Invited Review

Toxicologic Pathology, 43: 776-793, 2015

Copyright # 2015 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623315570339

Vaginal Cytology of the Laboratory Rat and Mouse:

Review and Criteria for the Staging of the Estrous Cycle
Using Stained Vaginal Smears
MICHELLE C. CORA1, LINDA KOOISTRA2, AND GREG TRAVLOS1
1
Cellular and Molecular Pathology Branch, National Toxicology Program, National Institute of
Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
2
Charles River Laboratories, Inc., Pathology Associates, Durham, North Carolina, USA
ABSTRACT
Microscopic evaluation of the types of cells present in vaginal smears has long been used to document the stages of the estrous cycle in laboratory
rats and mice and as an index of the functional status of the hypothalamic–pituitary–ovarian axis. The estrous cycle is generally divided into the four
stages of proestrus, estrus, metestrus, and diestrus. On cytological evaluation, these stages are defined by the absence, presence, or proportion of 4
basic cell types as well as by the cell density and arrangement of the cells on the slide. Multiple references regarding the cytology of the rat and mouse
estrous cycle are available. Many contemporary references and studies, however, have relatively abbreviated definitions of the stages, are in reference
to direct wet mount preparations, or lack comprehensive illustrations. This has led to ambiguity and, in some cases, a loss of appreciation for the
encountered nuances of dividing a steadily moving cycle into 4 stages. The aim of this review is to provide a detailed description, discussion, and
illustration of vaginal cytology of the rat and mouse estrous cycle as it appears on smears stained with metachromatic stains.

Keywords: estrus; proestrus; diestrus; metestrus.

INTRODUCTION estrus, and diestrus (Goldman, Murr, and Cooper 2007); alter-
natively, the four stages have been subcategorized into as many
Microscopic evaluation of the types of cells present in vagi-
as 13 stages (Thung, Boot, and Mühlbock 1956). On cytologi-
nal smears has long been used to document the stages of the
cal evaluation, these stages are defined by the absence, pres-
estrous cycle in laboratory rats and mice and as an index of the
ence, or proportion of the cells present as well as by the cell
functional status of the hypothalamic–pituitary–ovarian axis.
density and arrangement of the cells on the slide.
As such, assessment of the estrous cycle has been used both
Vaginal cytology can be evaluated immediately after collec-
as a principal measure in determinations of reproductive cycli-
tion as an unstained, wet mount preparation (direct cytology) or
city and as an ancillary test in reproductive toxicologic studies
as a fixed and stained slide preparation. Although some of the
(Goldman, Murr, and Cooper 2007).
discussion can be applied to the evaluation of direct wet mount
The estrous cycle in rats and mice averages 4–5 days and is a
preparations, this review will focus on the estrous cycle as eval-
repetitive but dynamic process whereby different cell types
uated on slide preparations that are dry-fixed and stained with
appear and recede in waves throughout the cycle, reflecting
metachromatic stains.
changes in the levels of estradiol and progesterone secreted
Multiple references regarding the cytology of the rat and
by the ovarian follicles. The estrous cycle is generally divided
mouse estrous cycle are available (Allen 1922; Byers et al.
into four stages—proestrus, estrus, metestrus, and diestrus.
2012; Caligioni 2009; Cooper and Goldman 1999; Goldman,
However, depending on the investigator and the objectives of
Murr, and Cooper 2007; Hubscher, Brooks, and Johnson
a study, it has been divided into as few as 3 stages—proestrus,
2005; Long and Evans 1922; Mandl 1951; Organization for
Economic Cooperation and Development [OECD] n.d.;
The author(s) declared no potential conflicts of interest with respect to the
Young, Boling, and Blandau 1941). Many of the contemporary
research, authorship, and/or publication of this article. references and studies, however, have relatively abbreviated
The author(s) disclosed receipt of the following financial support for the definitions of the stages, are in reference to direct wet mount
research, authorship, and/or publication of this article: This work was preparations, or lack comprehensive illustrations. This has led
supported (in part) by the National Institutes of Health, National Institute of to ambiguity and, in some cases, a loss of appreciation for the
Environmental Health Sciences.
Address correspondence to: Michelle C. Cora, 111 TW Alexander Dr.,
encountered nuances of dividing a steadily moving cycle into 4
PO Box 12233, Research Triangle Park, NC 27709, USA; e-mail: stages. The aim of this review is to provide a detailed descrip-
coramc@niehs.nih.gov. tion, discussion, and illustration of the vaginal cytology of the

776
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 777

rat and mouse estrous cycle and give the reader an appreciation
for the nuances and challenges in definitively characterizing
this dynamic process. For information on ovarian hormone
secretion and the subsequent impact on vaginal physiology as
well as how the evaluation of vaginal cytology applies to tox-
icological research, the reader is directed elsewhere (Butcher,
Collins, and Fugo 1974; Cooper and Goldman 1999; Goldman,
Murr, and Cooper 2007; Kovacic and Parlow 1972; Li and
Davis 2007; Nelson et al. 1981; Neill 2006). For a detailed
review of the histopathology of the estrous cycle, see West-
wood’s (2008) ‘‘The female rat reproductive cycle: a practical
histological guide to staging.’’

