Comp Clin Pathol (2006) 14: 186–190
DOI 10.1007/s00580-006-0596-4
ORIGINA L ARTI CLE
M. R. Ahmadi . S. Nazifi . H. R. Ghaisari . M. Damchy
Evaluation of the cytology of uterus, vagina, and clitoris
as predictors of uterine condition in the mare
Received: 31 October 2005 / Accepted: 10 January 2006 / Published online: 28 February 2006
# Springer-Verlag London Limited 2006
Abstract Detection of venereal disease and acute or
chronic endometritis can be achieved by cytological
examination. The presence of significant numbers of
neutrophils in the uterine smear is the best indication for
equine endometritis. Uterine function may be evaluated by
examining clitoral or vaginal smears. In this study, 20
nonpregnant mares were selected and their reproductive
functions were evaluated by rectal palpation. The levels of
progesterone were measured by radioimmunoassay (RIA).
Cytological smears were prepared from clitoris, vagina,
and uterus. A wet swab was applied to the clitoral fossa and
the collected material transferred to a microscope slide.
Vaginal samples were collected using 30 cm wet swabs.
Uterine samples were collected from uterine lavage fluid.
There was no significant difference (P≥0.05) between cell
densities in the estrus phase (p4<1 ng/ml) compared with
the interval between estrus phases (p4>1 ng/ml). Significant differences (P<0.05) were noted in epithelial cell,
lymphocyte, and neutrophil densities between diestrus
phase (p4≥3.5 ng/ml) and other phases (p4<3.5 ng/ml).
Epithelial, neutrophil, and lymphocyte densities were
significantly different between clitoral, vaginal and uterine
smears (P<0.05). There were significant differences in
epithelial cells and neutrophils of vaginal and uterine
smears in mares affected by metritis compared with repeat
breeder and normal mares. We concluded that the clitoral
cytology of mares could not predict uterine physiology but
evaluation of vaginal cytology showed more appropriate
potential. However, further study is needed to assess
vaginal cytology in comparison with other methods for
evaluating uterine reproductive physiology.
Keywords Mare . Endometritis . Cytology . Uterus .
Vagina . Clitoris
M. R. Ahmadi (*) . S. Nazifi . H. R. Ghaisari . M. Damchy
Department of Clinical Sciences,
School of Veterinary Medicine,
Shiraz University,
P.O. Box 1731, Shiraz, Iran
e-mail: rahmadi@shirazu.ac.ir
Introduction
Early studies performed by Knudsen (1964) indicated the
potential value of endometritic cytology. Cytological
examination of the endometrium is often used in the
mare to evaluate causes of infertility, specifically to detect
venereal disease and acute or chronic endometritis
(Ricketts et al. 1993; Roszel and Freeman 1988). Cytological diagnosis of equine endometritis is based on the
presence or absence of significant numbers of neutrophils
in the smear. Neutrophils are very rarely seen in normal
mares but can often be detected at the “foal heat” or the first
estrus of the breeding season in maiden mares although
there is no endometritis (Pycock and Newcombe 1996).
Also, in many cases, the uterine luminal fluid, which
accumulates before mating, is sterile and contains no
neutrophils (Pycock and Newcombe 1996). Results of
several researchers regarding the presence of neutrophils in
uterine fluid are shown in Table 1.
Troedsson (1999) suggests that a transient endometritis
is a normal physiological response to breeding. However,
in mares with impaired uterine defense mechanisms, the
condition may develop into a persistent endometritis and
subsequent subfertility. Mares with persistent and recurrent
endometritis are said to be susceptible to infection; in
particular, they are unable to resolve the acute endometritis
that always follows mating. It is thought, therefore, that
these mares have a local immunological defect in the uterus
that impedes the elimination of bacteria. Studies on
immunoglobulins, opsonins, and the functional ability of
neutrophils in the uterus of susceptible mares have not
confirmed the presence of an impaired immune response
(Allen and Pycock 1989).
