
having other genital malformations (Hjertkvist, Damber and Bergh, 1989;
Group 1992; Berkowitz et al., 1993; Berkowitz et al., 1995; Jones et al.,
1998; Thong, Lim and Fatimah, 1998; Akre et al., 1999; Weidner et al.,
1999; Ghirri et al., 2002; Boisen et al., 2004; Preiksa et al., 2005). The most
robust evidence of increased risk is associated with intrauterine growth
retardation and being small for gestational age. This was also evident in
Finnish newborns (Boisen et al., 2004). Prematurity is another risk factor,
but many of the premature newborns have a spontaneous descent of the
testes before the due date, reflecting normal physiology. Life style factors,
such as mothers smoking and alcohol consumption may also increase
the risk, although the evidence is less clear. In a prospective, clinical
cohort study, mothers alcohol consumption was associated with a dosedependent increase in the risk of cryptorchidism (Damgaard et al., 2007),
whereas in registry- and interview-based studies including persistent and
severe cases, i.e. those who usually needed treatment, only early gestation
binge drinking showed an association with a slightly increased risk (Jensen
et al., 2007; Mongraw-Chaffin et al., 2008; Strandberg-Larsen et al., 2009).
Most studies have not shown any effect of mothers smoking (MongrawChaffin et al., 2008; Damgaard et al., 2008), whereas the use of nicotine
patches was associated with an increased risk (Damgaard et al., 2008).
27

However, in one study heavy smoking was associated with an increased
risk of bilateral cryptorchidism (Thorup, Cortes and Petersen, 2006). Diettreated gestational diabetes was also found to increase the risk, possibly
by altering the hormone balance of the developing fetus (Virtanen et al.,
2006). Occupational risk factors include gardening and farming, putatively
due to pesticide exposure (Weidner et al., 1998), (Kristensen et al., 1997).
Many pesticides have been recognized as endocrine disrupters, but
there are not many studies linking direct exposure measurements and
cryptorchidism. Studies using occupational and job matrix analyses as
proxies for exposure have hinted at a possible association (Weidner et
al., 1998). Breast milk samples from mothers of cryptorchid boys had a
higher total amount of chlorinated pesticides than those from mothers of
boys without cryptorchidism (Damgaard et al., 2006), and these originated
from historical rather than recent exposures of the mothers as judged by
enantiomeric analysis (Shen et al., 2006). The levels of these chemicals
are declining, but because of the persistence of the compounds they
continue to add to the contaminant load of children in future generations.
The associations for individual chemicals, such as DDT or DDE, are not
apparent (Damgaard et al., 2006; Longnecker et al., 2002), emphasizing
the need to integrate data and use bioinformatic tools to analyze
complex data sets. Already rather simple principal component analyses
can demonstrate distinct chemical signatures between different regions
as exemplified by contrasts between Denmark and Finland (KrysiakBaltyn et al., 2010). However, some studies have identified differences
in individual compounds, e.g., higher levels of heptachloroepoxide and
hexachlorobenzene were found in fat samples of cryptorchid boys than in
controls (Hosie et al., 2000).
Polybrominated diphenyl ethers are used mainly as flame retardants and
they are also rather persistent in nature. Some of the PBDEs are antiandrogenic (Stoker et al., 2005). Mothers of cryptorchid boys had higher
breast milk concentrations of these compounds than mothers of control
boys (Main et al., 2007). Environmental contamination with PBDEs is
higher in the USA than in Europe, and many of these compounds have
been banned after initial introduction (Darnerud et al., 2001; Betts 2002;
Main et al., 2007).
Phthalate esters are ubiquitous environmental chemicals that are
everywhere in the modern milieu. They are used in plastics as softeners,
and they occur in packaging, tubing, surface materials, office and household
28
equipments. Humans are exposed mainly by food and drink, but also
through skin and indoor air. Diethyl hexyl phthalate and dibutyl phthalate
interfere with testosterone production and therefore have anti-androgenic
effects in developing rodents (Scott, Mason and Sharpe, 2009). In humans,
phthalate levels in mothers urine have been associated with the anogenital
index (defined as the anogenital distance (AGD) divided by the weight
of the boy at examination) of their sons, suggesting also anti-androgenic
effects (Swan et al., 2005). Phthalate levels in breast milk were positively
correlated with increased LH/testosterone ratios, compatible with an antiandrogenic effect forcing pituitary to exert a stronger stimulation to Leydig
cells to maintain normal androgen levels (Main et al., 2006a). Phthalate
levels in mothers breast milk were not directly associated with the risk
of cryptorchidism in the offspring (Main et al., 2006a). Different species
and strains show varying susceptibility to the testicular effects of in utero
phthalate exposure (Johnson et al., 2008; Scott, Mason and Sharpe, 2009).
b. Hypospadias
i. Epidemiology
In hypospadias the urethra has failed to fuse normally on the ventral side of
the penis and opens inappropiately to the end of the split (Figure 4). The
meatus can locate anywhere between the glans and perineum depending
on the severity of hypospadias (Kllen et al., 1986). If the urethra opens
to the glans or corona (sulcus), it is called distal, and this mild form of
hypospadias often does not necessitate any treatment. Therefore it is often
Hooded preputium
Normal
Glandular
Coronal
Penile
Penoscrotal
Scrotal
Perineal
Figure 4. Clinical classification of location of the urethral meatus in hypospadias.
not registered at all and malformation registries vary in their practices of

recording these defects. If the urethral meatus is located in the penile
29

shaft or penoscrotal area, the hypospadias is called proximal and these
require surgical management. A third category of middle hypospadias also
has been used to separate cases with penile shaft location of the urethral
meatus from distal and proximal defects (Brouwers et al., 2009). To make
it even more complicated, the distinction between distal and proximal
forms varies, because some studies include cases with mid shaft penile
hypospadias in distal forms (Cox, Coplen and Austin, 2008). Therefore
it is important to consider which types of hypospadias are included in
epidemiological studies before comparing the results and making any
conclusions. Physiological phimosis may hinder diagnosis of distal forms
at birth, and these may become visible only later when the foreskin can
be retracted behind the glans, as shown in Denmark where the birth rate
of hypospadias was 1% and the cumulative incidence at 3 years was 4.6%
(Boisen et al., 2005).
Registry-based studies on the incidence of hypospadias tend to underestimate the true rate (Toppari et al., 2001). The reasons include poor
ascertainment in routine clinical work, under-reporting to the registry, and
varying policies in recording distal cases. In many malformation registries
distal hypospadias are not considered at all, although these are very
common in population-based prospective clinical studies (Virtanen et al.,
2001; Pierik et al., 2002; Boisen et al., 2005). Several European studies have
shown higher prevalence rates than previous estimates of 0.4 and 2.4 per
1000 total births (Dolk et al., 2004). Despite the caveats in epidemiological
analyses of hypospadias, there is ample evidence of increased rates in
several regions of Australia, Europe, and the USA (Kllen et al., 1986;
Paulozzi, 1999; Toppari et al., 2001; Nassar et al., 2007). Many malformation
registries changed their approach to hypospadias to more active search in
1990s when it became evident that a large proportion of cases remained
unregistered (Hemminki, Merilainen and Teperi, 1993), which may also
explain many controversies in trend analyses (Aho et al., 2000; Carmichael
et al., 2003; Dolk et al., 2004; Porter et al., 2005; Fisch et al., 2009). An
increasing trend in the 1970s and 1980s in the USA was reported on the
basis of malformation registry data that showed an increase especially in
proximal hypospadias (Paulozzi, Erickson and Jackson, 1997). Hospital
discharge registries on operated cases of hypospadias reflect well the
prevalence of proximal hypospadias, but they do not include the mild
coronal and glanular forms that are not operated. In Denmark, the birth
rate of hypospadias was estimated to be 0.52% according to hospital
30
Table 4. Rate of hypospadias in boys in prospective or cross-sectional