HISTORICAL PERSPECTIVE
The term estrus was first used (as oestrus2) in Heape’s
1

(1900) publication, ‘‘The ‘sexual season’ of mammals and the

relation of the ‘pro-oestrum’ to menstruation,’’ in which he
defines estrus as the ‘‘special period of sexual desire in the
female.’’ Estrus is a Latin adaptation of the Greek word oistros,
which means gadfly, frenzy, or madness. It is from this base
word that Heape named and defined the other stages of the
mammalian estrous cycle: proestrus, an animal coming into
heat; metestrus, a short period in the absence of conception,
when ‘‘the activity of the [reproductive] organs gradually sub-
side during a definite period’’; diestrus, a period of short rest
(during the breeding season); and anestrus, a period of rest
(in the nonbreeding season). At that time, the stages were
defined and based on changes in behavior (e.g., the acceptance
of copulation) as well as gross morphological and histological
changes observed in the reproductive tract of cycling females.
Included in these were the macroscopic changes in the vulva
(e.g., swelling), vaginal excretions (e.g., hemorrhage, mucous),
and uterus (e.g., congestion) together with the microscopic
changes in the uterine tissue and ovaries. However, the macro-
scopic observations were not always reliable in small rodents,
and tissue pathology was and still is an invasive procedure and
not feasible for in-life estrous cycle staging. It was the need for
a more reliable, noninvasive method for defining the stages and
determining the time of ovulation in a laboratory setting that
lead to Stockard and Papanicolaou’s (1917) landmark article,
‘‘The existence of a typical estrous cycle in the guinea pig with
a study of its histological and physiological changes.’’ It was
with this article that, for the first time in any species, the
changes that occur in the vaginal canal during the estrous cycle
were thoroughly characterized both macroscopically and
microscopically (on histology and cytology). Additionally,

1.
Note that the word estrus is a noun, while the word estrous with an ‘‘o’’ is an
adjective, as in ‘‘the estrous cycle’’; the two words are not interchangeable.
2.
As is widely known, the British English, Greek-derived spelling of estrus FIGURE 1.—Historical representation of select stages of the rat estrous
with an ‘‘o’’ as in oestrus and the oestrous cycle and the use of an ‘‘o’’ in cycle as presented in Long and Evans (1922). These eloquent pencil
the spelling of the stages (pro-oestrus, metoestrus, dioestrus) is still used drawings illustrate proestrus (A), metestrus (B), and diestrus (C).
today in British-based publications. Additionally, older literature, regardless
of country of origin, generally used British English. This is important to note
these vaginal changes were correlated to the changes that occur
when undertaking a comprehensive literature search on the subject of the in other reproductive organs during the estrous cycle. This pub-
estrous cycle and its stages. lication influenced three other pioneers of reproductive
778 CORA ET AL. TOXICOLOGIC PATHOLOGY

physiology—Joseph Long, Herbert Evans, and Edgar Allen—

in their pursuit to study and then describe the rat (Long and
Evans 1922) and mouse (Allen 1922) estrous cycles (Figure
1). Long and Evans (1922) eloquently expressed the impor-
tance of Stockard and Papanicolaou’s (1917) findings with the
following:

By the fortunate discovery that in the guinea pig these

[vaginal] mucosal transformations are accompanied by
the dehiscence of epithelial cells so that at times the
lumen of the vagina has a characteristic cell content, it
has been possible for Stockard and Papanicolaou to
show us that we may discover with ease in the living
animal the exact occurrence and progress of these
cycles. (p. 6)

And so, it is from these early twentieth-century publications

that the present-day definitions of proestrus, estrus, metestrus,
and diestrus arose.
It would be a great disservice not to take this opportunity to
state that the abovementioned scientists were brilliant research-
ers and made many other significant contributions to the fields
of endocrine and reproductive physiology—laying the founda-
tion for many others working in these fields. The following sen-
tences highlight but a few of their contributions. The Pap smear
or test, developed by Papanicolaou and used as a screening test
for cervical cancer, is now recognized as the most significant
advancement in the control of cancer in the twentieth century
(Vilos 1998). In addition to developing the Long–Evans stain
of rat, Evans and colleagues went on to characterize the hor-
mones of the anterior pituitary (e.g., luteinizing hormone and
follicle-stimulating hormone) and define interactions between
this gland, the ovary, and uterus (Lindsey and Baker 2006).
Allen and Edward Doisy isolated the ovarian estrogenic hor-
mone (Wells 1945). Additionally, Allen and associates also
investigated the effects of estrogenic and androgenic hormones
on various reproductive and endocrine organs as well as the
relationship of estrogen concentrations to certain types of neo-
plasms (e.g., leukemia, mammary cancer; Wells 1945).

METHODOLOGY AND TECHNICAL CONSIDERATIONS

To elucidate the length and, subsequently, the stages of the
estrous cycle, vaginal cytology samples are collected over at
least 14 consecutive days (Cooper and Goldman 1999). Vagi-
nal cytology samples can be collected at any time of the day,
but the practice of most laboratories is to collect them early
in the morning after the lights are turned on. Regardless of
when samples are collected, it should be done at approximately
the same time of the day over the course of the collection FIGURE 2.—Several examples of poor quality smears. In the first
period to reduce variability. Collection of the samples can be example (A), the smear is understained, resulting in a lack of cellular
done either by vaginal lavage or swabbing. Lavage typically detail. Additionally, some cells are ruptured resulting in a background
yields a higher cellularity sample and is the preferred method of scattered cellular debris. Due to the poor quality, this smear was
of the authors. Regardless of the methodology employed, accu- ultimately deemed uninterruptable. Plate B has a background of eosi-
rate interpretation of vaginal cytology samples is dependent on nophilic streaming and debris (arrows), which most likely represents
the quality of the sample preparation (Figure 2). ruptured or disintegrated neutrophils. Depending on full evaluation
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 779

