Veterinary Immunology and Immunopathology 205 (2018) 65–71

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Endometrial transcripts of proinflammatory cytokine and enzymes in T

prostaglandin synthesis are upregulated in the bitches with atrophic
pyometra
⁎
Laishram Kipjen Singha, Manas Kumar Patraa, , Girish Kumar Mishraa, Vidya Singhb,
Vikramaditya Upmanyuc, Abhishek C. Saxenad, Sanjay Kumar Singha, Goutam Kumar Dasa,
Harendra Kumara, Narayanan Krishnaswamya
a
Animal Reproduction Division, ICAR - Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243 122, India
b
Pathology Division, ICAR - Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243 122, India
c
Division of Biological Standardization, ICAR - Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243 122, India
d
Division of Veterinary Surgery, ICAR - Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243 122, India

A R T I C LE I N FO A B S T R A C T

Keywords: Inflammatory markers of endometrial origin are valuable in order to differentiate the pyometra from cystic
Pyometra endometrial hyperplasia in the bitch. In the present study, we hypothesized that histological categorization
Cystic hyperplasia would distinguish the differential regulation of the proinflammatory genes in the endometrium of bitches with
Canine pyometra. Ovariohysterectomy was done on bitches with confirmatory diagnosis of pyometra (n = 18). Using
Endometrium
endometrium to myometrium ratio of 0.79 as threshold, the uteri (n = 8/group) were categorized into hyper-
Cytokines
Prostaglandins
plastic pyometra (HP) and atrophic pyometra (AP). Two samples were excluded as the diagnosis was incon-
clusive. In parallel, endometrial tissue was collected for total RNA extraction to study the differential expression
of TLR4, IL-6, IL-8, COX-2 and PGFS through real time PCR. Diestrus uterus of non-pyometra bitches (n = 6)
served as control. The mean fold change (2−ΔΔCt) for the target genes was determined using β-actin as en-
dogenous control and non-pyometra uterus as calibrator group. Except TLR4, other inflammatory genes were
upregulated significantly by 1.82 to 3.74 times in the AP as compared to HP with maximum upregulation of
COX-2 and PGFS. Further, correlation matrix with Spearman’s rho revealed that IL-8 had strong positive cor-
relation with COX-2 and PGFS in the AP group (P < 0.05). It is concluded that histological grading of pyometra
into HP and AP revealed differential regulation of inflammatory cytokines and enzymes in the PG synthetic
pathway in the canine endometrium that has diagnostic potential under clinical settings.

1. Introduction cascade that culminates in the secretion of pro-inflammatory cytokines

Pyometra is the common uterine pathology in the intact nulliparous
bitches of < 10 years (Niskanen and Thrusfield, 1998; Egenvall et al.,
2000). The onset is sly and insidious with subtle uterine changes that
fail to manifest clinically at the early stages; therefore, the diagnosis is
often made late resulting in potentially life-threatening medical emer-
gency (Fransson, 2003). Lipopolysaccharide (LPS) of the Gram negative
bacterial cell wall and exotoxin with super-antigenic properties of Gram
positive bacteria are absorbed through the compromised endometrial
mucosa. When the circulating LPS binds the receptor complex, cluster
of differentiation 14/ myeloid differentiation protein-2/ toll like re-
ceptor 4 (CD14/MD-2/TLR4), it initiates a downstream signalling
including interleukins (IL-1, IL-6, IL-8) and tumor necrosis factor-α
(TNFα) as well as secondary inflammatory mediators such as pros-
taglandins (PG), nitric oxide and reactive oxygen species (Fransson
et al., 1997; Dabrowski et al., 2007; Hagman et al., 2009; Karlsson
et al., 2012). The deregulated production of inflammatory mediators
result in cytokine storm to provoke systemic inflammatory response
syndrome (SIRS) followed by irreversible damage to the internal organs
and death (Fransson et al., 2007; Hagman et al., 2007).
The seminal work of Dow (1958) showed the importance of histo-
pathology of uterus in staging the canine cystic endometrial hyper-
plasia-pyometra complex. Subsequently, the histological grading of
pyometra was described by De Bosschere et al. (2001) to link the