clinical (non-register based) studies
Country
U.S., Rochester
Minnesota, St. Marys
Hospital
U.S., ante partum
clinic of the Sloane
Hospital, New York
City
U.S., Collaborative
perinatal project
Reference
(Harris and Steinberg,
1954)
Study type
Prospective study (n=4474)
Rate of hypospadias
0.70% (BW>2500g), 0.76%
of all live-born boys
(McIntosh et al., 1954)
0.54% of live-born boys
Korea, 38 hospitals
Southern Jordan
(Choi et al., 1989)

(al-Abbadi and Smadi,
2000)
(Virtanen et al., 2001)
prospective study on
pregnant women and
infants
(n=2793 live-born males)
prospective study (n=53394
consecutive single births
(boys and girls))
prospective study (n=7990)
Clinical study of 1748 boys
(aged 6 to 12 years)
Prospective cohort study
(n=1505)
Total hospital cohort
(n=5798)
Prospective study (n=7292)

(at 3 years: 4,64% of boys
(including also milder
cases detected when
physiological phimosis
dissolved))
0.29% of boys
Finland,Turku, Turku
University Hospital
(Myrianthopoulos and
Chung, 1974)
Netherlands,
Rotterdam
Denmark,
Copenhagen,
Rigshospitalet
(Pierik et al., 2002)

Prospective cohort study

(n=1072)
Bulgaria, 5 regions
(Kumanov et al., 2007)
Cross-sectional clinical
study (n=6200 boys aged 0
to 19 years)
0.80% of single-born boys

(76% of cases detected
at birth)
0.21% of newborn boys
0.74% of boys
0.73% of newborn boys
registries (Lund et al., 2009), whereas the prospective cohort study showed
the rate of 1.03% (Boisen et al., 2005). Interestingly, in Finland the birth
rate of hypospadias was only 0.3% in a parallel study to that of Boisen et
al. (Virtanen et al., 2001). Incidence data of hypospadias are presented in
Table 4.
ii. Mechanisms
Androgens regulate male urogenital differentiation. Defects in androgen

biosynthesis, metabolism or action can cause hypospadias. Genetic
mutations leading to disorders of testicular differentiation, testosterone
synthesis, conversion to dihydrotestosterone or androgen receptor
action may result in hypospadias (Kalfa, Philibert and Sultan, 2008).
Hypospadias is graded by the same Prader classification that is used for
description of the severity of androgen insensitivity (Quigley et al., 1995).
31

However, only about 20% of patients with isolated hypospadias have
signs of testicular dysfunction or other endocrine abnormalities (Rey et
al., 2005). Environmental effects on androgen action influence penile
development, as shown in experimental animals, in which anti-androgenic
compounds typically cause hypospadias (Wilson et al., 2008). The critical
role of androgens in both penile development and testicular descent is
another physiological link between cryptorchidism and hypospadias, and it
provides justification for the search for environmental etiologies for both
of these conditions.
The penis develops from the genital tubercle and several genes are known
to be involved in this, but only a few have been associated with human
hypospadias (Kalfa, Philibert and Sultan, 2008; Wang and Baskin, 2008).
Homeobox genes, HOXA and HOXD genes contribute to the development
of urogenital structures and loss of their function causes agenesis or
malformations of the genitalia (Morgan et al., 2003). HOXA13 mutations
have been found in the human hand-foot-genital syndrome (Mortlock and
Innis, 1997; Frisen et al., 2003). Expression of fibroblast growth factor
(FGF) 8 and bone morphogenetic protein 7 in the developing urethra
depend on HOXA13, which also influences vascularisation and androgen
receptor expression (Mouriquand and Mure, 2001). FGF 10 and FGF
receptor 2 have also been linked to the risk of hypospadias in humans
(Beleza-Meireles et al., 2007). Sonic Hedgehog (Shh) has been shown to be
crucial for normal genital development in the mouse models (Haraguchi et
al., 2001; Perriton et al., 2002; Yucel et al., 2004), but no human mutations
have been reported. Activating transcription factor (ATF) 3 was suggested
to be involved in the development of hypospadias, because its transcripts
were elevated in the foreskin samples in 86 % of operated hypospadias
patients, whereas only 13% of samples from circumcision patients had
elevated levels (Liu et al., 2005). ATF3 is influencing TGF-beta signalling
and it is estrogen-responsive, which might give one explanation why
estrogens increase the risk for hypospadias (Liu et al., 2006; Willingham
and Baskin, 2007). In addition to hand-foot-genital syndrome, hypospadias
can be found in many other multi-organ syndromes, which suggests
genetic causes. Genes that are identified may also be targets of endocrine
disrupters that can disturb their regulation during critical developmental
windows.
Mutations in MAMLD1 (or CXORF6) cause hypospadias and testicular
dysgenesis (Fukami et al., 2006). The defect appears to cause disruption of
32
androgen production, because the gene affects hormone synthesis and has
the NR5/SF1 target sequence (Fukami et al., 2008). Mutation in NR5/SF1
cause testicular dysgenesis, too (Bashamboo et al., 2010) and this gene may
be an important target for endocrine disrupters (Suzawa and Ingraham,
2008). MAMLD1 mutations are rare in patients with hypospadias, but this
mutation can be a part of the cascade of events leading to this disorder
(Ogata, Wada and Fukami, 2008; Ogata, Laporte and Fukami, 2009).
Genetic polymorphisms in androgen and estrogen receptors have been
associated with the risk of TDS disorders including hypospadias (Aschim
et al., 2004b; Yoshida et al., 2005; Beleza-Meireles et al., 2006; Watanabe
et al., 2007). However, contradictory results have been published and the
associations with the single nucleotide polymorphisms will need to be
replicated in larger populations (van der Zanden et al., 2010b ; Wang et al.,
2008). A genome-wide association study revealed a common variant of
DGKK, encoding diacylglycerol kinase, to be linked to an increased risk of
hypsopadias (van der Zanden et al., 2011).
Cryptorchidism and hypospadias share risk factors, such as being smallfor-gestational age (Akre et al., 1999; Aschim et al., 2004a; Pierik et al.,
2004; Akre et al., 2008). Anti-androgens and estrogens can cause both
conditions, as demonstrated in epidemiological studies that followed the
children of women who used diethyl stilbestrol (DES) during pregnancy
(for review see (Toppari et al., 1996)). There is also evidence of secondgeneration effects of DES, because the sons of women exposed in utero
have a higher prevalence of hypospadias than other men (Klip et al.,
2002; Brouwers et al., 2006; Kalfa, Philibert and Sultan, 2008), suggesting
epigenetic effects by DES. The adverse developmental effects of DES in
humans are very similar to those described in animals (McLachlan et al.,
2001).
Epidemiological studies on hypospadias have used many different ways
to assess exposures, including direct measurements in biological samples
from mothers or children, environmental measurements, and job-exposure
matrices. Pesticides have been high on the list of suspected chemicals
because of their endocrine disrupting properties. A meta-analysis of 9
studies assessing the association of pesticide exposure with hypospadias
found elevated but marginally significant risks associated with maternal
occupational exposure [pooled risk ratio (PRR) of 1.36, CI = 1.04-1.77],
33