Materials
To perform vaginal lavage, one needs a pipette or eyedrop-
per, saline, and glass slides. The pipette tips or eyedroppers
need to be smooth and tapered with a recommended 1.5 mm
internal tip bore for pipette tips (OECD n.d.). It is a best prac-
tice to use a new tip or dropper for each animal. If a single tip
or dropper is to be used on more than 1 animal, it should be
thoroughly rinsed with fresh distilled or sterile water between FIGURE 3.—Example of a commercially available 100  300 glass slide
animals. Thorough rinsing is very important to prevent with a predrawn numbered grid for vaginal cytology sample collec-
between-animal sample contamination that may lead to inac- tion. This slide contains samples that were collected daily for 8 con-
curacies in staging or, rarely, infection of the reproductive secutive days from an individual animal and, after dry fixation,
tract. were stained with a metachromatic stain.
Phosphate-buffered saline or normal saline are the pre-
ferred fluid to use for vaginal lavage. Some publications Collection and Processing
also list distilled water or tap water as options, and many
To collect cells from the vaginal canal, approximately 0.1
research studies have used these in lieu of saline. Saline
ml (mice) or 0.2 ml (rats) of saline is drawn into the pipette
is an isotonic solution while water is hypotonic. The use
or dropper. The tip of the dropper is gently inserted into the
of water can cause distorted epithelial cell morphologies
vaginal orifice at a depth of approximately 1–2 mm in mice and
and will invariably rupture neutrophils, which can lead to
5–10 mm in rats and then the saline flushed into the vagina and
confusion or inaccuracies in the evaluation of vaginal cytol-
back out 2 or 3 times. If after the first flush the fluid is cloudy,
ogies. Water has seemingly been successfully used when
subsequent flushings may not be necessary (OECD n.d.). When
cells are evaluated on direct preparations that are evaluated
inserting and flushing into the vaginal orifice, care must be
shortly after collection. Nevertheless, to avoid any issues
taken not to insert the tip too deep so as to avoid cervical stimu-
with cell morphology and for the purposes of preparing
lation. Excessive stimulation can induce pseudopregnancy,
stained slides, saline should be used.
which appears as a persistent diestrus for up to 14 days (Gold-
For archiving purposes and ease of sample evaluation, it
man, Murr, and Cooper 2007).
is advantageous to place 6–8 consecutive samples from 1
After the lavage, a small drop of the sample is then placed
animal onto a basic 1  3 inch glass slide. Grids that form
evenly on the slide in a thin layer (smear) and allowed to air-
‘‘boxes’’ or ‘‘squares’’ representing each day may be drawn
dry. Crystals may form on the dry unstained slides; these will
on the slides with a black, solvent-resistant marker. Alterna-
be removed as part of the staining process. Several different
tively, glass slides with pre-drawn and numbered grids rep-
metachromatic stains may be used to stain the dry fixed slides.
resenting 8 days are also available at an additional cost
Romanowsky-type stains (e.g., Modified Wright’s, Wright’s
(Figure 3). Some investigators use wax pencils to draw the
Giemsa) and Toluidine blue O are the commonly used cytolo-
grids on glass slides. An advantage of using a wax pencil is
gical stains offering good coloration for the identification of the
that the wax forms a barrier that will prevent 1 day’s sample
different cell types and are recommended for staining vaginal
from ‘‘bleeding’’ into another day’s sample. However, if an
smears. It is important to avoid heavy staining and to rinse the
automated coverslipping machine is used to process the
slides thoroughly. Dry fixed slides do not require a spray fixa-
slides, the wax markings can lead to an irregular slide sur-
tive (e.g., Spray-CyteTM) prior to staining. A spray fixative is
face that may result in an uneven distribution of permount
used when a stain requires wet fixation (i.e., application of the
or prevent direct contact between the slide and coverslip,
spray fixative onto the sample before the cells air-dry), such as
resulting in air bubbles and inadequate preservation. With
is needed with the Papanicolaou stain.
care and practice, slides can be easily and successfully made
Papanicolaou stain has also been used to stain vaginal cytol-
without the use of a wax pencil. If wax pencils are to be
ogy samples, especially in humans as a screening procedure for
used, removal of the wax by soaking the slides in xylene
cervical cancer (i.e., Pap smear or test). This stain is multichro-
(or equivalent) prior to coverslipping with an automated
matic involving the use of 5 different dyes and requires a wet
machine is recommended.
fixation of the sample before staining. Use of this stain offers
better chromatin detail and stains the cytoplasm with shades
FIGURE 2—(Continued). of the smear and the experience of the evalua- of orange, pink, or blue, depending on the degree of cornifica-
tor, this smear could be misclassified as proestrus (instead of diestrus)
tion. This level of detail, however, is often not needed to
or could be deemed an insufficient sample. Plate C has a large amount
identify the stages of the estrous cycle in rodents.
of eosinophilic material in the background, making visualization of
the neutrophils that are present challenging. The background material
either was due to poorly maintained lavage fluid or is stain precipitate Slide Evaluation
due to the poorly maintained staining solutions. Modified Wright–
Giemsa stain (A, B) and Toluidine blue O stain (C). Original objective In general, cells can be identified using a 10 objective;
magnification of 10 (A) or 20 (B, C). however, in some cases the use of higher objectives may be
780 CORA ET AL.
needed (e.g., to better visualize neutrophils). Care should be
taken to examine the whole smear as cell types and numbers
may vary in different areas on the slide. Evaluating daily
smears with knowledge of the preceding or succeeding days’
stages (vs. ‘‘blindly’’ reading each day) is important for the
accurate interpretation of the stages and cycling status because
it puts each day into context. This is of particular importance
when a smear is a transition between two consecutive stages
(transitional stage) when it may be difficult to make an accurate
determination without knowledge of the preceding or succeed-
ing day’s smears. For example, in a rat, a smear transitioning
from proestrus to estrus will have combinations of round
nucleated cells and keratinized anucleated cells. Knowing the
previous day’s findings will help one to identify the day in
question as proestrus rather than late estrus, where similar cell
types may be observed.
For the best viewing of stained cytology preparations, it is
important that the microscope is properly configured for Köh-
ler illumination. In laboratories where wet mount preparations
or unstained specimens are evaluated, the microscope conden-
ser and field diaphragm are usually adjusted for greater contrast
with a resulting loss in image illumination and resolution; with
these types of adjustments the microscope does not have proper
Köhler illumination. When proper Köhler illumination is
applied, a stained cytology image will have an even illumina-
tion with optimal resolution and contrast and minimal artifact
or glare. Adjusting a microscope for Köhler illumination is rel-
atively simple. The reader is directed elsewhere for details on
Köhler illumination (Rottenfusser 2013).
One of the single most important factors in the evaluation of
vaginal cytologies is proper training and experience
The evaluator needs to be aware of the inherent variations
that may be encountered, be able to recognize artifactual
changes (e.g., balled-up or ruptured neutrophils, understained
cells), and understand that the smears represent a moment in
time of a dynamic process and that the total impression of the
smear is more important than the exact cell counts. Practice slide
sets, whereby the trainee follows groups of individual animals