⁎
Corresponding author.
E-mail address: drmanas01@gmail.com (M.K. Patra).

https://doi.org/10.1016/j.vetimm.2018.10.010
Received 4 April 2018; Received in revised form 16 October 2018; Accepted 21 October 2018
0165-2427/ © 2018 Elsevier B.V. All rights reserved.
L.K. Singh et al.
uterine lesions with the clinical stage of the disease. Accordingly,
pyometra was classified into hyperplastic (HP) and atrophic (AP) ca-
tegories based on the endometrium to myometrium ratio (E:M), se-
verity of degree of inflammatory reaction and fibroblast proliferation as
well as presence or absence of cysts.
The expression of certain upregulated genes in the uterine tissue and
the corresponding products in the circulation may be useful for diag-
nosis and prognosis of canine pyometra (Hagman, 2014). Pyometra-
associated E. coli endotoxin release stimulates the up-regulation of the
rate limiting enzyme in prostaglandin (PG) synthesis, cyclooxygenase 2
(COX-2) as well as the tissue specific terminal synthases such as PGF
synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) in the
endometrium of bitches compared to anestrus and diestrus control
(Silva et al., 2009). Further, an increased expression of transcripts of the
genes encoding TLR2 and TLR4, PG synthesis enzymes upon stimula-
tion by LPS and lipoprotein of E. coli constituents in the endometrium of
pyometra affected bitch results in a higher endometrial concentration of
PGE2and PGF2α, which may further regulate the local inflammatory
response (Silva et al., 2010). Global endometrial transcriptomic pro-
filing by microarray with network analysis revealed that many genes
including IL-8, IL-1α, IL-1β, interleukin 18 receptor, interleukin 1 re-
ceptor antagonist and IL-6 were upregulated about 10–77 folds in the
uteri of bitches with pyometra (Bukowska et al., 2014). In another
study, transcriptomic profile of the canine endometrium revealed that
genes like secretory leukocyte protease inhibitor (SLPI), COX-2, matrix
metalloprotenase 1 (MMP1) and IL-8 were upregulated in the canine
pyometra (Voorwald et al., 2015). Analyses of the plasma or serum
levels of inflammatory mediators revealed that prostaglandin F meta-
bolite (PGFM), endotoxin, IL-6 and IL-8 are elevated in the bitches with
pyometra (Fransson et al., 1997; Hagman et al., 2006a, b; Nakamura
et al., 2008; Karlsson et al., 2012; Jitpean et al., 2014).
The clinical presentation of pyometra in a bitch depends on the
degree and severity of endometrial inflammatory response and onset of
SIRS is the end stage of disease. Accordingly, the molecular lesions are
more likely to be different at early and advanced stages of the disease
and will be a useful tool when endometrial transcervical biopsy is op-
timized in the bitch (Christensen et al., 2012). Thus, in the present
study, we defined the pyometra histologically as hyperplastic (HP) or
atrophic (AP) based on the established criteria and studied the differ-
ential expression of endometrial genes such as TLR4, IL-6, IL-8, COX-2
and PGFS, that are known to be upregulated.
2. Materials and methods
2.1. Case history
The study was done at Veterinary Gynaecology and Obstetrics unit
of Referral Veterinary Polyclinic, Indian Veterinary Research Institute,
Izatnagar during November 2016 - June 2017. A tentative diagnosis of
pyometra was made based on the history and clinical signs.
Confirmatory diagnosis was based on the sonographic signs of pyo-
metra (Bigliardi et al., 2004 and Smith, 2006) and leukocytosis in the
complete blood cells count (Hagman et al., 2009). Briefly, ultra-
sonography of lower abdomen was done with a curvilinear probe of
5 MHz frequency. The appearance of uterine lumen with anechoic to
hypo-echoic content was the diagnostic criteria which may or may not
accompany thickened uterine wall with or without cystic changes
(Fig. 1 A–D). A total of 18 bitches of different breeds such as Spitz,
Labrador, German Shepherd, Pug, Dalmatian and non-descript were
confirmed to have pyometra and ovariohysterectomy was done at small
animal surgery unit. The age varied from 3 to 11 years (average 8.28
years) with 66.7% (10/15) nulliparous animals. Based on the elective
ovariohysterectomy schedule, six diestrus uteri of adult bitches (3
Labrador, 2 Spitz and 1 non-descript) were collected to serve as control.