and paternal occupational exposure was not statistically significant (PRR
of 1.19, CI= 1.00-1.41) (Rocheleau, Romitti and Dennis, 2009). Vegetarian
diets of mothers were associated with an increased risk for hypospadias
in the ALSPAC study (North and Golding, 2000), and a somewhat similar
finding showed a decreased risk for mothers having fish or meat in their
diet during pregnancy (Akre et al., 2008). Subfertility and the use of assisted
reproductive techniques are risk factors for hypospadias (Sweet et al.,
1974; Czeizel, 1985; Wennerholm et al., 2000; Klemetti et al., 2005; Kllen
et al., 2005). The causes can be both genetic and epigenetic, including
environmental effects. The role of pharmaceutical sex steroids other than
DES is controversial. Use of progestins was associated with an increased
risk of hypospadias (Czeizel, Toth and Erodi, 1979; Calzolari et al., 1986),
but a meta-analysis of fourteen studies showed no association between
exposure to sex steroids (excluding DES) during the first trimester and
external genital malformations (Raman-Wilms et al., 1995).
c. Timing of puberty
i. Epidemiology
Age at menarche has been approximately 13 years for several decades,

whereas 200 years ago it was around 17 years (Aksglde et al., 2008 and
2009a). Improved nutrition, health and better living conditions may have
caused the decline of the age at menarche (Parent et al., 2003). Now there
appears to be a new downwards trend; breast development that normally
occurs about two years before menarche appears much earlier than before.
Three American studies (PROS, NHANES III, BCERC) and studies
from Europe report earlier breast development in girls (Biro et al., 2010;
Herman-Giddens et al., 1997; Sun et al., 2002; Wu, Mendola and Buck,
2002; Chumlea et al., 2003; Aksglaede et al., 2009b; Semiz et al., 2008;
Castellino et al., 2005), as compared to previous data (Foster et al., 1977;
Lee 1980; Juul et al., 2006; Euling et al., 2008; Reynolds and Wines 1948;
Nicolson and Hanley 1953). The American PROS and NHANES III
studies both showed approximately 0.6-1.2 years advancement in entering
breast stage 2 in the 1980s and 1990s compared to earlier data from the
1930s and 1940s (Herman-Giddens et al., 1997), and the most recent study
confirmed this development in the 2000s (Biro et al., 2010). However,
there was no change in age at menarche (12.9 years in PROS) or only
small advancement (0.3 years) (12.6 years in NHANES III) compared to
34
the previous studies. The girls were assessed only by visual inspection in
the NHANES III, which has been criticized because this may have caused
some misclassification of some girls as having breast development when
there was just fat around the mammary gland. In the PROS study, 39%
of the girls were also palpated in addition to visual inspection to detect
breast tissue (Kaplowitz and Oberfield 1999), which demonstrated only
limited bias compared to visual assessment alone. An international expert
panel concluded in 2003 that the available data for girls were sufficient to
suggest a secular trend toward earlier onset of breast development among
American girls (Euling et al., 2008). At that time there were not yet studies
supporting such a trend in age at breast development among European
girls (Mul et al, 2001; Juul et al, 2006). However, recent European data
support the US findings of a decline in age at pubertal onset. The age at B2
was 10.3 years in 1638 Italian girls (Castellino et al., 2005), and 10.2 years
in 1562 Turkish girls (Semiz et al., 2008). In Denmark, two similar cohort
studies in which breast development was judged by palpation of glandular
breast tissue showed 12 months earlier age at B2 in 2006-8 (mean age at
B2 was 9.9 years) than in 1991-93 (Aksglaede et al., 2009b; Juul et al., 2006).
As in the US studies, age at menarche advanced only slightly (Aksglaede
et al., 2009b).
Several outbreaks of precocious puberty have been reported, e.g., in
Puerto Rico and in Italy (Comas, 1982; Fara et al., 1979). These have
appeared to be peripheral, i.e. not central, precocious puberty, and the real
causes remained elusive despite many exposure measurements. There are
also some areas with a high incidence of central precocious puberty, e.g. in
Northwest Tuscany (Massart et al., 2005). Pollution from greenhouses and
several small navy yards in that area were suspected to contribute to the
problem, but no causal relationships have been demonstrated.
Adopted and immigrant children from developing countries have an
increased susceptibility to central precocious puberty, which has been
reported in several Western countries (for references see Parent et al.,
2003). The reason is not known, but endocrine disrupters may contribute
(Krstevska-Konstantinova et al., 2001). Relatively high levels of p,p-DDE
were found in 26 immigrant girls with precocious puberty in Belgium,
whereas only two of 15 native Belgian patients had detectable serum
DDE concentration (Krstevska-Konstantinova et al., 2001), which lead
to a hypothesis that early and temporary exposure to weakly estrogenic
dichlorodiphenyltrichloroethane (DDT, parent compound to DDE) in
35

certain developing countries could stimulate hypothalamic and pituitary
maturation at the same time that it inhibits the pituitary gonadotrophin
secretion via a negative feedback that prevents manifestation of central
maturation. After migration, the exposure dramatically decreases and
the negative feedback disappears allowing the onset of puberty (Rasier
et al., 2006). The problem in this hypothesis is the long half life of DDT
that makes the sudden decline in exposure unlikely. Experimental work
on DDT, however, has shown its capability to influence GnRH activity
(Rasier et al., 2006).
ii. Mechanisms
Regulation of pubertal onset occurs at the central nervous system where

several neuronal and humoral inputs act in the neuronal network controlling
GnRH neurons. The puberty starts when these cells start to secrete GnRH
in a pulsatile manner, which in turn activates pituitary gonadotropes to
secrete gonadotropins FSH and LH that act on the gonads. After the
testes and ovaries have started to secrete sex steroids, secondary sexual
characteristics start to appear. Endocrine disrupters can interfere with
pubertal onset on several levels. They may influence the neuronal network
in the brain, GnRH neurons, the pituitary gland, the gonads, and they
may exert direct peripheral effects as hormone agonists or antagonists or
both, depending on the dose and background hormone levels. The same
compound can have an agonistic effect when the endogenous hormone
level is very low (childhood), whereas it can be an antagonist when the real
hormone is available (adulthood). Kisspeptin and its receptor in GnRH
neurons was found to be a central upstream signal triggering GnRH
neuron activity, and therefore much interest has recently been focused on
the regulation of Kisspeptin producing neurons as targets of endocrine
disruption (Tena-Sempere, 2010).
Exposure of children to pharmaceuticals containing sex steroids or any