For reliable cytological results, a collection technique is
required that yields many well-preserved cells representative of a large uterine surface area (Roszel and Freeman
1988). Endometrial cells for cytological evaluation may be
collected in uterine aspirations, washings, or by using
guarded cotton swabs. A technique using the plastic cap of
a guarded culture swab was also described (LeBlanc 1991).
187
Table 1 Quantitative estimation of neutrophils in endometrial smears
Authority (Year)
Method of quantification
Knudsen (1964)
Wingfeild Digby (1978)
Asbury et al. (1982)
Asbury (1984)
Couto and Hughes (1984)
Brook (1985)
Positive if >1 neutrophil in 5 fields (×200)
Estimates ratio of neutrophils to epithelial cells
Positive if ratio of epithelial cells to neutrophils >10:1
Positive if more than 1 neutrophil in 5 high-powered fields
Graded according to ratio of neutrophils to epithelial cells in 8 fields (×100)
Negative if no PMNs in 10 fields (×400)
Doubtful if 1 to 5 PMNs in 10 fields
Positive if >5 PMNs in 10 fields
Used a 0 to 5 grading method depending on ratio of PMNs to epithelial cells
Positive if >2% of cells were neutrophils
Positive if ≥1 neutrophil per field (×400)
Negative 0% neutrophils
± less than 0.5% PMNs
+0.5 to 5% PMNs
2+5 to 30% PMNs
3+ >30% PMNs
La Cour and Sprinkle (1985)
Ball et al. (1988)
Purswell et al. (1989)
Rickets and Mackintosh (1989)
PMN Polymorphonuclear
The activity of uterine-derived cells of susceptible mares
was unaffected by stage of cycle. Susceptibility to endometritis was not associated with a defect in the phagocytic
function of uterine neutrophils. Also, the function of
uterine cells from resistant mares was greater during estrus
than diestrus (Asbury and Hansen 1987). However, there
are no reports regarding cytological evaluation of clitoris
and vagina in comparison with uterine cytology for mares.
The aim of this study was (1) to compare the density of
reactive cells in clitoral, vaginal, and uterine smears of
mares; (2) to detect differences in cells densities between
different progesterone levels; and (3) to evaluate the use of
clitoral or vaginal smears for the detection of uterine status.
Materials and methods
Twenty nonpregnant mares were selected randomly from
riding stud horses around Shiraz. Reproductive functions
were evaluated by rectal palpation in middle breeding
season and mares had not been mated in the preceding
30 days. Cytological smears were prepared from clitoris,
vagina, and uterus. The tail was bandaged and held to one
side and the clitoris sample was collected by bacteriological swab. The wet swab was applied to the clitoral fossa
and the collected was material transferred to a microscope
slide. The vulva and perineum were washed thoroughly
with clean water. The area was dried using a disposable
paper towel. The vaginal sample was collected using a 30cm wet swab. The wet swab was applied to the vagina and
the collected material transferred to a microscope slide. The
uterine sample was collected, using sterile technique, from
uterine lavage fluid. The uterus fluid was collected using an
equine uterus catheter (Equine Uterus Catheter, Sopa,
Iran). The balloon catheter was placed through the cervix,
which was inflated with 60 ml of air to ensure retention
within the uterus. Normal saline (100–200 ml at 40°C) was
injected and the uterus was manipulated by rectal palpation
to distribute the fluid into both uterine horns. As much of
the saline as possible was then recovered and collected into
a 250-ml cylinder for 30–40 min, after which the supernatant was removed to leave approximately 20 ml. This
was centrifuged at 250×g for 5 min. The resultant sediment
suspension was used to prepare smears to be stained with
Diff-Quik for cytologic examination.
The smears were labeled, air-dried, fixed with methyl
alcohol at five min, and stained with Giemsa for 20 min
followed by washing in distilled water and subsequent
drying. At least 100 to 200 cells were evaluated by light
microscope and were counted over 20 microscopical fields
(×900). Differential cellular counts were carried out on
Giemsa-stained smears of the mucosa. The counted cells
were epithelial, neutrophil, lymphocyte, macrophage, and
monocyte cells.