The age of the bitches in the control group was 3–5 years (Suppl.
Table 1).
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Veterinary Immunology and Immunopathology 205 (2018) 65–71
2.2. Ovariohysterectomy
The ovario-hystectectomy operation and post-operative care was
performed following the standard operating guidelines of the institute
animal ethics committee. Pre-anesthesia was given with atropine
(0.045 mg/kg subcutaneous), butorphanol (0.2 mg/kg intravenous) and
diazepam (@ 0.5 mg/kg intravenous). Anesthesia was induced with
ketamine (10 mg/kg intravenous) and maintained with the mixture of
ketamine and diazepam at 1:1 ratio (v/v). The bitch was secured in
dorsal recumbency and a 10–15 cm midline abdominal incision was
made depending on the body size. The uterine horn was located and a
clamp was placed on the proper ligament of the ovary to retract it,
while the suspensory ligament was stretched or broken with index
finger. A window was made in the mesovarium caudal to the ovarian
vessels. The ovarian pedicle was triple clamped and the pedicle was
severed closest to the ovary. Three clamps were placed just cranial to
the cervix and the uterine body was severed between the proximal and
the middle clamps after ligating the uterine arteries. The pedicle was
gently replaced into the abdomen after ensuring hemostasis. The ab-
dominal muscles were apposed using simple interrupted suture pattern
with vicryl and skin was closed by cross mattress suture pattern using
polyamide. Post-operative care was given for 7 days with ceftriaxone
(20 mg/kg) with anti-inflammatory and analgesic drugs.
Owner consent was obtained to use the uterine tissues for histo-
pathology and real-time PCR studies. Immediately after ovariohyster-
ectomy, the uterus was brought to the laboratory in ice, washed in
chilled phosphate buffer saline and gross observation was recorded.
Tissue sampling for RNA extraction and histopathology were done apart
from sterile swabbing of the endometrial wall for routine bacteriology.
2.3. Gross and histopathology
The uterine morphometry, degree of fluid accumulation, colour and
consistency of the uterine contents and the gross changes of the en-
dometrial mucosa were recorded (Fig. 2A-F). Sterile swabs were
scraped on the endometrial wall and transported to lab in peptone
water. The swabs were directly inoculated on blood agar plates in du-
plicate and incubated at 37 °C for 48 h aerobically and anaerobically.
The isolates were then subjected to standard morphological and bio-
chemical tests to identify the bacteria (Holt et al., 1994). A 3 cm long
and 3 cm wide thick sections of the uterus was collected from either one
of the horns and fixed in 10% neutral buffered formal saline (v/v) so-
lution. A 5 μm thick section was made and stained with haematoxylin
and eosin and screened as per the criteria laid (De Bosschere et al.,
2001) and the thickness of endometrium and myometrium was mea-
sured with micrometry to determine the E:M ratio (Fig. 3A-C). The
mean E:M ratio of 0.79 in the non-pyometra group served as cut-off to
discriminate HP and AP groups as it was comparable with the reported
value of 0.78 in the diestrus bitch (De Bosschere et al., 2001). Receiver
operating characteristic curve (ROC) using the E:M ratio of the HP and
AP samples revealed that the value of 0.7698 (closest to the cut-off)
showed a diagnostic sensitivity of 86.67% and specificity of 83.33%
with a likelihood ratio of 5.2 (Suppl. Fig. 2). Further, the E:M ratio of
0.79 was close to the mid-point of the two standard deviations (2 SD),
which covers 95% of sample dispersion of HP and AP groups. En-
dometrial glandular atrophy, degree of cystic changes and fibrosis were
included to arrive at a histological diagnosis when the E:M ratio was at
borderline. Based on the histopathology, the uteri were retrospectively
categorized into HP or AP (n = 8/group). Two samples were not pro-
cessed for transcriptional analysis as the histopathology was incon-
clusive.
2.4. Differential expression of certain endometrial transcripts
Differential expression of endometrial transcripts viz., TLR4, IL-6,
IL-8, COX-2 and PGFS was analyzed by real time PCR. A small piece of
L.K. Singh et al. Veterinary Immunology and Immunopathology 205 (2018) 65–71

