Download Guidelines for Histopathological Evaluation

4. NORMAL BACKGROUND VARIATION OF STRUCTURE
Introduction
4.1
This section illustrates several examples of normal morphological changes in the female reproductive
tract that can complicate the evaluation of reproductive tissues from TG 407 studies. These alterations may be
developmental in nature or simply reflect the normal physiology of the oestrous cycle. Artefacts caused by
suboptimal sampling and/or sectioning of the reproductive organs may also occur. Oblique sections through
epithelia, for example, can mimic focal areas of epithelial hyperplasia or squamous metaplasia. Similarly,
inadequate ovarian sections may not be representative of the organ in terms of the follicular and luteal stages
present.
4.2
An awareness of these normal background and artefactual changes is important in order to avoid
erroneously labelling them as treatment-related. The situation is complicated by the fact that the incidence of
many common spontaneous variations may be influenced by substances with endocrine activity. In TG 407
studies the normal background variation for the test animal population is based on five control animals per sex.
Given this small group size, it is important that small increases in the incidence of spontaneous lesions are
not over-interpreted as treatment-related.
4.3
As TG 407 studies use young, sexually mature, non-mated adult rats, spontaneous age-related changes
and alterations associated with pregnancy should not be encountered and are not discussed here.
Spontaneous changes
.
Figure 4.1 – Vagina: dioestrus (rat, H&E). Minimal neutrophil infiltration (N) of the stratum
germinativum. This is a normal but inconsistent feature of dioestrus and should not be misdiagnosed as
vaginitis.
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Figure 4.2 – Uterus: late oestrus (rat, H&E). A marked neutrophil infiltration (N) of the endometrial
glands may be observed during late oestrus/metoestrus, reflecting the dominance of oestrogen during these
stages. This is a normal finding and should not be mistaken for endometritis.
Figure 4.3 – Uterus: early oestrus (rat, H&E). Early squamous metaplasia (SM) of the endocervix,
affecting the luminal and/or glandular epithelium, may be occasionally observed in young control rats.
This morphological alteration may also develop following treatment with oestrogenic compounds.
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4.4
Ovarian luteal cysts (Figure 4.4) are occasional spontaneous findings in young rats that form when
tertiary and Graafian follicles fail to ovulate but undergo lutenisation. Increased numbers of cystic corpora
lutea may be observed following administration of compounds with gonadotrophic activity (Table 5.1).
4.5
Luteal cysts should be differentiated from follicular cysts; these develop from secondary follicles that
fail to progress through folliculogenesis. Follicular cysts are fairly common spontaneous findings in aged
rodents but are not typically observed in young rats. They may develop after dosing with antioestrogenic
substances such as tamoxifen and CGS 18320B (Table 5.1). Cysts of the ovarian bursa or rete ovarii may also be
occasionally encountered in young rodents as spontaneous developmental lesions.
Figure 4.4 – Ovary: corpus luteum (rat,
H&E). Cyst (C) formation within a
corpus luteum (CL). Encircling the cyst
are small, granulosa-like cells admixed
with large, plump luteal cells – the latter
differentiate this structure from a
follicular cyst. The fluid-filled cyst cavity
is lined by a simple squamous epithelium
(not visible).
Figure 4.5 – Ovary: corpus luteum (rat,
H&E). A patchy, often marked,
mononuclear inflammatory cell
infiltration (I) may be observed in
degenerating corpora lutea. This is a
normal feature of luteal regression.
Consistent absence of this change in
treated animals may reflect persistence of
corpora lutea – an effect of some endocrine
disrupting substances.
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Common artefacts
Figure 4.6 – Normal vulva (rat, H&E). This may be inadvertently sampled and included in histological
sections of the vagina. The vulval epithelium is stratified squamous in type and covered by a thin,
permanent stratum corneum (SC). Numerous small rete pegs (RP) project into the underlying dermis.
Sweat glands and occasional hair follicles (not shown) may also be visible in the dermis.
Figure 4.7 – Uterus: dioestrus (rat, H&E). Oblique sections (Ob) through the normal endometrial