Blood samples were collected from the jugular vein and
serum was separated by centrifugation at 750×g for 15 min.
Sera were frozen and stored at −20°C. Progesterone level
was measured by RIA (Orion Diagnostica, Finland) with
sensitivity of 0.2 ng/ml and the intra- and interassay were
10.2 and 6.5%, respectively. The mares were divided to
two groups by progesterone levels. First, mares were
separated by progesterone level below 1 ng/ml (estrus
phase) and above 1 ng/ml. Second, the mares were divided
to bellow 3 ng/ml and above 3.5 ng/ml (diestrus phase)
(Ginther 1993). Mares were also grouped as repeat breeder
mares (infertile for at least one breeding season) and mares
who showed signs of endometritis on clinical examination.
Data were analyzed by SPSS software, version 11.5. The
independent sample t test was used to detect significant
differences between clitoral, vaginal, and uterine cell
densities in mares with different progesterone levels. Cell
densities of clitoris, vagina, and uterus in normal, clinical
endometritis and repeat breeder mares were compared
using one-way ANOVA and Duncan’s multiple range tests.
188
Results
There was no significant difference (P≥0.05) in cell
densities of mares at estrus phase (p4<1 ng/ml) compared
to mares at other phases (p4>1 ng/ml) (Table 2). There was
significant difference (P<0.05) in epithelial cell, lymphocyte, and neutrophil densities when mares were divided to
midluteal phase (p4>3.5 ng/ml) and other phases
(p4<3.5 ng/ml). The neutrophil densities in vaginal and
uterine smears were significantly more than in clitoral
smears (P<0.05). Also, epithelial cells and lymphocytes in
clitoral and vaginal smears were significantly increased
compared to uterine smears (P<0.05) (Table 3).
Neutrophils in vaginal smears and uterine lavage smears
of mares affected by endometritis were significantly higher
(P<0.05) than repeat breeder and normal mares but
epithelial cells in repeat breeder mares were significantly
lower than in other groups (P<0.05). There was significant
difference in monocytes of clitoral smears between the
three groups (P<0.05), but in clitoral smears, there was no
significant difference in other cells between the groups
(P>0.05) (Table 4).
Discussion
The results of this study did not show significant differences in cellular densities in smears prepared from clitoris,
vagina, and uterus between mares divided by being above
or below 1 ng/ml of progesterone. When mares were
divided to above and below 3.5 ng/ml of progesterone,
there were differences in the densities of epithelial cells and
lymphocytes in smears from clitoris and vagina. Also, there
was significant difference in neutrophil densities in vaginal
and uterine smears when the mares were divided to diestrus
(p4>3.5 ng/ml) and other phases. These finding are in
agreement with reports by Asbury and Hansen (1987).
Significant differences were found in the means of
neutrophils in cervical mucosa smears at different estrus
cycle phases of ten Holstein dairy heifers. The means
(±SD) of neutrophil densities were 0.10±0.10, 17.60±8.83,
0.50±0.30, and 3.60±1.44 at estrus, metestrus, diestrus, and
proestrus, respectively (Ahmadi et al. 2000). Ahmadi et al.
(2004) found no significant difference in cell densities
between cervical mucosa and uterine swab in cattle.
The ability of uterine fluid samples to attract neutrophils
was measured using chemotactic chambers. There was no
significant difference between distances migrated by
neutrophils toward fluids obtained during estrus or diestrus. Chemotaxis scores tended to be higher with samples
from infected uteri and the difference was significant for
samples from two mares. Chemotaxis was neither
significantly correlated with protein concentration of
uterine fluid nor with serum estrogens or progesterone
(Blue et al. 1984).
Saltiel et al. (1987) considered cervico-endometrial
cytology in mares. The number of neutrophils was
positively correlated with the percentage of eosinophils,
bacterial flora, and cellular necrosis but had a negative
association with the presence of ciliocytopholia.