Fig. 1. Representative ultrasonographic images of the uterus of bitches with pyometra.

A&B. Proliferative changes in the endometrium with (Black bold arrow) accumulation of anechoic to echogenic particles in the lumen indicating hyperplastic
pyometra (HP). C &D. Multiple anechoic pockets (Thin white arrow) separated by thick fibrinous bands along with thickened uterine wall in a case of atrophic
pyometra (AP).

endometrial tissue weighing about 100 mg was excised from either of
the horn using a diethylpyrocarbonate (DEPC) treated sterile scalpel
and taken into a DEPC treated storage vial containing 200 μL RNAlater®
(Sigma – Aldrich, USA). The tissue samples were transferred im-
mediately to −20 °C till extraction of total RNA. Total RNA was ex-
tracted using Trizol solution (Invitrogen, USA). The A260/280 of total
RNA was within 1.8 and 2.0 (UV-VIS 3000, LabIndia, India) and used
for cDNA synthesis using RevertAidTM M-MuLV Reverse Transcriptase
enzyme (Fermentas Life Science, USA). Both forward and reverse pri-
mers for target genes including β-actin were designed using online
Primer Quest tool of Integrated DNA technologies from the published
coding DNA sequences available with the NCBI. The primer sets used
for amplification of β-actin, TLR-4, IL 6, IL 8, COX-2 and PGFS were
custom synthesized by Europhin, India (Table 1).
The PCR cyclic condition of target genes was standardized using
different concentration of magnesium chloride and annealing tem-
perature in a thermal cycler (Agilent Technologies, California, USA).
Agarose gel electrophoresis (1.5% w/v) was done and the product size
was checked in gel documentation system (Suppl. Fig. 1A–D). The
target genes were amplified using Quantitect SYBR green master mix
(Qiagen, GmbH, Germany). The relative fold change of target genes in
the bitches with HP or AP were calculated using the diestrus en-
dometrium of non-pyometra group as calibrator and β-actin as en-
dogenous control. The relative fold change (n-fold) for each target gene
was determined using 2−ΔΔCt method (Livak and Schmittgen, 2001).
2.5. Statistical analysis
The data obtained from the histopathology conformed to equality of
variance as evidenced by Levene’s test (P > 0.05); thus, one-way
Anova was done with Tukey as post-hoc test. The relative fold change
was analyzed with unpaired t-test with Welch correction as the variance
was unequal. As AP group showed significant upregulation for most of
the genes, correlation coefficient was calculated using Spearman’s rho.
Significance was set at 95%. Data was analyzed by GraphPad Prism
Version 4.0.
3. Results and discussion
Cystic endometrial hyperplasia (CEH) - pyometra complex is a
continuum; however, the range of clinical signs, response to treatment
and prognosis depend on the severity of the lesions in the uterus. The
prototype work of Dow (1958) showed the histological correlates to
define CEH-pyometra continuum, which include endometrial hyper-
plasia with or without cystic changes, endometritis, and pyometra. In
the present study, the difference in the uterine length and diameter,
thickness of the endometrium and myometrium with their ratio is
presented in Table 2 and Fig. 3A-C. In HP group, the horns were dis-
tended with little congestion of the perimetrium (Fig. 2A). Upon inci-
sion, endometrial thickening (Fig. 2B) with multiple translucent cysts
(Fig. 2C) were recorded. The congestion of the serosa was more fre-
quent in AP group apart from distension of the uterus (Fig. 2D). Upon
incision, copious purulent exudate was drained; the luminal surface