for several cycles, is the best way to develop proficiency in read-
ing vaginal smears. To reduce variability and ensure consis-
tency, it is also important to limit the number of people
evaluating the slides for a given study or within a given labora-
tory and to make certain there is agreement on the criteria for the
classification of each stage. This is particularly important in the
inevitable event of transitional stages, when the evaluation is
prone to increased subjectivity despite established objective cri-
teria. If more than one observer is involved in reading smears,
discrepancies can be reduced by initially having them simultane-
ously evaluate the same slides to ensure consistency of stage
interpretation (Cooper and Goldman 1999).
THE CELLS AND STAGES OF THE ESTROUS CYCLE
As previously stated, estrous cycle length averages 4–5 days
in both rats and mice, but occasional 6-day cycles may be
TOXICOLOGIC PATHOLOGY
FIGURE 4.—Many neutrophils (arrows), characterized by their very
small size and multilobulated nuclei, and two large nucleated epithe-
lial cells (arrowhead) are present on this diestrous vaginal smear from
a Sprague-Dawley rat. Toluidine blue O stain. In Elmore et al. Pro-
ceedings of the 2014 National Toxicology Program satellite sympo-
sium. Toxicol Pathol, 43:10-40, 2015.
observed in some individuals (Nelson et al. 1982; Mandl
1951; Morrissey et al. 1988; Long and Evans 1922; Goldman,
Murr, and Cooper 2007; Parkes 1928; Blandau, Boling, and
Young 1941). Compared to rats, mice have less regularity in
cycle length or cycle contiguity; that is, the likelihood of a 4-
day cycle being followed by a 5-day cycle instead of another
4-day cycle is higher in mice (Nelson et al. 1982). Many factors
influence cycle length including light, age, temperature, noise,
nutrition, stress, and social relationships (Li and Davis 2007;
Nelson et al. 1981; Campbell, Ryan, and Schwartz 1976; Camp-
bell and Schwartz 1980; Nelson et al. 1982; Whitten 1956; Gold-
man, Murr, and Cooper 2007). For example, mice housed singly
cycle more regularly and about 1 day shorter than mice housed
in multiples (Nelson et al. 1982). Female rodents cycle more reg-
ularly when males are present in the room (i.e., the Whitten
effect), although mice are more sensitive to this phenomenon
than rats (Whitten 1956; OECD n.d.).
The rodent estrous cycle is very sensitive to changes in the
light–dark cycle. A 12:12 light–dark cycle is standard in most
laboratories. In laboratories using a 14:10 light–dark cycle, a
different ratio in the number of 4- and 5-day cycles may occur
in comparison to a 12:12 cycle (OECD n.d.).
The length of the four stages varies between 6 and 72 hr
depending on the stage and individual rodent, therefore, some
short stages may be ‘‘missed’’ especially if samples are col-
lected very early or late in the day. For example, proestrus, with
an average length of 14 hr in rats, could be missed with an early
morning sample collection because in many females proestrus
does not start until midmorning and would end before the next
collection. Sample collection can be shifted to late morning or
early afternoon in an effort to minimize the incidences of
missed stages.
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE
FIGURE 5.—Several condensed or ‘‘balled-up’’ neutrophils (A, arrows) are
interspersed among the epithelial cells of a diestrous smear from a Spra-
gue-Dawley rat. The pale pink background is due to the presence of
mucous. Plates B and C demonstrate ruptured neutrophils (arrows) from
vaginal cytology smears of a Sprague-Dawley rat (B) and B6C3F1/N
mouse (C). Modified Wright–Giemsa stain (A) and Toluidine blue O stain
(B, C). Original objective magnification of 20 (A, B) or 40 (C).
781
FIGURE 6.—Small nucleated epithelial cells from a proestrous vaginal
smear of a Sprague-Dawley rat. Occasional nucleated epithelial cells
containing small cytoplasmic vacuoles (arrow) and a few anucleated
epithelial cells are also present. Toluidine blue O stain. Original objec-
tive magnification of 40.
FIGURE 7.—Several large nucleated epithelial cells (arrows) are inter-
mixed with anucleated epithelial cells (arrowheads) in a vaginal smear
of late estrus from a Sprague-Dawley rat. Numerous bacteria are
observed adhered to many of the epithelial cells. Modified Wright–
Giemsa stain. Original objective magnification of 40. In Elmore
et al. Proceedings of the 2014 National Toxicology Program satellite
symposium. Toxicol Pathol, 43:10-40, 2015.
Cell Types of the Estrous Cycle
For this discussion, cell descriptions are based on cytology
preparations that were dry fixed and stained with a metachro-
matic stain.
Neutrophils
Neutrophils are also known as leukocytes or polymorpho-
nuclear cells. These cells are round, very small, and possess
multilobulated nuclei (Figure 4). During collection and
782 CORA ET AL.
FIGURE 8.—A large nucleated epithelial cell containing many small
cytoplasmic granules (arrow) from a Sprague-Dawley rat. Modified
Wright–Giemsa stain. Original oil objective magnification of 100.
FIGURE 9.—Anucleated epithelial cells from a vaginal smear of estrus
in a B6C3F1/N mouse. Some cells possess ghost nuclei (arrows).
Modified Wright–Giemsa stain. Original objective magnification of
40. In Elmore et al. Proceedings of the 2014 National Toxicology
Program satellite symposium. Toxicol Pathol, 43:10-40, 2015.
preparation, these cells may condense or ‘‘ball-up,’’ making
visualization of their nuclei difficult, but with experience, they
can still be recognized as neutrophils (Figure 5A). If at low power
cells are suspected to be condensed neutrophils, the magnification
can be increased to a 20 or 40 objective. Usually on higher
magnification, condensed neutrophils can be identified as such
and neutrophils with a more normal appearance can sometimes
be found on the periphery of the smear. Neutrophils are relatively
delicate cells and can sometimes rupture during collection or pro-
cessing (Figure 5B and C). It is important to recognize the appear-
ance of ruptured neutrophils to avoid misinterpretation of a
particular stage.
TOXICOLOGIC PATHOLOGY
FIGURE 10.—A keratin bar (arrow) is observed in a vaginal smear of
estrus from a Sprague-Dawley rat. Note that many bacteria are either
adhered to the cells or free in the background. Modified Wright–
Giemsa stain. Original objective magnification of 40.
Small Nucleated Epithelial Cells
These cells are small, round to oval, and nonkeratinized
(Figure 6). They have a high nuclear to cytoplasmic (N:C)
ratio relative to large epithelial cells, a round nucleus, and
blue cytoplasm. They may stain very dark or basophilic,
precluding visualization of the nucleus. Small cytoplasmic
vacuoles may be seen in occasional small epithelial cells
of proestrus.
Large Nucleated Epithelial Cells
These cells are larger with moderate to abundant amounts of
blue to sky blue cytoplasm and a lower N:C ratio in comparison
to small epithelial cells (Figures 4 and 7). They are round to
polygonal and may have irregular, jagged, or angular borders.
Large epithelial cells can have some degree of keratinization
and possess nuclei that may be intact, degenerate, or pyknotic.
Occasionally, large nucleated epithelial cells will contain
finely stippled cytoplasmic granules (Figure 8).
Anucleated Keratinized Epithelial Cells
Anucleated epithelial cells are also known as squames or
‘‘cornflakes.’’ They are aged cells and are characterized by
an abundant blue to sky blue cytoplasm with jagged or angular
edges (Figures 7 and 9). As the name infers, they lack nuclei,
although pale round areas representing where a nucleus had
existed (nuclear ‘‘ghost’’) may be visualized (Figure 9). These
epithelial cells can fold onto themselves or break apart, creat-
ing jagged elongated structures referred to as ‘‘keratin bars’’
(Figure 10).
It should be noted that the distal one-fourth to one-third of
the vaginal lumen is covered by squamous epithelial cells and
is permanently keratinized. Therefore, it is possible that dur-
ing sample collection, cells from this area of the vagina may
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 783