The significantly higher percentages of monocytes in
clitoral smears in mares affected by metritis in comparison
with repeat breeder mares revealed that the presence of
leukocytes in the clitoris may originate from uterine
infections if the mare has discharge. The significantly
higher neutrophil densities in mares affected by clinical
endometritis shown in vaginal swabs and uterine lavage
smears compared with normal and repeat breeder mares
showed a role for cytological evaluation of vagina and
uterus.
The mean percentage of neutrophils in normal and repeat
breeder mares were lower than 5% in vaginal and uterine
smears, but were higher than 30% in mares affected by
endometritis. Significantly higher neutrophil densities in
vaginal smear and uterine lavage smears of mares affected
by endometritis revealed that neutrophil-positive vaginal
cytology can be useful in reproductive tract evaluation.
This was supported by the lack of significant difference
between cytology evaluation of vagina and uterus. However, cytological evaluation of vagina and uterus is not
usually useful in repeat breeder mares because repeat
breeder syndrome in mares is due to many factors and
Table 2 Comparison of mean (±SD) cytology of clitoris, vagina, and uterus of mares with different progesterone (p4) levels
Cells
Groups
Number
Clitoris
Vagina
Uterus
Epithelial
A
B
A
B
A
B
A
B
A
B
A
B
10
3
10
3
10
3
10
3
10
3
10
3
96.50±2.22
95.67±0.58
0.00±0.00
0.33±0.58
0.40±0.70
0.33±0.58
0.10±0.31
0.33±0.58
2.40±1.07
3.00±1.00
0.60±0.84
0.33±0.58
96.30±2.31
95.33±1.53
0.40±0.70
1.00±1.00
0.30±0.67
0.00±0.00
0.10±0.32
0.67±1.15
2.30±1.25
2.33±1.15
0.70±0.82
0.67±0.58
96.90±1.79
97.33±1.15
0.40±0.70
0.67±1.15
0.20±0.42
0.00±0.00
0.10±0.32
0.33±0.58
1.80±1.03
1.33±1.15
0.60±0.97
0.33±0.58
Neutrophil
Basophil
Eosinophil
Lymphocyte
Monocyte
A=P4>1 ng/ml and B=P4<1 ng/ml
189
Table 3 Comparison of mean (±SD) cytology of clitoris, vagina, and uterus of mares with different progesterone (p4) levels
Cells
Groups
Number
Clitoris
Vagina
Uterus
Epithelial
C
D
C
D
C
D
C
D
C
D
C
D
5
8
5
8
5
8
5
8
5
8
5
8
97.80±1.30a
95.38±1.77b
0.00±0.00a
0.13±0.35a
0.20±0.45a
0.50±0.76a
0.00±0.00a
0.25±0.46a
1.80±0.84a
3.00±0.93b
0.20±0.45a
0.75±0.89a
97.60±1.52a
95.13±1.96b
0.00±0.00a
0.88±0.83b
0.60±0.89a
0.00±0.00a
0.00±0.00a
0.38±0.74a
1.40±0.89a
2.88±0.99b
0.40±0.55a
0.88±0.83a
97.60±1.14a
96.63±1.85a
0.00±0.00a
0.75±0.89b
0.20±0.45a
0.13±0.35a
0.00±0.00a
0.25±0.46a
1.80±0.84a
1.63±1.19a
0.40±0.55a
0.63±1.06a
Neutrophil
Basophil
Eosinophil
Lymphocyte
Monocyte
Values of each cell with different superscripts in each column are those which differ significantly (P<0.05)
C=P4>3.5 ng/ml and D=P4<3 ng/ml
endometritis in one of several factors that can cause
infertility in mares. These results are in agreement with
reports of Ricketts and Mackintosh (1989).