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L.K. Singh et al. Veterinary Immunology and Immunopathology 205 (2018) 65–71

Fig. 2. Representative images of gross pathological changes in the uterus of bitches with hyperplastic (upper panel) and atrophic pyometra (lower panel).
Upper panel: A. Distended uterine horns with little congestion of the serosal surface. B. Pus with extensive thickening of the endometrium (Thin black arrow), C.
Presence of multiple cysts in the endometrium (black dashed arrow).
Lower panel: D. Distended uterine horns with severe congestion and haemorrhage in the perimetrium (Black diamond arrow). E. Extensive haemorrhage, ulceration
and purulent exudate in the endometrial lumen (white arrow). F. Note the multiple ulcers in the endometrial surface (white arrow).

showed ulceration, extensive haemorrhage (Fig. 2E and F) and highly
corrugated mucosa (Fig. 2E). The average horn diameter in the AP
group was significantly higher (P < 0.01) than those of the HP and
non-pyometra. However, the endometrial thickness and the ratio of E :
M thickness showed three- fold increase in the HP as compared to the
non-pyometra control. In contrast, an increase in the thickness of
myometrium was a consistent feature of AP due to distinct hypertrophy
of muscle fibre. An E : M ratio of 0.79 ± 0.11 in the non-pyometra
group is comparable with the reported value of 0.78 in the diestrus
bitch (De Bosschere et al., 2001). The histological changes in HP, AP
and non-pyometra control are presented in Fig. 4A-H. In HP, the mu-
cosa was thrown into finger-like folds into the lumen (Fig. 4A) and
excessive proliferation of the hyperplastic endometrial glands gave the
typical labyrinthine appearance (Fig. 4B-C). Further, periglandular in-
filtration by mononuclear cells was a characteristic feature (Fig. 4D) in
most of the cases in HP, which is in accordance with the study of De
Coster et al. (1979).
Histologically, the uterus of AP group showed severe thinning of the
endometrial mucosa with concurrent hypertrophy of the myometrium
(Fig. 4E and 4 G). Endometrial glands were lined by thin cuboidal
epithelia and the luminal debris contained desquamated and in-
flammatory cells with exudate (Fig. 4E). In a few cases, haemorrhagic
endometritis was observed with severe atrophy and necrosis of the
endometrium (Fig. 4F). Multiple foci of suppurative lesions scattered

Fig. 3. Representative photomicrographs showing the thickness of the endometrium and myometrium in the bitch (H & E).
A. Note that the myometrium (Double headed yellow arrow) thickness (880.33 μm) is higher than that of endometrium (762.76 μm; Double headed green arrow) in a
normal diestrus bitch; B. Hyperplastic endometrium (Double headed green arrow) in a HP bitch. C. Atrophic endometrium with concurrent hypertrophy of the
myometrium (Double headed yellow arrow) in an AP bitch.

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L.K. Singh et al. Veterinary Immunology and Immunopathology 205 (2018) 65–71

Table 1
Primers used for amplification of endometrial transcripts in the bitches with pyometra.
S. N. Target genes Primer sequence (5’ > > > > > 3’) Product size (bp) Accession No. Annealing temp. (°C)