other products with such endocrine activities cause typically peripheral
precocious puberty, which has been described in many case reports.
Estrogens stimulate breast development, whereas androgens cause growth
of pubic hair and changes in skin (oily skin and hair, adult-type sweat
odour). Ointments and salves containing estrogenic compounds have
been linked to prepubertal gynecomastia (Henley et al., 2007). If the
36
Table 5 Overview of epidemiological studies investigating the effects of

endocrine disrupters on onset of human puberty
Contaminant
Sex
Male
Chlorinated pesticides
(DDT and DDE)
Female
Male
Dioxins
Female
Female
Polychlorinated
biphenyls (PCBs)
Male
Polybrominated
biphenyls (PBBs)
Female
Bisphenol-A
Female
Female
Lead
Male
Cadmium
Female
Observation
No association with pubertal
development
References
Gladen et al., 2000
Younger age at menarche
Vasiliu et al., 2004
Precocious puberty
Krstevska-Konstantinova et al., 2001
No association with breast stage or

pubic hair development
development
No association with sexual maturation
Later onset of breast development
No association with the onset of
menarche
Wolff et al., 2008

Gladen et al., 2000
Den Hond et al., 2002
Leijs et al., 2008
Warner et al., 2004
Lower stage of breast development
Den Hond et al., 2002
Slowed breast development
Staessen et al., 2001
No association with menarche or

pubertal stages
Den Hond et al., 2002; Vasiliu et al.,

2004

Wolff et al., 2008

development
Gladen et al., 2000
Late first ejaculation
Leijs et al., 2008
Reduced penile length
Guo et al., 2004
Slowed genital development
Den Hond et al., 2002; Staessen et

al. 2001
No association with the development

of puberty
development
Earlier age at menarche and pubic hair
development
Delayed breast and pubic hair
development
Delayed menarche and pubic hair
development
Inversely associated with inhibin B
levels
Delayed breast development, pubic
hair growth and age of attainment of
menarche
Delayed onset of puberty on the basis
of testicular volume of > 3 ml, genitalia
staging and pubic hair staging
High levels of both cadmium and lead
is inversely associated with inhibin
B levels
Mol et al., 2002

Gladen et al., 2000
Blanck et al., 2000
Wolff et al., 2008
Selevan et al., 2003
Wu et al., 2003
Gollenberg et al., 2010
Naicker et al., 2010
Williams et al., 2010
Gollenberg et al., 2010
37

source of exposure can be recognized and eliminated, peripheral puberty
does not advance and breast tissue disappears slowly. Peripheral puberty
also may stimulate central puberty, which presents a complex problem.
Table 5 summarizes epidemiological studies on the exposure-outcome
relationships in pubertal development.
Timing of puberty among 151 daughters of fish-eating mothers and their
controls was studied in the Michigan anglers cohort in which exposure
to DDT was measured (Vasiliu, Muttinemi and Karmaus, 2004). Early
age at menarche was associated with fetal exposure to high levels of
DDE. In contrast, in the North Carolina infant feeding study of 316
girls and 278 boys, pubertal timing was not significantly associated with
exposure to DDE (Gladen, Ragan and Rogan, 2000). No association of
DDE exposure and breast development was found in 9-year-old inner
city girls in New York (Wolff et al., 2008). Higher serum DDT levels were
associated with earlier age at menarche in 466 Chinese textile workers,
(Ouyang et al., 2005).
High exposure to endosulfan was associated with later puberty in a study
comparing 117 boys from a highly contaminated area to 90 matched
control boys from an uncontaminated area (Saiyed et al., 2003). It was
suggested that the antisteroidogenic properties of endosulfan could have
contributed to the effect.
Polychlorinated biphenyls (PCBs)
Epidemiological studies on exposure to PCBs in relation to the timing of
puberty have yielded somewhat controversial results. In a Belgian study, a
delay of puberty was found among boys in urban areas and in association
with high serum PCB levels (PCB congeners 138, 153 and 180), whereas
no association of PCB levels to pubertal timing was found among girls
(Staessen et al., 2001; Den Hond et al., 2002). The study included 120 girls
and 80 boys, examined by trained physicians, from rural and urban areas. In
the North Carolina infant feeding study, no association of PCB exposure
to the self-reported timing of puberty (including age at menarche) was
found among 316 girls and 278 boys, although there was a tendency to
early maturation among the girls in the highest prenatal exposure group
(Gladen, Ragan and Rogan, 2000). No association of PCB exposure with
self-reported timing of puberty was found in 327 (Blanck et al., 2000)
or 151 girls (Vasiliu, Muttinemi and Karmaus, 2004) in studies from the
38
Great Lakes area, Michigan in USA, or in 196 boys from the Faroe Islands
(Mol et al., 2002). High PCB levels in boys were correlated with late first
ejaculation among 14 Dutch boys in a longitudinal cohort study, but no
other pubertal sign was associated with PCB concentration (Leijs et al.,
2008). In the Yucheng accident, 55 boys were exposed to high levels of
PCB and polychlorinated dibenzofuran (PCDF) levels, and in the followup studies they had shorter penile length than the control boys at the same
age, suggesting pubertal delay (Guo et al., 2004). Among girls in the inner
city of New York, PCB levels were associated with a smaller likelihood
of having breast development among lean 9-year-old girls, whereas no
associations were found with DDE, lead and bisphenol A concentrations
(Wolff et al., 2008). The girls with breast development in that study had
lower levels of urinary biomarkers of phytoestrogens than control girls.
In a small longitudinal cohort study in the Netherlands, no association
was found between PCB and polybrominated diphenyl ether levels and
pubertal development either in boys or girls (Leijs et al., 2008). In summary,
there are two studies suggesting a correlation with delayed puberty and
two studies showing no effect of PCB exposure on the timing of puberty
among boys, whereas there are no consistent associations found among
girls.
Polybrominated biphenyls (PBBs)
An animal feeding accident in Michigan in the 1970s caused a secondary
exposure to polybrominated biphenyls (PBBs) in thousands of people
using the products from the farm. In the follow-up studies some years
later, PBBs were measured in the serum of mothers. These measurements
were then used to approximate perinatal exposure of their children. High
exposure through breast feeding was associated with earlier pubic hair
development and an earlier age at menarche among the girls, whereas
breast development was not associated with exposure levels. This study
was based on self-assessment of pubertal development, which might have
caused more inaccuracy in detection of breast development than that of
pubic hair appearance and age at menarche (Blanck et al., 2000).
Phthalates
Children are ubiquitously exposed to phthalate compounds. Animal studies
have shown clear endocrine disrupting properties of many phthtalates,
but there are not many human studies on their possible effects on pubertal
39