Results of uterine culture are best interpreted in relation
to numbers of neutrophils detected at the endometrial
surface by exfoliative cytology. However, mares with
chronic endometritis may have little surface irritation
(Roszel and Freeman 1988). If neutrophils are present but
there is an absence of pathogen growth on the culture, this
most likely indicates the presence of a pathogen that was
missed during sampling (Ball et al. 1988). Ball et al. (1988)
considered a low volume (60-ml) uterine flush using
phosphate buffered saline via a transcervical balloon
catheter in barren or subfertile mares. In practice, this
more complicated sampling method (compared with the
guarded swab) would only be appropriate for those mares
that were previously proven to be subfertile or where a
previous sample using a guarded swab has resulted in
absence of a pathogen in culture but the presence of
neutrophils in the cytology smear.
Collection of a surface sample of the endometrial lining
of the uterus with the guarded endometrial swab is
undoubtedly the easiest method of sampling. Bourke et
al. (1997) compared cytological findings from cotton swab
and cytology brush to histopathology findings. The
cytology brush technique provided samples with higher
cell yields and fewer distorted epithelial cells, providing a
more satisfactory sample for cytological evaluation.
Although polymorphonuclear (PMN) numbers were not
significantly different by either technique, more studies are
Table 4 Cytologic comparison of clitoris, vagina, and uterus of normal mares, clinical endometritis, and repeat breeder mares (mean±SD)
Cells
Groups
Number
Clitoris
Vagina
Uterus
Epithelial
N
E
R
N
E
R
N
E
R
N
E
R
N
E
R
N
E
R
13
4
3
13
4
3
13
4
3
13
4
3
13
4
3
13
4
3
96.31±1.97
92.25±7.54
96.67±3.06
0.28±0.08
3.00±1.00
0.00±0.00
0.38±0.65
0.00±0.00
0.00±0.00
0.15±0.38
0.75±0.96
0.33±0.58
2.54±1.05
2.00±0.00
2.00±2.00
0.54±0.78a
2.00±1.41b
0.67±0.58a
96.08±2.14a
57.00±8.16b
93.33±5.69a
0.36±0.78a
36.25±4.19b
1.67±2.08a
0.23±0.60
0.00±0.00
0.00±0.00
0.23±0.60
0.96±0.75
0.33±0.33
2.54±1.05
5.00±4.50
2.00±2.00
0.54±0.78
2.00±1.41
0.67±0.58
97.00±1.63a
47.50±5.45b
72.92±5.69a
0.15±0.38a
47.25±9.46b
2.00±3.46a
0.15±0.38
0.25±0.50
0.00±0.00
0.15±0.38
0.25±0.50
0.33±0.58
1.69±1.03
4.75±4.86
2.00±1.73
0.54±0.88
0.00±0.00
0.33±0.58
Neutrophil
Basophil
Eosinophil
Lymphocyte
Monocyte
Values of each cell with different superscripts in each column are those which differ significantly (P<0.05)
N Normal Mares, E mares affected by endometritis, and R repeat breeder mares
190
needed to establish criteria for the interpretation of
cytobrush samples in the mare.
Endometrial cytological examination is a rapid reliable
aid for diagnosing acute endometritis in the mare (Reiswig
et al. 1993). Reiswig et al. (1993) reported the correlation
between cytology and culture comparable to other reports.
Cytology was determined to be as acceptable as biopsy in
the determination of acute inflammatory responses associated with bacterial endometritis; however, it was not
possible to detect the chronic inflammatory or fibrotic
changes by cytology that were revealed by histopathology.
Results of this study show that the combination of
cytological evaluation and progesterone level detection is
probably more useful than cytological evaluation alone.
The presence and density of neutrophils in clitoral, vaginal,
and uterine flush smears at all phases of normal mares was
consistent with almost all previous publications regarding
cytological evaluation of equine uteri. We concluded that
clitoral cytology could not predict uterine physiology but
evaluation of vaginal cytology could add significantly to
diagnostic robustness. It should also be noted that false
negatives can occur in the evaluation of equine uterine
cytology (Wingfield Digby 1978). Further study is needed
to explore the potential for vaginal cytology as a predictor
for equine uterine conditions.
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