1 β- actin F: CCA TCT TGC GTC TGG ACC T 162 NM_001003200.1 53.0
R: GCC ATC TCC TGC TCG AAG T
2 TLR-4 F: GAG AAA CTG GAC CTG AGC TTTA 142 NM_001002950.2 54.7
R: GGT GGT TTA GGC CCT GAT ATG
3 PGFS F: GGA CAT CAT CCT GAC TGC ATA C 101 NM_001012344 55.2
R: CAT TGA GAAC TGG GTC CTT CAG
4 COX-2 F: ACA GGA GAG AAG GAA ATG GC 250 NM_001003354.1 52.5
R: GGA TTG AGG CAG TGT TGA TG
5 IL-6 F: AAC CTA CAT CTT CCC AAA CTG G 110 NM_001003301.1 54.8
R: TGT AGC TGA AAC TCC ACA AGA C
6 IL-8 F: ACA CTC CAC ACC TTT CCA TC 120 NM_001003200.1 54.2
R: TCC AGG CAC ACC TCA TTT C

deep in the myometrium indicating the spread of the infection through
the blood and/or lymphatics (Fig. 4H). The histological observations in
the HP and AP group concurred with the earlier reports (De Bosschere
et al., 2001 and De Cock et al., 2002). The persistence of the infectious
stimuli for a longer time facilitates the invasion of the lesion into deeper
layers of the uterus, and with time, the inflammatory pattern changes to
fibrosis and calcification (Kochhar et al., 1996; Sadashiv, 1996). Pre-
sence of anechoic to echogenic particles in the uterine lumen with
thickening of endometrium in the HP group (Fig. 1A and C) and
thickened uterine wall with accumulation of anechoic to hypoechoic
fluid (Fig. 1B and D) in the ultrasonography is consistent with earlier
report (Bigliardi et al., 2004). In present study, E. coli was isolated from
50% cases followed by Enterobacter spp. (33.33%), Staphylococcus
aureus (16.67%) and Proteus spp. (16.67%). However, no anerobic
bacteria could be isolated. The result is in accordance with previous
report (Henriques et al., 2016). Taken together, the sonographic ob-
servation, gross and histopathological findings formed the basis for
categorization of pyometra to study the differential expression of in-
flammatory transcripts of endometrium origin.
3.1. Differential expression of inflammatory cytokines in the endometrium
The relative fold change of TLR4, IL-6, IL-8, COX-2 and PGFS
transcripts in the endometrial tissues of HP and AP as compared to the
non-pyometra as calibrator is presented in Table 3 and the dispersion of
ΔCt is indicated in the form of whisker plot (Suppl. Fig. 3 A–E). The
expression of β-actin as endogenous control gene in the endometrium
was comparable among the groups with the mean Ct value of
24.85 ± 1.05, 24.80 ± 0.88 and 24.59 ± 1.41 (P = 0.985), in the
HP, AP and non-pyometra, respectively. The mean difference in the fold
change of endometrial TLR4 between HP and AP was 2.17 fold
(P = 0.285). However, it is reported that the endometrial TLR4 tran-
script was upregulated by 2.4 fold in the bitches with pyometra as
compared to diestrus control (Silva et al., 2010).
Pro-inflammatory cytokine genes such as IL-6 and IL-8 were up-
regulated in HP as well as AP pyometra relative to the non-pyometra
control (Table 3). The result is supported by the studies of Hagman
et al. (2009), who reported upregulation of IL-6 and IL-8 transcripts in
the endometrium of bitches with pyometra. The relative fold change of
IL-6 was 2.39 times higher in the AP than that of HP (P < 0.01). The
upregulation of IL-6 in the AP as compared to HP is supported by a
recent review in which it is reported that it upregulates monocyte-at-
tracting chemokines and causes a switch from acute to chronic in-
flammation (Ataie-Kachoie et al., 2014). An increase in the serum acute
phase proteins such as C-reactive protein (Fransson et al., 2004), serum
amyloid A (Jitpean et al., 2014) and haptoglobin in the bitches with
pyometra Dąbrowski et al. (2009) might be, in part, due to increase in
the IL-6. An increase of 1.82 times in the relative fold change of IL-8
was seen in the AP group as compared to HP (P < 0.01, Table 3). The
IL-8 is produced by many cell types, including mononuclear phago-
cytes, antigen–activated T cells, endothelial and epithelial cells, and
even neutrophils (Miller and Krangel, 1992). The main inflammatory
impact of IL-8 lies in its chemotactic effects on neutrophils and its
ability to stimulate granulocyte activity by up-regulating cell surface
adhesion molecule expressions, thereby mediates recruitment and ac-
tivation of neutrophils in the inflammed tissue (Warren, 1990; Huber
et al., 1991).
The expression of COX-2 as well as PGFS was significantly upre-
gulated in AP by 3.75 and 3.15 times, respectively as compared to HP
(P < 0.01,Table 3). The LPS of E. coli strongly stimulates the PG
synthesis by up-regulating COX-2, PGES and PGFS genes in the en-
dometrium (Silva et al., 2009). This observation does not contradict the
non-modulation of TLR4 in AP group as IL-6 and IL-8 can exacerbate
the inflammation by upregulating COX-2 (Karlsson et al., 2012). This is
supported by the strong positive correlation of IL-8 with COX-2
(P < 0.05) and PGFS (P < 0.01, Table 4). Our finding is in accordance
with the previous reports, where bitches with pyometra had sig-
nificantly higher endometrial PGFS gene transcript (Silva et al., 2010).
In fact, elevated plasma PGFM is shown to have diagnostic and prog-
nostic utility in the canine pyometra (Hagman et al., 2006 a, b; Karlsson
et al., 2013). In another study, marked upregulation of endometrial
COX-2 and PGFS transcripts were reported in the pyometra as com-
pared to healthy diestrus or anestrus uterus (Silva et al., 2009). How-
ever, no past reports are in place to correlate our finding on PGFS and