development. The epidemic of early breast development in Puerto Rico
was followed by many studies on putative endocrine disrupters, including
phthalates (Colon et al., 2000). Phthalates were linked to gynecomastia,
because two thirds of 41 girls with early breast development and only 14
% of 35 controls had measurable phthalate levels in serum. However, the
phthalate measurements were criticized for technical inconsistensies and
the serum exposure profile raised a serious concern about possible sample
contamination or technical problems, because the levels of unmetabolized
diethyl hexyl phthalate were high as compared to other phthalates (McKee
et al., 2004).
Dioxins
Dioxins are a group of well-characterized endocrine disrupters, whose
mechanisms of action are at least partly known: they act through aryl
hydrocarbon receptors and thereby interact with other nuclear receptors
(Wormke et al., 2003). In July 1976 an explosion occurred in a chemical
company in Medina, Italy. A toxic cloud with high concentrations of
dioxins affected neighbouring communties, including the village of Seveso.
After the Seveso accident, large amounts of dioxins were spread to the
environment. In a retrospective analysis of the age of menarche and the
level of exposure, no association was found, but uncertainty remained
whether the timing of exposure was relevant for pubertal effects in these
girls (Warner et al., 2004). In the Yucheng (Taiwan) accident, children
were exposed to both PCBs and PCDFs (furans) via contaminated rice
oil. The exposed boys had signs of delayed puberty as described earlier
(Guo et al., 2004). In a small (n=18) cohort study in the Netherlands,
later onset of breast development was correlated in girls with higher
prenatal dioxin exposure (Leijs et al., 2008). Total dioxin-like activity in
serum was assessded by the Calux assay among the children from rural
and two urban areas in Belgium (Staessen et al., 2001; Den Hond et al.,
2002). Dioxin-like activities in childrens serum were higher in urban areas
than in the rural area. The age at menarche and pubic hair development
showed no correlation with exposure, but slow breast development to
the adult stage was associated with high dioxin activity (Den Hond et al.,
2002). Among boys there was no significant exposure-pubertal outcome
relationship found. However, the testes of boys living in the urban areas
were significantly smaller than those of the boys in the rural area (Den
Hond et al., 2002). Dioxins are known to have both estrogenic and antiestrogenic effects, because dioxin-AhR-nuclear translocator complex
40
interacts with estrogen receptors (Ohtake et al., 2003). These effects could
have contributed to breast development in highly exposed girls.
Lead
Studies on the association of lead exposure with the timing of puberty
have given the most consistent results of the epidemiological puberty
studies. Lead exposure is associated with a delay in pubertal onset. High
lead levels in blood were associated with a delayed age at menarche and
delayed pubic hair development in two studies from the National Health
and Nutrition Examination Survey in U.S. (NHANES III) (Selevan et al.,
2003; Wu et al., 2003). In the study of 2186 girls, breast development was
also delayed (Selevan et al., 2003). Similar findings were reported from
South Africa (Naicker et al., 2010). In a cross-sectional study including
705 10-11 years old girls, blood lead levels were inversely correlated with
inhibin B levels, suggesting a delay of the onset of puberty that is marked
by increasing inhibin B levels (Gollenberg et al., 2010). The correlation
was even stronger when the urinary cadmium concentration was high
(Gollenberg et al., 2010). Lead exposure also is associated with delayed
puberty and growth in boys. Even rather low lead levels in blood were
associated with growth and pubertal development among boys in Central
Russia (Hauser et al., 2008).
d. Thyroid effects
i. Epidemiology
Hypothyroidism is the most frequent thyroid disease, the incidence of

which is influenced by both sex and age (Fatourechi, 2009). Clinical
hypothyroidism is a relatively frequent disease in fertile women, thus
potentially affecting the fetus. Among children, the incidence of
hypothyroidism is highest in adolescence. Furthermore, subclinical
hypothyroidism is a condition probably affecting a considerable number
of both children and adults, and that may be more relevant with respect to
effects of endocrine disrupting chemicals.
Estimating effects on levels of circulating thyroid hormones is dependent
on well-defined population-based reference ranges, which are, however,
quite large compared to intra-individual variations in thyroid hormone
levels (Feldt-Rasmussen et al., 1980). Thus, minor changes in thyroid
hormone levels due to exposure to endocrine disrupting chemicals may not
41

be detected in small cross-sectional human studies, in which the expected
inter-individual variations may camouflage real differences associated with
exposure.
During different life stages levels of both TSH and thyroid hormone
levels vary greatly. In pregnancy, endocrinological and physiological
alterations, including an estrogen-induced increase in TBG, result in an
additional stimulation of the maternal thyroid gland. Accordingly, total
thyroid hormone levels increase, and free thyroid hormone levels decrease
in the first half of pregnancy until a new steady-state is reached. In the
neonate, TSH increases dramatically immediately after birth peaking at 30
minutes, followed by an increase in both T4 and T3. All of these hormone
levels subsequently decrease, leaving evaluation of TSH and thyroid
hormone levels highly dependent on exact age and individual factors.
Thus, evaluation of especially TSH, but also thyroid hormone levels, in
pregnancy, the neonatal period and early childhood for use in statistical
associations with exposure to levels of environmental chemicals should
allow for age as a critical confounder. In particular, TSH measured in cord
blood may not be appropriate as a stable marker of thyroid function.
Thyroid hormone levels influence not only neurological development
but also metabolic processes in the body, including elimination processes
serving to eliminate endocrine disrupting chemicals from the body. Thus,
persons with high TH-levels may have a better capacity to eliminate
endocrine disrupting chemicals and thus lower levels of endocrine
disrupting chemicals in biological samples. This may be misleading in the
interpretation of research results as a high level of endocrine disrupting
chemicals may be causally linked to the levels of thyroid hormones.
However, these questions have not yet been addressed directly by
experimental or human studies.
Effects on cognitive function resulting from exposure to thyroiddisrupting chemicals are extremely difficult to estimate. It is not yet clear
which specific cognitive functions, or methods of testing, may be the most
representative of thyroid function during development. Furthermore, as
in the case of hypothyroidism, effects may be subclinical and require very
thorough testing to detect.
42
Boas M., Feldt-Rasmussen U, Skkakebaek N., Main K. Toppari J. European Journal of Endocrinology,
2006, 154:599-611. Society of the European Journal of Endocrinology (2006). Used with permission.
Figure 5. Possible mechanisms of action of environmental chemicals on the hypothalamic-pituitary-thyroid axis. (1) Synthesis of
thyroid hormones (TH): interference with NIS, TPO or TSH receptor. (2) Transport proteins. (3) Cellular uptake mechanisms. (4)
The TH receptor. (5) Iodothyronine deiodinases. (6) Metabolism of THs in the liver. TRH, thyrotropin-releasing hormone; TSH, thyroid stimulating hormone; NIS, sodium-iodide symporter; T4, thyroxine; T3, triiodothyronine; TPO, thyroid peroxidise. From: Boas
M., Feldt-Rasmussen U, Skkakebaek N., Main K. Toppari J. European Journal of Endocrinology, 2006, 154:599-611. Society of
the European Journal of Endocrinology (2006). Used with permission.
ii. Mechanisms
The mechanisms involved in thyroid homeostasis are numerous and

complex. Consequently, environmental chemicals can act at many levels in
the thyroid system. (See Figure 5.)
Synthesis of thyroid hormones: interference with the sodium iodide symporter, thyroid peroxidase activity or TSHreceptor
The basic synthesis of thyroid hormones may be compromised by
substances interfering with the processes in the thyroid gland, e.g. uptake
of iodine and the function of thyroid peroxidase (TPO). Thus, both
perchlorate and the phthalates DIDP, butyl benzyl phthalate (BBP) and
Di-n-octylphthalate (DnOP) have been shown to interfere with the activity
of the sodium iodide symporter (NIS) (Tonacchera et al., 2004; Breous,
Wenzel and Loos, 2005). Thyroid peroxidase (TPO) activity was in vitro
inhibited by nonylphenol (NP), BPA and BP2 (Schmutzler et al., 2004;
Schmutzler et al., 2007). The activity of the thyroid gland is stimulated
by TSH and may thus be altered by environmental chemicals affecting
the function of the TSH receptor. DDT and the PCB-mixture Aroclor
43