FIGURE 11.—Estrous cycle wheels. Visual representation of the cell types and relative proportion of each cell type present during the 4 stages of the
estrous cycle in the mouse and rat. The size of each quadrant does not directly correlate to the length of each stage. Adapted from Byers et al.
(2012). The mouse wheel was modified from its original form to create the rat wheel. Adaptations and modifications by David Sabio.

TABLE 1.—Basic classification of the stages of estrous cycle based on cell types and relative numbers of these cell types in vaginal smears.

Stage Neutrophils Small nucleated epithelial cells Large nucleated epithelial cells Anucleated keratinized epithelial cells Relative cell density

Proestrus 0 to þ þþ to þþþ 0 to þ 0 to þ Low to moderate

Estrus
Rat 0 to þ 0 to þþ 0 to þþ þþ to þþþ Moderate to high
Mouse 0 to þ 0 to þ 0 to þ þþ to þþþ Moderate to high
Metestrus
Rat þ to þþþ þ to þþ þ to þþ þ to þþþ Moderate to high
Mouse þ to þþþ 0 to þ 0 to þ þþ to þþþ Moderate to high
Diestrus þþ to þþþ þ to þþ þ to þþ 0 to þ Low to moderate

Note: 0 ¼ none; þ ¼ few; þþ ¼ moderate; þþþ ¼ high.

be collected and is one reason why low numbers of anucleated
keratinized cells can be seen in proestrus and diestrus. Addi-
tionally, glass slides may be contaminated with keratin if not
handled properly (i.e., touching the surface of the slide with
bare hands).
Stages of the Estrous Cycle
The stages of the estrous cycle are identified by the absence,
presence, or proportion of the described four basic cell types as
well as by the cell density and arrangement of the cells on the
slide. Conventionally, the cycle is divided into the four stages
of proestrus, estrus, metestrus, and diestrus; however, some
researchers use a simple proestrus–estrus–diestrus classifica-
tion, while others subdivide the four stages, record transitional
stages, or roughly quantify the individual cell populations
(Goldman, Murr, and Cooper 2007). The following will define
and characterize the range in appearance of the four classic
stages, including species differences and a brief discussion of
transitional stages, from which criteria for stage subdivisions
or transitional stages can be based.
One general approach to staging vaginal smears is to first
assess for the presence of neutrophils—are there a good num-
ber of neutrophils observed or just an occasional one? If neu-
trophils are a dominant feature, or consistently observed, the
stage is either metestrus or diestrus; if neutrophils are rare to
absent, the stage is either proestrus or estrus. Figure 11 and
Table 1 are graphical and tabular representations of the estrous
cycle adapted from various sources and serve as good adjuncts
to the subsequent narrative.
784 CORA ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 12.—Vaginal smears of proestrus from Sprague-Dawley rats characterized by high numbers of small nucleated epithelial cells found
individually and in cohesive clusters (arrows). Proestrus appears the same in mice. Plate D shows the appearance of a so-called epithelial
‘‘strand’’ that is typical of proestrus. Modified Wright–Giemsa stain (A, D) and Toluidine blue O stain (B, C). Original objective magni-
fication of 10 (A, B), 20 (C), or 40 (D). Plate C is in Elmore et al. Proceedings of the 2014 National Toxicology Program satellite
symposium. Toxicol Pathol, 43:10-40, 2015.