columnar epithelium (CE) should not be confused with foci of squamous metaplasia, a potential
treatment-related effect.
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Figure 4.8 – Ovary: prooestrus (rat, H&E). Preovulatory (Graafian) follicles (GF); the absence of
free-floating primary oocytes within the follicular lumina is a sectional artefact.
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5. MORPHOLOGICAL PATTERNS OF ENDOCRINE DISRUPTION
Introduction
5.1
Endocrine disrupting substances can affect the hypothalamic-pituitary-ovarian (HPO) axis in several
ways, producing alterations in the female reproductive tract that may be detected histologically. The updated
TG 407 is concerned with detecting substances with oestrogenic, antioestrogenic, androgenic and
antiandrogenic activity. Potent endocrine disruptors typically cause overt histological changes in the vagina,
uterus and ovary that are relatively straightforward to identify. Ethinyl oestradiol (a potent oestrogenic
compound), for example, induces ovarian atrophy in association with hyperplastic and hypertrophic changes in
the uterus and vagina.
5.2
Detection of substances with weak endocrine activity is more problematic. Compounds such as
genistein and nonylphenol do not usually cause profound histopathological changes in individual reproductive
tract organs; they may, however, disrupt the synchrony of the normal oestrous cycle-associated morphological
alterations in the reproductive tract. Thus, whilst individual tissues may be histologically normal, they fail to
correlate in terms of oestrous cycle stage when collectively evaluated as a unit. The challenge of identifying
asynchronous morphological changes in the female reproductive tract when assessing TG 407 studies is
discussed further below.
Overview of morphological patterns of endocrine disruption
5.3
A useful classification of the morphological alterations that may be observed in the female rodent
reproductive tract following xenobiotic administration has been described by Yuan (1998). This scheme
comprises three types of morphological response, based on the combined histological appearance of the vagina,
uterus and ovary and is summarised below:
 Type I
 Type II
 Type III
atrophic vagina, uterus and ovary
atrophic ovary with hyperplastic/hypertrophic uterus and vagina
hyperplastic/hypertrophic ovary, uterus and vagina
5.4
Using this classification scheme, Table 5.1 provides an overview of the morphological alterations likely
to be encountered in the female reproductive tract following administration of (anti)oestrogenic or androgenic
substances. The expected histopathological changes and underlying mechanisms of endocrine disruption
associated with each type of response are summarised. Although compounds with (anti)oestrogenic or
androgenic activity typically induce type I and II responses, the type III response is included here for reference.
Figures 5.1 to 5.13 illustrate examples of the morphological alterations that may be observed. This part of the
guidance document concludes with a summary of recommended terminology for the recording of these
changes.
5.5
Note that overt histopathological alterations associated with short-term antiandrogen administration are
typically limited to the male reproductive tract (Kunimatsu et al, 2004), although ovarian interstitial stromal cell
hyperplasia/hypertrophy has been described in rats following subacute oral dosing with flutamide, a potent
androgen antagonist (Andrews et al, 2001).
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Table 5.1 – Overview of the morphological responses associated with endocrine disruption (modified after Yuan, 1998).
EXPECTED MORPHOLOGICAL ALTERATIONS
MORPHOLOGICAL
PATTERN
VAGINA
Thin, atrophic epithelium
comprising 2-3 cell layers
UTERUS
OVARY
Thin, atrophic endometrial
and glandular epithelium
consisting of low columnar
cells
Atrophy with inactive
interstitial glands; glandular
cells are small and spindleshaped
Sparse endometrial glands
and atrophic myometrium
Cystic anovulatory follicles
may be present
Reduction in numbers of
follicles and corpora lutea
may not be observed in
short-term studies
Type I
(Atrophic vagina, uterus and
ovary)
EXAMPLE
SUBSTANCE
MECHANISM OF
ENDOCRINE
DISRUPTION
Antioestrogenic
effect
Aromatase inhibition
impairs the conversion of
androgens (produced by
theca interna cells) to
oestrogens by the follicular
zona granulosa
CGS 18320B
(non-steroidal
aromatase
inhibitor)
Endogenous production of
ovarian oestrogen is thus
reduced, initiating
widespread atrophic
reproductive tract changes
Note