1254 interfered in vitro with post-receptor signalling by inhibition of the
adenylate cyclase activity and cAMP production (Santini et al., 2003).
Transport proteins
In serum, the hormones T3 and T4 are transported to the tissues bound
to transport proteins. Thyroxine binding globulin (TBG) is the most
important thyroid hormone transport protein in humans, but albumin and
transthyretin (TTR) also play a role. Competitive binding of environmental
chemicals to thyroid hormone transport proteins may result in increased
bioavailibility of endogenous thyroid hormones. The investigation of this
mechanism of action is restrained by interspecies differences, as TTR
is the principal transport protein in rodents and TBG in humans. It is
unlikely that enough T4 could be displaced from TTR to be toxic in adult
humans (Purkey et al., 2004). However, TTR is the major thyroid hormone
transport protein in the human brain, presumably playing an essential role
in the determination of FT4 levels in the extracellular compartment, which
is independent of the T4 homeostasis in the body. Furthermore, TTR may
mediate the delivery of T4 across the blood-brain barrier and the maternal
to fetal transport through the placenta. Thus, environmental chemicals
bound to TTR may be transported to the fetal compartment and fetal
brain, and be able to decrease fetal brain T4 levels (Ulbrich et al., 2004).
In experimental studies, PCBs (Meerts et al., 2002; Purkey et al., 2004),
flame retardants (Meerts et al., 2000), phenol compounds (Yamauchi et
al., 2003; Kudo and Yamauchi, 2005) and phthalates (Ishihara et al., 2003)
competitively bind to transthyretin (TTR). Metabolites and derivatives
of PCBs, several brominated flame retardants and phenol compounds
had remarkably stronger binding affinity than their parent compounds,
indicating an important role for hydroxylation and halogenation in thyroid
toxicity (Meerts et al., 2000). In contrast to the interference with TTR, no
environmental chemicals have been demonstrated to compete with thyroid
hormones for binding to TBG or albumin with significant strength (van
den Berg, 1990; Lans et al., 1994).

Cellular uptake mechanisms
Bioavailibity of thyroid hormones to the nuclear thyroid hormone
receptors may become compromised as TH are probably actively
transported across the cell surface via membrane bound transporters.
44
Several environmental chemicals, including di-n-butyl phthalate (DBP)

and n-butylbenzyl phthalate (BBP) inhibited [125I]T3 uptake in red blood
cells from bullfrog tadpoles (Shimada and Yamauchi, 2004).
The thyroid hormone receptor
Environmental chemicals can change thyroid hormone-stimulated gene
transcription, but it is still not clear through which mechanisms these
changes are induced.
In experimental studies, BPA, and hydroxylated PCBs acted as antagonists
to T3 (Moriyama et al., 2002; Sun et al., 2009; Kitamura et al., 2005a;
Arulmozhiraja et al., 2005; Iwasaki et al., 2002). Similarly, the derivatives
TBBPA and TCBPA competed for binding to the receptor (Kitamura et al.,
2005b; Jagnytsch et al., 2006; Fini et al., 2007; Hofmann, Schomburg and
Kohrle, 2009). A possible pathway of interference with TR is regulation
of TR-genes. Studies indicated that BPA, Dicyclohexyl phthalate (DCHP),
BBP and PCP inhibit the expression of the TR beta gene (Seiwa et al.,
2004; Sugiyama et al., 2005).
Environmental chemicals may also alter the expression of TH-responsive
genes. PCB and HCB induced several TH-responsive genes (Gauger et al.,
2004; Bansal et al., 2005 Zoeller et al., 2000; Loaiza-Perez et al., 1999).
Neural growth
Oligodendrocyte development and myelination are under thyroid
hormone control, as well as the extension of Purkinje cell dendrites, which
is essential for normal neuronal circuit formation (synaptogenesis) and
subsequent behavioral functions. PCBs, PBDE and BPA caused abnormal
development of Purkinje cell dendrites, human neural progenitor cells
or mouse oligodendrocytes (Sharlin, Bansal and Zoeller, 2006; KimuraKuroda, Nagata and Kuroda, 2005; Seiwa et al ., 2004).
Metabolism of circulating thyroid hormones
Peripheral iodothyronine deiodinases are controlling the conversion
of thyroid hormones in different organs and are thus essential in the
regulation of levels of the biologically active T3 by activation of T4 and
inactivation of T4 and T3. In the liver, several enzymes are involved in the
metabolism of thyroid hormones.
45

Type I 5deiodinase (5DI) in the liver was in vitro decreased by
several environmental chemicals: octyl-methoxycinnamate (OMC),
4-methylbenzylidene-camphor (MBC) (Schmutzler et al., 2004),
methoxychlor (Zhou et al., 1995), dioxins (Viluksela et al., 2004) and a
mixture of organochlorines, lead and cadmium (Wade et al., 2002).
Mechanistic studies indicated that PCBs, dioxins, PBDEs and PFOS may
act through interference with hepatic glucuronidation (Nishimura et al.,
2002; Hallgren et al., 2001; Yu, Liu and Jin, 2009; Nieminen et al., 2002a)
or sulfation (Schuur et al., 1998c; Schuur et al., 1998b; Schuur et al., 1998a).
PCBs
Multiple studies of PCB exposure and effects have been carried out in
human populations, several of which raise concern that environmental
levels of PCBs may reduce peripheral thyroid hormone levels (Hagmar et
al., 2001b; Persky et al., 2001; Abdelouahab et al., 2008; Turyk, Anderson
and Persky, 2007; Schell et al., 2008). A few studies also demonstrated a
positive correlation between PCB-exposure and TSH (Osius et al., 1999;
Schell et al., 2008).
Alterations in fetal and infant thyroid homeostasis due to environmental
exposures are of special concern, as it is well known that normal thyroid
function is crucial for neurological development. In recent years, several
studies have aimed at elucidating the potential toxic effects of environmental
levels of PCBs on human thyroid function in developmentally-important
age groups. Thus, environmental levels of PCBs are associated with
reduced thyroid hormone levels and/or positive associations with TSH
in pregnant women in several studies (Takser et al., 2005; Chevrier et al.,
2008), but not in all (Wilhelm et al., 2008). This indicates that maternal
thyroid function, which is important for the neurological development in
the fetus, may be altered by PCBs or other organochlorine compounds.
Studies of newborn babies and infants have been performed in different
settings, but the results are not consistent. This may be due to difficulties in
obtaining sufficiently large populations as well as obtaining blood samples
for evaluation of thyroid hormone levels. Serum levels of especially
thyroid-stimulating hormone, and to a lesser degree peripheral thyroid
hormones, change dramatically over the first few days of life, influenced
by various factors related to pregnancy, delivery and perinatal health
(Herbstman et al., 2008). An optimal evaluation of thyroid hormones in
46
the newborn infant therefore relies on the timing of blood samples.