Proestrus low numbers of large epithelial cells and keratinized anu-

Proestrus is a short stage, lasting an average of 14 hr in rats
and less than 24 hr in mice (Astwood 1939; Long and Evans
1922; Mandl 1951; Allen 1922; Grasso, Rozhavskaya, and Rie-
chert 1998). The predominant feature of this stage is the pres-
ence of small, round, nucleated epithelial cells of relatively
uniform appearance and size (Figures 6 and 12); these cells
may stain deeply basophilic. Often they will be seen in cohe-
sive clusters (grape clusters), sheets, or strands. However,
cohesive clusters, sheets, and strands are not always observed,
especially in low cellularity samples, and should not be thought
of as a prerequisite in the determination of proestrus; cells may
only be seen individually throughout the smear. Typically, no
neutrophils will be seen. That said, rare to occasional neutro-
phils can be found in early proestrus as the rodent would have
recently transitioned from diestrus into proestrus. Relatively
cleated cells may also be observed. As the cycle approaches
estrus, keratinized cells will become more abundant. The pres-
ence of low numbers of neutrophils, or large and anucleated
epithelial cells, does not preclude the diagnosis of proestrus
when the predominating feature of the smear are the small, round
epithelial cell population.
Estrus
Estrus duration ranges between 24 and 48 hr in rats and
between 12 and 48 hr in mice (Astwood 1939; Long and Evans
1922; Mandl 1951; Allen 1922; Grasso, Rozhavskaya, and Rie-
chert 1998). It is characterized by the presence of predomi-
nately anucleated keratinized epithelial cells (Figures 13 and
14). Numerous bacteria may be observed adhered to the cells
or free in the background. Occasional nucleated epithelial cells
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 785

FIGURE 13.—Representative photomicrographs of the first (A, C) and second (B, D) ‘‘phases’’ of estrus in mice. The first phase typically has
smaller anuclear epithelial cells that are arranged in loose clusters reminiscent of proestrus. The anuclear epithelial cells of the second phase are
generally larger, more evenly dispersed, and higher in numbers. Plates C and D are 2 consecutive days of estrus from the same B6C3F1/N mouse.
Modified Wright–Giemsa stain (A, B) and Toluidine blue O stain (C, D). Original objective magnification of 10.