Table 2
Uterine morphometry, endometrium to myometrium ratio in the bitches with and without pyometra.
Type of pyometra Length of uterine horn Uterine diameter (cm) Endometrium thickness (μm) Myometrium thickness (μm) Ratio of endometrium to
(cm) myometrium

Hyperplastic (HP, 12.22 ± 1.16 5.01 ± 0.82a 3187.48 ± 496.82 a

1558.49 ± 193.43a 2.53 ± 0.65a
n = 8)
Atrophic (AP, n = 8) 14.80 ± 1.24 20.68 ± 4.27b 1434.37 ± 219.86b 2785.83 ± 392.68b 0.56 ± 0.05b
Control (n = 6) 11.42 ± 1.04 2.59 ± 0.28c 1160.14 ± 225.08b 1450.22 ± 195.63a 0.79 ± 0.11c
P value 0.189 0.001 0.001 0.008 0.0001

Data was analysed with one-way Anova with Tukey’s as post-hoc test. Levene’s test revealed equality of variance (P > 0.05). Values (mean ± SEM) with different
superscripts (a, b and c) within a column indicate significant difference (P < 0.05).

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L.K. Singh et al. Veterinary Immunology and Immunopathology 205 (2018) 65–71

Fig. 4. Representative photo-

micrographs of the uterus from the
bitches with hyperplastic (A through
D) or atrophic (E through H) pyo-
metra.
A. The endometrium is thrown into
folds (black arrow) with vacuolatory
changes (black dashed arrow) of the
epithelia. B. Severe cystic dilation of
the glandular endometrium with in-
folding of the lining cells indicating
the initiation of labyrinthine change
(black arrow). Note the accumulation
of basophilic material composed of
secretions and cell debris. C. Typical
labyrinthine appearance of the en-
dometrial glands (black arrow head)
due to extensive infolding of the epi-
thelia. D. Pseudostratification of the
endometrial glands by cuboidal to co-
lumnar epithelium (black arrow) with
periglandular infiltration by mono-
nuclear leukocytes (black dashed
arrow). E. Apparent thinning of the
endometrium with desquamation of
the lining epithelia (black arrow). F.
Haemorrhagic endometrium with dis-
ruption of the lining (black arrow) and
atrophic endometrial glands (black
dashed arrow). G. Hypertrophy of the
myometrial layer (black arrow). H.
Multiple foci of suppurative lesions
scattered deep in the myometrium
(black arrow) suggesting the spread of
the infection through the blood and/or
lymphatics.