In 1994 a study of 105 mother-infant pairs analysed associations between
PCBs and dioxin-like toxicants in breast milk with thyroid hormones in
maternal serum samples and infant serum samples obtained at two weeks
and 3 months of age. PCB levels were significantly correlated with lower
maternal T3 and T4 in late pregnancy and postpartum, with higher TSH
in infants at two weeks and three months of age. Infants with high toxic
equivalents levels had lower FT4 and total T4 at the age of two weeks
(Koopman-Esseboom et al., 1994).
Darnerud et al measured PCBs and dioxin in breast milk and thyroid
hormones in infant blood samples from 150 mother-infant pairs. After
adjustment for confounding factors, they found a negative correlation
between PCBs and total T3 at 3 weeks of age (Darnerud et al., 2010). In
a study of 98 mother-infant pairs in a polluted area, PCB levels in cord
blood were positively correlated with TSH in 3 days old infants. Peripheral
thyroid hormones were not analysed in this study (Ribas-Fito et al., 2003).
Other studies of newborns have confirmed these associations (Herbstman
et al., 2008; Chevrier et al., 2007), but several other studies did not find any
associations between PCB levels and levels of TSH and thyroid hormones
in cord blood (Wilhelm et al., 2008; Longnecker et al., 2000; Dallaire et al.,
2008; Dallaire et al., 2009; Wang et al., 2005; Steuerwald et al., 2000; LopezEspinosa et al., 2009).
Focusing on long-term effects of perinatal exposure, Matsuura et al. found
no associations between PCB levels in breast milk and thyroid hormone
levels at the age of 1 year (Matsuura et al., 2001). Similarly, Su et al. found
no associations between dioxins/furans in placentas and TH at 2 years of
age, but at 5 years T3 levels were higher in highly exposed individuals (Su
et al., 2010).
In older children, several studies have found negative correlations between
PCB levels in serum and thyroid hormone levels at the age of 4 years
(T3 and FT4) (Alvarez-Pedrerol et al., 2007), 7-10 years (FT3) (Osius et al.,
1999), and 10-15 years (T4 and FT4) (Schell et al., 2004).
Flame retardants
Few human studies exist regarding flame retardants and thyroid function.
These compounds accumulate in animal fat, (in fish, for instance), therefore
bio-accumulating through the food chain. However, recently a large
47

study of consumers of fish from the Great Lakes (US) reported negative
associations between concentrations of PBDE congeners in serum and
serum levels of T3 and TSH, as well as a positive relation with T4 (Turyk,
Anderson and Persky, 2008). However, a previous study of men exposed
through Baltic fish consumption showed negative associations between
TSH and PBDE (Hagmar et al., 2001a).
Recently, a study among 270 pregnant women (in gestational week 27)
showed negative associations between serum levels of PBDEs and
TSH (Chevrier et al., 2010). In a small study of 12 mother-infant pairs,
PBDE levels in pregnancy were not significantly associated with thyroid
hormones in cord blood (Mazdai et al., 2003). Thus, evidence on the effect
of flame retardants on human thyroid function is very limited, and current
results are conflicting.
Perfluorinated chemicals
Recently, a substudy of the NHANES study in the US found that women
with high levels of PFOA and men with high levels of PFOS were more
likely to report current treated thyroid disease (Melzer et al., 2010). A large
study of 506 employees in a PFC manufacturer company showed negative
associations between PFOA and FT4 (Olsen and Zobel, 2007), but
epidemiological human studies of effects of environmental PFC levels
are lacking. These studies indicate that exposure to high levels of PFOS
may interfere with human thyroid function. No studies among pregnant
women or children have been identified.
Phthalates
One study examined the associations between urinary levels of phthalates
in 76 pregnant women and thyroid function and found a significant
negative association between DBP-levels and T4 and free T4 (Huang et
al., 2007). Likewise, negative associations between DEHP-exposure and
FT4 and T3 have been reported in adult men (Meeker, Calafat and Hauser,
2007b), but studies of smaller populations did not find any relationships,
probably due to lack of statistical power (Janjua et al., 2007; Rais-Bahrami
et al., 2004).
48
Pesticides
Some human studies of HCB exposure have reported an inverse association
with thyroid hormone levels (Meeker, Calafat and Hauser, 2007a; Schell et
al., 2010).
BPA, UV-filters
No studies of effects of BPA and ultraviolet filters on thyroid function in
humans have been identified.
4. Data gaps and research needs

Recent trends in the frequency of reproductive problems and other
endocrine disorders among children and adolescents are a matter of great
concern and suggest that our modern environment can interfere with
endocrine systems. Particularly noteworthy is that even adult reproductive
disorders may have a fetal origin, although onset of the clinical problem
may not be noted until the reproductive age has been reached. However,
although these trends are established our understanding of their causes
is quite poor. Animal experiments have clearly demonstrated that there
are sensitive developmental periods when endocrine disruption causes
permanent organizational changes that may appear as structural and
functional anomalies much later. Mixture studies in animals have shown
the dose-additive effects of chemicals acting on the common endocrine
pathways. This challenges all our estimates of dose-response relationships
when the fact is that we are exposed to a wide variety of chemicals at the
same time. We should gain more knowledge on the endocrine disrupting
properties and mechanisms of action of all those chemicals that have not
yet been analyzed and to which we are potentially exposed. We need to
know more about the influence of mixtures. These should be analyzed
both experimentally in animals and in vitro, and by methods of systems
biology combining data from different sources.
Human studies of endocrine disrupters are still largely missing, because
either the exposure data are weak or the outcome data are vague. Thus,
human studies with proper exposure data from a relevant exposure
window and reliable ascertainment of the outcome are of vital importance.
Long term cohort studies with standardized examination methods can
give valuable information. It is important to harmonize both clinical and
49

environmental measurements. Development of good biomarkers would
be useful for health surveys. The prime targets of endocrine disrupters
are naturally endocrine systems, such as reproductive organs and their
function. Since adult reproductive health depends on normal fetal and early
childhood development, the focus on exposure measurements should be in
these periods without forgetting about contemporary exposure. Outcome
variables, such as genital abnormalities (cryptorchidism, hypospadias)
should be diagnosed using defined criteria, and in adult studies e.g. semen
analyses should be performed with good external quality control. Genetic
susceptibility may vary and this should be taken into account in these
analyses. This will require new genetic studies including genome-wide
association analyses, deep sequencing and rigorous testing of candidate
susceptibility genes. Genetic data need to be integrated with exposure
data. New findings on epigenetic effects of endocrine disrupters need to
be tested both in experimental animals and in human studies. Exposome
data and new omics data on genome, epigenome, metabolome etc. should
be integrated for versatile analysis of exposure outcome relationship.
Environmental monitoring and follow-up of reproductive development
and health, frequency of congenital hypothyroidism and other endocrine
endpoints should be made systematic. Cancer registries in many countries
are reliable especially for testicular germ cell cancers, but malformation
registries give data on hypospadias that cannot be compared between
countries and data on cryptorchidism are largely missing. There are no
international or even national systems that would give information on
semen quality in general population, although in some countries followup studies have been performed. All these data would be needed to follow
up the trends that might alert us to environmental problems. Puberty
is an important transition period from childhood to adulthood when
endocrine systems mature to a terminally differentiated state. This process
and its regulation remain poorly understood, and translational studies
extending from molecular mechanisms of neuronal control in the brain to
epidemiological studies on timing of puberty and environmental effects
on it are needed. The ultimate goal is to recognize any adverse effects
of environmental factors, which would give the opportunity to develop
preventive measures to avoid future health problems. Child health is the
basis of adult health and these two should not be separated in a larger
context. The European Science Foundation recently published a science
policy briefing on male reproductive health, its impacts in relation to general
wellbeing and low European fertility rates (ESF Science Policy Briefing
50
40, September 2010; www.esf.org). Its conclusions and recommendations