can be observed throughout the stage of estrus, and neutrophils
are absent or occasionally observed in late estrus. The first and
second half of estrus, while consisting mostly of anucleated
keratinized cells, have distinct differences in their appearances
and also differ between rats and mice. These differences were
distinct enough that Young, Boling, and Blandau (1941) and
Allen (1922) had different terminology or subdivisions for the
first and second ‘‘phases’’ of estrus in rats and mice, respec-
tively. Long and Evans (1922) described these different phases
in rats but did not distinguish them as subdivisions of estrus.
In a mouse when 2 consecutive days of estrus are observed,
these different phases are generally appreciated; however, the
second phase in rats occurs late in estrus and will not always
be seen (i.e., it will be missed) even if 2 days of estrus are
recorded. In mice, at the start of estrus, the anucleated cells are
smaller and are typically arranged in loose clusters or sheets
reminiscent of proestrus (Figure 13A and C). As estrus
progresses, the cell numbers generally increase, and the cells
appear larger and more evenly dispersed and can be seen in
stacks or layers (Figures 9 and 13B, D). Allen (1922) notes this
distinction in mice as follows:
By standardizing the technique of smear preparation, . . .
[the different phases of estrus] are easily differentiated,
although the basis for this division is the number and
clumping rather than any change in the [anucleated,
keratinized] character of the cells themselves. (p. 306)
In late estrus of rats, before the emergence of neutrophils and
metestrus, higher numbers of nucleated epithelial cells appear
(Figures 7 and 14C–E). These cells are small to large; round,
oval, or spindle shaped with smooth to irregular borders; and
sometimes stain deeply basophilic. Long and Evans (1922)
describe this phase as such:
786 CORA ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 14.—Vaginal smears of estrus (A, B) and late estrus (C–F) in several Sprague-Dawley rats. Plates A and B represent a typical estrus stage
with high numbers of anucleated epithelial cells. Vaginal smears of the late estrus phase (C–F) are characterized by the presence of round-, oval-,
and spindle-shaped nucleated epithelial cells interspersed among anucleated epithelial cells. Modified Wright–Giemsa stain (A–C, E) and Tolui-
dine blue O stain (D, F). Original objective magnification of 10 (A, C, D), 20 (F), or 40 (B, E). Plate A is in Elmore et al. Proceedings of the
2014 National Toxicology Program satellite symposium. Toxicol Pathol, 43:10-40, 2015.
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE
It is interesting that in a certain proportion of animals the
epithelial desquamation of the mucosa may proceed
somewhat further than to the cornified cell layer before
leucocytes come in, and hence we may have . . . Stage
Three [estrus] immediately succeeded by a few hours of a
stage in which rather large, spindle-like, nucleated
epithelial cells are shed. In such cases, however, vestiges
of the cornified cell are always present and when leuco-
cytes appear three cell types area actually found. (p. 21)
Young, Boling, and Blandau (1941) referred to the large oval
nucleated epithelial cells of late estrus in the rat as ‘‘pavement
cells’’ and their presence became an indication that metestrus
was fast approaching. This phase of late estrus should not be
mistaken for proestrus. In general, the nucleated cells of proes-
trus have a higher N:C ratio, are round to slightly oval, and
have smooth cell borders.
In conclusion, estrus is the stage predominated by kerati-
nized anucleated epithelial cells. Neutrophils may be occasion-
ally seen in late estrus, as the rodent is transitioning to
metestrus; but once they are observed with regularity through-
out all fields, the stage should be interpreted as metestrus.
Relatively high numbers of nucleated epithelial cells are
interspersed with the keratinized cells in late estrus of rats.
Metestrus
It is well documented that metestrus is a short stage of 6–8
hr in rats (Astwood 1939; Long and Evans 1922; Mandl 1951).
Based on the limited reports in the literature and personal expe-
rience, the duration of metestrus in mice seems to be longer
than rats and can last up to 24 hr (Allen 1922; Grasso, Rozhavs-
kaya, and Riechert 1998) and, although infrequent, metestrus
(as described herein) has been identified for 2 consecutive days
in control mice on reproductive studies (personal experience;
Figure 15A and D).
Metestrus is characterized by a combination of anucleated
keratinized epithelial cells and neutrophils. In mice, nucleated
cells may appear occasionally throughout metestrus (Figure
15). In rats, the small and large nucleated cells of late estrus are
present in low to moderate numbers throughout the stage (Fig-
ure 16).
In early metestrus, neutrophils are interspersed with the
epithelial cells and are sometimes tightly packed together or
clumped around the cells; the epithelial cells usually predomi-
nate but may be in equal proportion to the neutrophils. As
metestrus progresses, neutrophils become very high in num-
ber—outnumbering epithelial cells by as much as 10-fold
(Hubscher, Brooks, and Johnson 2005)—and the smear will
be highly cellular and dense. Neutrophil and epithelial cell
numbers decrease by late metestrus, with a decrease in smear
cellularity, before the transition to diestrus. Allen (1922) gives
the following simple description of metestrus in mice:
. . . the smear shows polymorphonuclear leukocytes
among the red, horny elements [anucleated epithelial
787
cells]. A few polymorphs and many cornified cells is an
early [metestrus] stage, and a heavy leucocytic infiltra-
tion and decrease in number of the cornified cells is a late
one. (p. 306)
The separation between where metestrus ends and diestrus
begins is not always obvious, as they (i.e., the end of metestrus
and start of diestrus) are very similar in appearance and are
defined by the same cell types. Early and mid metestrus, how-
ever, are easily identified if and when sample collection hap-
pens to occur at these times. Some investigators who are
only interested in cycle length and number of days in estrus and
diestrus usually count metestrus as a ‘‘diestrus’’ day (Goldman,
Murr, and Cooper 2007). While distinguishing and recording
metestrus is recommended, if it cannot be certain if a smear
is (late) metestrus or diestrus, then it is best to be as consistent
as possible and pragmatically ‘‘error’’ on the side of diestrus;
there is minimal value in overly scrutinizing a late metestrus/
early diestrus smear.
Diestrus
Diestrus is the longest stage of the estrus cycle with an aver-
age duration of 48–72 hr in both mice and rats (Astwood 1939;
Long and Evans 1922; Mandl 1951; Allen 1922; Grasso, Roz-
havskaya, and Riechert 1998). This stage is characterized by a
substantial decrease in the number of anucleated keratinized
epithelial cells (but not necessarily an absence of) as the animal
transitions out of metestrus. Overall cellularity is moderate to
low with a combination of neutrophils, small and large
nucleated epithelial cells, and low numbers of anucleated ker-
atinized cells (Figures 4 and 17). Neutrophil numbers can vary
but are usually higher in number relative to the epithelial cells
with smears sometimes being exclusively neutrophilic. Occa-
sionally, in early diestrus, neutrophils may still appear in
clumps. It is not unusual for diestrus smears to have a very low
cellularity, especially on day 2 or 3 of diestrus, with only a
sparse scattering of neutrophils and epithelial cells (Figure
17E). In late diestrus, the epithelial cells may become more
round or be organized in small clumps, indicating proestrus the
next day; however, neutrophils will still be consistently
observed (Figure 17F).
During diestrus, macroscopic vaginal excretions are low.
When extrections are present, however, they can be viscous
and stringy (i.e., mucous) and this is especially true during
times of persistent diestrus. This excretion is sometimes aspi-
rated during vaginal cytology sample collection. It will stain
and appear as a thick pink to blue-violet material with
entrapped neutrophils and epithelial cells. The cells may be dis-
torted or elongated in the strings of mucous (Figure 18).
Transitional stages
For any given study, transitional stages will be encoun-
tered (Figure 19). There are several strategies or paradigms
that can be used for the recording of a transitional stage. The
strategy used would depend on the reason or purpose for
788 CORA ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 15.—Metestrous smears from several B6C3F1/N mice. Metestrus begins with the emergence of neutrophils interspersed or clumped
(arrow) among the anucleated epithelial cells (A–D). Occasional nucleated epithelial cells may also be present. As metestrus progresses, neutro-
phil numbers increase resulting in smears of very high cellularity (E, F). Neutrophil and epithelial cell numbers decrease as the mouse transitions
to diestrus (G, H). Plates A and D illustrate 2 consecutive days from a control B6C3F1/N mouse, representing the uncommon occurrence of a
metestrus stage being recorded for 2 days in a row in a mouse. Modified Wright–Giemsa stain (A–D, G, H) and Toluidine blue O stain (E,
F). Original objective magnification of 10 (A, C–E, G), 20 (F, H), or 40 (B). Plate F is in Elmore et al. Proceedings of the 2014 National
Toxicology Program satellite symposium. Toxicol Pathol, 43:10-40, 2015.
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE
FIGURE 15.—Continued
documenting the stages of the estrous cycle; for example, a
study designed to document potential changes in the fre-
quency or duration of a particular estrous stage may require
a different transitional stage paradigm than a study evaluating
the estrous cycle in a group of young rodents to determine
sexual maturity or their first ovulation. Following are 2 of the
more common approaches for dealing with or recording tran-
sitional stages.
In studies where only the 4 classic stages are to be used to
identify the stages of the estrous cycle, then it is recom-
mended that with a transitional smear the predominating fea-
ture(s) of the slide be used to make the final call. For instance,
when confronted with a proestrus–estrus transition smear, if
the impression is that more than 50% of the cells are small and
nucleated, then the smear would be classified as proestrus.
Another strategy for the classification of transition stages is
to use uppercase and lowercase letters. For example, the pre-
viously described smear would be designated as P(e) indicat-
ing that while the day was called proestrus (denoted by ‘‘P’’),
anucleated keratinized cells were also present in significant
numbers, indicating that a transitioning toward estrus was
occurring (denoted by ‘‘e’’). Similarly, if clumps of small
nucleated cells are observed in late diestrus, the smear can
be recorded as D(p), indicating a transition from diestrus into
proestrus. Regardless of what classification paradigm is used,
to stay consistent and thus minimize variability, it is impor-
tant that a concerted effort be made to ensure agreement on
the criteria for stage definitions.
Cellularity
The cell density or cellularity of a cytology preparation may
aid in defining a stage. Cellularity of a vaginal cytology, how-
ever, should not be used in isolation because it can vary for
numerous reasons including those independent of the actual
stage of the estrous cycle. The overall cellularity and ratio of
cells during the estrous cycle can differ between individual
789
rodents and this is particularly true for diestrus. Long and
Evans (1922) reference this with the statement that ‘‘both types
of cell [neutrophils, nucleated epithelial cells] may occur [dur-
ing diestrus] in considerable numbers in some animals or may
be both of them very sparse in others.’’ (p. 18). For a given ani-
mal, cellularity and cellular composition is usually consistent
from cycle to cycle. For example, one animal may consistently
have almost exclusively neutrophilic diestrus smears, while
another will have a good mixture of neutrophils and epithelial
cells. Thus, comparison of one animal to another should be
avoided, and comparison of day-to-day changes for each ani-
mal individually is the best approach for identifying and pre-
dicting in advance a given animal’s cyclic pattern (Hubscher,
Brooks, and Johnson 2005).
Further, there are preanalytical sources of variation that can
affect cell density on a vaginal cytology preparation, including,
collection technique, compliance of the rat or mouse during
collection and preparing more than one smear for each collec-
tion day. More specifically, preparation of more than one smear
for each day can alter the cellularity enough as to cause a
metestrus smear to be classified as diestrus, for example.
Although this is not a common issue, one should be aware that
this phenomenon could occur when making more than 1 smear
for each day.
CONCLUSION
Evaluation of sequential vaginal cytology smears is a time-
honored tool for the assessment of the estrous cycle in labora-
tory rats and mice. Collection and preparation of vaginal cytol-
ogy samples is an essentially noninvasive procedure that, with
proper training, is easily mastered. For reliable and consistent
data generated from vaginal cytology studies, evaluations of
the cytology smears must be based on well-established standar-
dized criteria. Smear review and, ultimately, the data obtained,
however, can only be as ‘‘good’’ as the technical expertise and
experience of the evaluator performing the microscopic
790 CORA ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 16.—Metestrous vaginal smears from several Sprague-Dawley rats. Metestrus begins with the emergence of neutrophils interspersed or
clumped among the nucleated and anucleated epithelial cells (A, B). Note that the appearance of metestrus differs in rats and mice, as rats have
much higher numbers of nucleated epithelial cells. As metestrus progresses, neutrophil numbers increase resulting in smears of high cellularity
(C–F). Toluidine blue O stain. Original objective magnification of 10 (A, C, E) or 40 (B, D, F).
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE 791