Atrophic changes are more readily detected in the vagina compared with the uterus and ovary

Ovarian atrophy may be particularly difficult to detect in short-term studies. Early (primordial
to tertiary) follicular stages develop independently of hormonal stimulation and are thus
frequently observed in the atrophic ovaries of non-cycling rats from subacute studies. Similarly,
corpora lutea formed during previous cycles may also be present

Therefore, in short-term studies, the presence of normal follicular and luteal structures does
necessarily imply unpeturbed reproductive function
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Modulation of feedback
control on the hypothalamus
and pituitary by reduced
endogenous oestrogen levels
may suppress
gonadotrophin secretion,
resulting in the formation of
follicular cysts from
anovulatory follicles
Table 5.1 (cont.) – Overview of the morphological responses associated with endocrine disruption (modified after Yuan, 1998).
MORPHOLOGICAL
PATTERN
EXPECTED MORPHOLOGICAL ALTERATIONS
VAGINA
UTERUS
OVARY
Hyperplasia and
hypertrophy of
epithelium
Hyperplasia and
hypertrophy of luminal
and glandular epithelium
Atrophy with inactive
interstitial glands
Keratinisation of
superficial epithelium;
may be incomplete with
retention of nuclei in cells
of stratum corneum
Neutrophil infiltration of
endometrium
Cystic follicles may be
observed (tamoxifen)
Areas of epithelial
mucification may be
noted; superficial
epithelial cells are
hypertrophied and
contain large mucinfilled vacuoles.
Foci of squamous
metaplasia present in
luminal and glandular
epithelium
Epithelial mucification
typically predominates
over keratinisation
following androgen
administration
Cystic endometrial
glands may be observed
EXAMPLE
SUBSTANCES
Oestrogenic effect
Ethinyl oestradiol
(oestrogen agonist)
Type II
(Hyperplastic/hypertrophic
vagina and uterus with
atrophic ovary)
Reduction in numbers of
follicles and corpora
lutea may not be
observed in short-term
studies
Interstitial stromal cell
hypertrophy/hyperplasia
may be noted (tamoxifen
& flutamide)
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MECHANISM OF
ENDOCRINE
DISRUPTION
Tamoxifen
(mixed oestrogen
agonist/antagonist)
Methyl
testosterone
(androgen agonist)
Administration of potent exogenous
sex steroids results in negative
feedback at the hypothalamicpituitary level, inhibiting
gonadotrophin release and
inducing ovarian atrophy
By contrast, sex steroids directly
stimulate the uterus and vagina
resulting in organ hyperplasia and
hypertrophy. Exogenous
androgens most likely undergo
aromatisation in the ovary before
exerting oestrogenic effects on these
tissues
The formation of ovarian follicular
cysts by tamoxifen may reflect its
antioestrogenic activity in rats,
modulating feedback control by
oestrogen on the hypothalamus and
pituitary and reducing
gonadotrophin secretion.
Proliferative changes in the uterus
and vagina are consistent with the
oestrogen agonist activity of
tamoxifen in these tissues
Table 5.1 (cont.) – Overview of the morphological responses associated with endocrine disruption (modified after Yuan, 1998).
MORPHOLOGICAL
PATTERN
EXPECTED MORPHOLOGICAL ALTERATIONS
VAGINA
Hyperplasia and
hypertrophy of
epithelium
Keratinisation of
superficial epithelium;
may be incomplete with
retention of nuclei in cells
of stratum corneum
Type III
(Hyperplastic/hypertrophic
vagina, uterus and ovary)
UTERUS
OVARY
Hyperplasia and
hypertrophy of luminal
and glandular epithelium
Enlarged with increased
numbers of corpora lutea
and tertiary follicles.
These changes are less
pronounced with prolactin
analogues
EXAMPLE
SUBSTANCES
Gonadotrophic/luteotrophic
effect
Substances with gonadotrophic
activity trigger continuous follicle
maturation and corpora luteal
formation.
Cystic/incompletely
lutenised corpora lutea
may be observed
Areas of epithelial
mucification may be
noted; superficial
epithelial cells are
hypertrophied and
contain large mucin-filled
vacuoles.
MECHANISM OF
ENDOCRINE
DISRUPTION
Analogues of
LH, LHRH, FSH
and prolactin
The increased ovarian activity
results in elevated oestrogen and
progesterone levels that initiate
proliferative changes in the uterus
and vagina.
Prolactin analogues (luteotrophs)
prolong the lifespan of corpora
lutea; this results in persistent
progesterone secretion and
development of a
pseudopregnancy-like state
Proliferative changes
associated with prolactin
analogues are typically
limited to mild epithelial
hypertrophy and
mucification
Dopamine-depleting compounds
(e.g. reserpine) impair the release
of dopamine (a prolactininhibiting factor) from the
hypothalamus. By increasing
endogenous prolactin secretion,
such substances have an indirect
luteotrophic effect
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Selected examples of morphological alterations associated with endocrine disruption
A. Vagina
x20
Figure 5.1 – Vagina: atrophy (rat, H&E). Detection of
chemically induced atrophy in the vagina is
straightforward. Note the marked attenuation of the
vaginal epithelium which comprises 2-3 layers of small
cells. Generalised atrophy of the female reproductive tract
occurs normally in aged, reproductively senescent rats,
but should not be encountered as a background finding in
the young, sexually mature animals specified by the TG
407.
Figure 5.2 – Vagina: hyperplasia/hypertrophy with
mucification (rat, H&E). Superficial epithelial cells are
hypertrophied with large, intracytoplasmic, mucin-filled
vacuoles (V). The stratum germinativum (SGerm)
consists of the two most basal cell layers. Mucification of