are also very valid for child health. International conventions, such as
the Stockholm Convention, call for the ban of certain persistent organic
pollutants (POPs) (including some endocrine disrupters), the list of which
is updated as new evidence arises. The updated list is available at http://
chm.pops.int/default.aspx
5. Summary
Several reproductive and other endocrine disorders have reached epidemic
frequencies and birth rates are extremely low in many countries. The
background for these trends is poorly understood. One of the main
reasons for low birth rates in the increased use of contraception, but
increased infertility might be partially attributed to environmental factors.
Some of the disorders such as undescended testis and hypospadias often
lead to early surgery of affected infants, who nevertheless have increased
risk of infertility and testis cancer later in life. Fetal development is a
critical period for all these disorders, also for testis cancer and some cases
of infertility and it is likely that the same factors can lead to all of them,
albeit not necessarily all at the same time. This quadrad (cryptorchidism,
hypospadias, testis cancer and failure of spermatogenesis) has been
called testicular dysgenesis syndrome (TDS). Exposure to antiandrogenic
compounds at a critical developmental window leads to a TDS-like
phenotype in the rat. These chemicals have additive effects, and adverse
effects in mixture studies appear at chemical doses that are below noadverse-effect levels for individual compounds. Therefore it is difficult
to estimate, whether current safety margins for allowed daily intakes
are adequate. In epidemiological studies, exposure to some endocrine
disrupter groups, such as polybrominated flame retardants and chlorinated
pesticides, has been associated with an increased risk of cryptorchidism.
However, much more work is needed to expand the information on
exposure-outcome relationships both for different chemicals and for
different outcomes. Normal thyroid function is crucial for development,
and any disruption of thyroid hormone action may have disastrous
consequences in childrens health. The first two years of life when the
central nervous system is rapidly developing are the most critical period.
It is therefore very important to recognize any endocrine disrupters that
can interfere with thyroid function or thyroid hormone action. The most
51

subtle effects would appear only as a small decline in intellectual capacity.
However, for society such changes would have far reaching ramifications.
Similarly, subtle adverse effects on reproductive health can appear as a
reduced sperm production capacity in the adulthood, which may have
dramatic effects on a mans personal life if a couple is suffering from
infertility. For a society it can be reflected in an increased demand for
expensive assisted reproductive techniques and extremely low fertility
rates, which are now seen in several parts of the industrialized world,
including many European Countries and Asia. International and national
efforts are needed to pursue multiple unresolved research questions. This
necessitates intensive interdisciplinary and translational research targeting
the developmental processes with all means that we have from chemistry
and genetics to epidemiology and modern systems biology. Improving
fetal and child health will influence the whole life of an individual and
reflect the wellbeing and future of our society.
52
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84
PUBLIC HEALTH AND ENVIRONMENT

The present document is a short summary of the current
knowledge of the effects of endocrine disrupters on child
health. The main focus is on the congenital disorders,
cryptorchidism and hypospadias, which have an endocrine
connection, on thyroid hormone-related problems, and on
puberty. There is ample evidence of endocrine disruption in
wildlife, and the mechanisms of action of endocrine disrupters
have been elucidated in experimental animals, but there is
limited knowledge of the association of human disorders
with exposure to endocrine disrupters. Accumulating data
suggest that many adult diseases have fetal origins, but the
causes have remained unexplained. Improving fetal and
child health will influence the whole life of an individual and
reflect the wellbeing and future of our society.
ISBN 978 92 4 150376 1

Public Health & Environment Department (PHE)
Health Security & Environment Cluster (HSE)
World Health Organization (WHO)
Avenue Appia 20, CH-1211 Geneva 27, Switzerland
www.who.int/phe/en/
www.who.int/ceh
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Endocrine disrupters and child health

WHO Library Cataloguing-in-Publication Data

Possible developmental early effects of endocrine disrupters on

1.Endocrine disruptors. 2.Disorders of sex development. 3.Sex

ISBN 978 92 4 150376 1

(NLM classification: WK 102)

World Health Organization 2012

Possible developmental early effects of endocrine disrupters on child health

This document was initially planned by an international working group

World Health Organization

International working group of experts

Georg Becher, Norwegian Institute of Public Health, Oslo, Norway

ke Bergman, Stockholm University, Sweden

Poul Bjerregaard, University of Southern Denmark, Denmark

Riana Bornman, Pretoria Academic Hospital, South Africa

Ingvar Brandt, Uppsala University, Sweden

Taisen Iguchi, National Institutes of Natural Sciences, Okazaki, Japan

Susan Jobling Eastwood, Brunel University, United Kingdom

Karen Kidd, University of New Brunswick, Canada

Andreas Kortenkamp, University of London, United Kingdom

Derek Muir, Environment Canada, Canada

Roseline Ochieng, Aga Khan University Hospital, Kenya

Niels Erik Skakkebaek, University of Copenhagen, Denmark

Hans-Christian Stolzenberg, Federal Environment Agency, Germany

Jorma Toppari, University of Turku, Finland

Thomas Zoeller, University of Massachusetts, US

Tracey Woodruff, University of California San Francisco, US

Possible developmental early effects of endocrine disrupters on child health

The development and publication of this document was funded by the

2. Endocrine disrupters (recognized on the basis of

3. Early effects, child health problems putatively associated

4. Data gaps and research needs

Possible developmental early effects of endocrine disrupters on child health

World Health Organization

Possible developmental early effects of endocrine disrupters on child health

in the brain, the pituitary gland connected to the hypothalamus and

in the brain controlling the GnRH neurons. In an analogous fashion,

Figure 2. Schematic representation of the

World Health Organization

Possible developmental early effects of endocrine disrupters on child health

Exposure of female fetuses to androgens leads to their masculinization,

It is well established that thyroid hormones are of special importance in

World Health Organization

Possible developmental early effects of endocrine disrupters on child health

hyperthyroidism or the presence of thyroid autoantibodies. However, it

2. Endocrine disrupters (recognized on the

World Health Organization

Increased risk of hypospadias

Brouwers et al., 2006; Klip et

Tendency towards smaller testes

Bibbo et al., 1977; Gill et al.,

Palmer et al., 2009

Capsular induration of testis

Bibbo et al., 1977; Gill et al.,

Severe sperm abnormalities

Bibbo et al., 1977; Gill et al.,

Bibbo et al., 1977; Gill et al.,

Palmer et al., 2009

Increased risk of breast cancer