FIGURE 17.—Diestrous vaginal smears from B6C3F1/N mice (A–E) and a Sprague-Dawley rat (F) characterized by a moderate to low cellularity with
predominately neutrophils that are mixed with lower numbers of epithelial cells. A small clump of round epithelial cells (arrow) is present on a diestrous
smear taken the day before the emergence of proestrus. Modified Wright–Giemsa stain (C–E) and Toluidine blue O stain (A, B, F). Original objective
magnification of 10 (A, C, E, F) or 40 (B, D). Plate E is in Elmore et al. Proceedings of the 2014 National Toxicology Program satellite symposium.
Toxicol Pathol, 43:10-40, 2015.
792 CORA ET AL.
FIGURE 18.—Diestrous vaginal smears from several Sprague-Dawley
rats. Basophilic to eosinophilic staining mucous has entrapped and
distorted the epithelial cells and neutrophils. A classic mucous
‘‘swirl’’ is seen in plate B (arrow). Modified Wright–Giemsa stain
(B) and Toluidine blue O stain (A, C).
TOXICOLOGIC PATHOLOGY
FIGURE 19.—Lower (original objective magnification of 10) and
higher (20) magnifications of a vaginal cytology smear taken
from a Sprague-Dawley rat representing a proestrus-to-estrus tran-
sition. Individual and clumps of small epithelial cells are seen
among anucleated epithelial cells. Based on complete review of
this smear, the recorded stage (based on the 4-stage paradigm) was
proestrus or ‘‘P.’’ Using the other classification paradigm dis-
cussed in the article, it could also be classified as ‘‘P(e).’’ Please
refer to Transitional Stages section for further details. Modified
Wright–Giemsa stain.
examination. Thus, it is recommended that individual labora-
tories define and codify their respective processes via labora-
tory procedures, and the proficiency of the personnel
reviewing the cytology materials must be established and is
achieved through appropriate training, the use of vaginal cytol-
ogy study sets of known diagnoses, and experience. As high-
lighted in this review, the cytological appearance of the rat
and mouse estrous cycle differs in several ways. Thus, regard-
less of general similarities, when it comes to the estrous cycle, a
‘‘mouse is not a rat’’ and proper training is required for each
species to garner full understanding of the differences.
Vol. 43, No. 6, 2015 VAGINAL CYTOLOGY IN RATS AND MICE
ACKNOWLEDGMENTS
The authors would like to thank Drs. Mark Cesta, NTP,
NIEHS, and Barry McIntyre, NTP, for their helpful suggestions
concerning the article, and Beth Mahler, EPL, for her help with
all the photomicrographs.
AUTHOR CONTRIBUTION
Authors contributed to conception or design (MC, LK, GT);
drafting the article (MC); and critically revising the article
(MC, LK, GT). All authors gave final approval, and agreed
to be accountable for all aspects of work in ensuring that ques-
tions relating to the accuracy or integrity of any part of the
work are appropriately investigated and resolved.
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