COX-2 gene expression in the endometrium in relation to the histo-
pathological changes. Thus, excess and unabated production of in-
flammatory mediators with progression of the disease culminates in
SIRS, followed by multiorgan dysfunction syndrome and death
(Fransson et al., 1997; Hagman et al., 2006b). Exaggerated histological
lesions in the AP group support the upregulation of all the inflammatory
transcripts studied except TLR4. The results of the study have diag-
nostic potential once transcervical endometrial biopsy is optimized in
the bitch (Hagman, 2016). In conclusion, histological grading of pyo-
metra into HP and AP revealed differential regulation of inflammatory
cytokines and enzymes in the PG synthetic pathway in the canine en-
dometrium that has diagnostic potential under clinical settings.

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L.K. Singh et al. Veterinary Immunology and Immunopathology 205 (2018) 65–71

Table 3 Ann. Med. Vet. 123, 233–247.

Fold change (2-ΔΔCt) of different inflammatory gene transcripts in the en- Dow, C., 1958. The cystic endometrial hyperplasia - pyometra complex in the bitch. Vet.
dometrium of bitches with hyperplastic (HP) and atrophic pyometra (AP)a. Rec. 70, 1102–1108.
Egenvall, A., Bonnett, B.N., Olson, P., Hedhammar, A., 2000. Gender, age and breed
Proinflammatory gene HP AP P value pattern of diagnoses for veterinary care in insured dogs in Sweden during 1996. Vet.
Rec. 146, 551–557.
TLR4 2.68 ± 0.507 0.51 ± 1.84 0.285 Fransson, B.A., Karlstam, E., Bergström, A., Park, J.S., Evans, M.A., Ragle, C.A., 2004. C
IL-6 16.85 ± 0.331 40.36 ± 1.685 0.002 -reactive protein in the differentiation of pyometra from cystic endometrial hyper-
plasia/mucometra in dogs. J. Am. Anim. Hosp. Assoc. 40, 391–399.
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a
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TLR4 0.071 0.333 0.238 0.619 cometra in bitches by prostaglandin F2α metabolite analysis. Theriogenology 66,
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IL-6 0.262 0.262 0.167
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IL-8 0.833* 0.905**
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COX-2 0.738* 55–68.
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Acknowledgement Henriques, S., Silva, E., Silva, F.M., Carvalho, S., Diniz, P., Lopes-da-Costa, L., Mateus, L.,
2016. Immunomodulation in the canine endometrium by uteropathogenic
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Referral Veterinary Polyclinic, ICAR-IVRI for facilitating the work. Determinative Bacteriology, 9th ed. Lippincott, Williams & Wilkins, Baltimore,
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Conflict of Interest Huber, A.R., Kunkel, S.L., Todd, R.F., Weiss, S.J., 1991. Regulation of trans- endothelial
neutrophil migration by endogenous interleukin-8. Science 254, 99–102.
Jitpean, S., Pettersson, A., Hoglund, O.V., Holst, B.S., Olsson, U., Hagman, R., 2014.
The authors declare that there is no conflict of interest that could be Increased concentrations of serum amyliod A in dogs with sepsis caused by pyometra.
perceived as prejudicing the impartiality of the research reported. BMC Vet. Res. 10, 273.
Karlsson, I., Hagman, R., Johannisson, A., Wang, L., Karlstam, E., Wernersson, S., 2012.
Cytokines as immunological markers for systemic inflammation in dogs with pyo-
Appendix A. Supplementary data metra. Reprod. Domest. Anim. 47, 337–341.
Karlsson, I., Wernersson, S., Ambrosen, A., Kindahl, H., Sodersten, F., Wang, L., Hagman,
R., 2013. Increased concentrations of C-reactive protein but not high-mobility group
Supplementary material related to this article can be found, in the
box 1 in dogs with naturally occurring sepsis. Vet. Immunol. Immunopath. 156 (1-2),
online version, at doi:https://doi.org/10.1016/j.vetimm.2018.10.010. 64–72.
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