the vaginal epithelium is a potential alteration associated
with oestrogenic substances, and is the expected
morphological response of the vagina to administration of
large doses of androgens. Luteotrophic compounds (e.g.
prolactin analogues) typically cause mild epithelial
hypertrophy and mucification.
Figure 5.3 – Vagina: hyperplasia/hypertrophy with
mucification associated with pregnancy (rat, H&E).
This figure clearly illustrates the effects of progesterone on
the vaginal epithelium following oestrogen priming. Note
the marked vacuolation (V) and hypertrophy of the
superficial stratum germinativum cells.
x40
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B. Uterus
x20
x20
Figure 5.4 – Uterus: atrophy of luminal epithelium (rat, H&E). A low columnar epithelium lines the uterine lumen; endometrial
glands (not shown) are reduced in number.
x40
Figure 5.5 – Uterus: hypertrophy/hyperplasia of luminal epithelium (rat, H&E).
Proliferative changes in the luminal and glandular epithelium are features of the type
II (oestrogenic) and type III (gonadotrophic/luteotrophic) morphological responses.
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Figures 5.6 and 5.7 – Uterus: squamous metaplasia (rat, H&E). Figure 5.6 (above) shows
squamous metaplasia (SM) in the luminal epithelium of the endocervix – a spontaneous
background finding in a control animal. Figure 5.7 (below) illustrates hyperplasia/hypertrophy
of the luminal epithelium (HE) together with a focus of squamous metaplasia (SM) in a rat treated
with the synthetic oestrogen diethylstilbestrol.
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C. Ovary
x4
Figures 5.8 and 5.9 – Ovary: atrophy (rat, H&E). Figure 5.8 (above) shows an obvious reduction
in the number of follicles and corpora lutea; this characterises ovarian atrophy as observed in
long-term studies and aged female rats. In subacute studies, this alteration may not be observed,
reducing the sensitivity of the ovary as an endpoint for the detection of (anti)oestrogenic effects.
Changes in the ovarian stroma and interstitial glands, however, may be induced by the short-term
administration of endocrine disrupting substances (Figures 5.10 and 5.11). Figure 5.9 (below) shows
a normal ovary from a young, sexually mature control animal for comparison.
x4
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Figure 5.10 – Ovary: atrophy (rat, H&E).
Hyperplasia and hypertrophy of stromal interstitial
cells is a common feature of ovarian atrophy in ageing
rodents, and has been reported following short-term
dosing of young adult rats with tamoxifen and
flutamide. Pale-staining, plump stromal cells are
arranged in variably sized islands and clusters (IC).
This ovarian section is from an aged control animal;
note the orange-brown lipofuscin pigment present in
some stromal cells, a normal finding in the ovaries of
old, reproductively senescent rats.
Figure 5.11 – Ovary: atrophy (rat, H&E). High
power view of a cluster of large, pale-staining stromal
cells supported by a fine fibrovascular stroma. The
ovarian surface epithelium (OSE) and tunica albuginea
(TA) are also visible.
Figure 5.12 – Ovary: luteal cyst (rat, H&E). Early
cyst (C) formation within a corpus luteum (CL).
Encircling the cyst are small, granulosa-like cells
admixed with large, plump luteal cells – the latter
differentiate this structure from a follicular cyst (Figure
5.13). The fluid-filled cyst cavity is lined by a simple
squamous epithelium (not visible).
x20
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ZG
SE
SE
Figure 5.13 – Ovary: follicular cyst (rat, H&E). Cyst (C) formation within a
secondary follicle. The surrounding zona granulosa (ZG) is attenuated;
lutenisation of the granulosa cells is not a feature of follicular cysts. The cyst
cavity is lined by a simple squamous epithelium (SE, inset).
Reproductive tract asynchrony
5.6
As previously noted, weak endocrine disruptors are difficult to detect as they fail to significantly
perturb the HPO axis of mature female rats and produce overt histopathological changes in the vagina, uterus
and ovary. Such substances may, however, be associated with asynchrony of the oestrous cycle-associated
morphological changes that normally occur in the reproductive tract.
5.7
Observation of potential reproductive tract asynchrony in short-term studies is a difficult and
ambitious enterprise, even for an experienced toxicological histopathologist. A good working knowledge of
the normal histological appearance of the reproductive tract at each stage of the oestrous cycle is essential.
Careful evaluation of the female control rats (limited to 5 animals in TG 407 studies) should be performed in
order to gain an appreciation of the range of physiological and background spontaneous changes that are
normal for the strain and age of rat being utilised.
5.8
For asynchrony to be confidently diagnosed, a profound lack of correlation in the histological
appearance of the reproductive tract tissues should be observed. As previously discussed, reproductive tract
staging is initially based on the appearance of the vagina; the presence of markedly incompatible
morphological alterations in the uterus and ovary is consistent with asynchrony.
5.9
Given the highly dynamic nature of the female reproductive system and small group sizes utilised in
TG 407 studies, great care should be taken by the study pathologist to avoid erroneously interpreting minor
normal variations in morphology as evidence of reproductive tract asynchrony.
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SUMMARY OF RECOMMENDED TERMINOLOGY
A. VAGINA

Epithelial atrophy
 attenuated vaginal epithelium comprising 2-3 cell layers

Epithelial hyperplasia/hypertrophy
 thickened vaginal epithelium comprising increased number of cell layers/enlarged cells; may occur with:
—
keratinisation
- superficial epithelial cells are variably keratinised
—
mucification
- superficial epithelial cells are variably vacuolated
—
keratinisation and mucification - combination of changes is present
B. UTERUS

Atrophy
 attenuated, low columnar luminal and glandular epithelium
 sparse endometrial glands and atrophic myometrium

Epithelial hyperplasia/hypertrophy
 thickened luminal and/or glandular epithelium comprising increased number of cell layers/enlarged cells

Endometrial inflammation
 inflammatory cell (neutrophil) infiltration of endometrial stroma and/or glands

Cystic endometrial glands
 dilation/cystic change in endometrial glands

Squamous metaplasia
 transformation of luminal/glandular columnar epithelium into a stratified squamous epithelium
C. OVARY

Atrophy
 inactive interstitial glands +/- reduced numbers of follicles/corpora lutea; may occur with
—
follicular cysts
—
interstitial stromal cell hypertrophy/hyperplasia

Increased numbers of follicles/corpora lutea

Luteal cysts
 cystic change in corpora lutea; cyst cavity lined by a simple squamous epithelium and surrounded by lutenised and nonlutenised granulosa cells

Incomplete lutenisation of corpora lutea
 corpora lutea are composed of lutenised and non-lutenised granulosa cells
D. FEMALE REPRODUCTIVE TRACT

Type I response
 generalised reproductive tract atrophy

Type II response
 proliferative changes in uterus and vagina accompanied by ovarian atrophy

Type III response
 generalised reproductive tract proliferation

Asynchrony
 marked lack of correlation in the histological appearance of the uterus and/or ovary relative to the vagina
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