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mucometra in bitches by prostaglandin F2α metabolite analysis
Theriogenology 66 (2006) 198–206 www.journals.elsevierhealth.com/periodicals/the
Differentiation between pyometra and cystic endometrial hyperplasia/mucometra in bitches by prostaglandin F2a metabolite analysis R. Hagman a,*, H. Kindahl b, B.A. Fransson c, A. Bergstro¨m a, B. Stro¨m Holst d, A.-S. Lagerstedt a a Department of Small Animal Clinical Sciences, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 7037, SE-75007 Uppsala, Sweden b Department of Clinical Sciences, Division of Comparative Reproduction, Obstetrics and Udder Health, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 7037, SE-75007 Uppsala, Sweden c Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164-7060, USA d Department of Small Animals, National Veterinary Institute, SE-75189 Uppsala, Sweden
Received 1 October 2004; received in revised form 2 November 2005; accepted 4 November 2005
Abstract Bitches with pyometra are potential emergency cases which may be clinically difficult to differentiate from cases of cystic endometrial hyperplasia (CEH) in combination with mucometra. In the present study plasma prostaglandin F2a, as measured by its main metabolite 15-keto-13,14-dihydro-PGF2a (PG-metabolite) concentrations, blood biochemical and hematological parameters were measured in 59 bitches with pyometra, 10 bitches with CEH and nine controls to determine if PG-metabolite could differentiate between the three uterine conditions. Bitches with pyometra had significantly higher plasma levels of PG-metabolite than bitches with CEH (P = 0.002) and the controls (P = 0.002). PG-metabolite analysis alone had a high sensitivity (98.3%) and a high specificity (80.0%) for the differentiation of pyometra versus CEH in bitches where fluid in the uterus was diagnosed. When a combination of PG-metabolite and percentage band neutrophils (PBN) was used for differentiation of the two diagnoses, a sensitivity of 100% and specificity of 90.0% was obtained. This means that the combination of PG-metabolite and PBN analysis allows for differentiation between cases of pyometra and CEH. If the PG-metabolite level in a bitch is 4524 pmol l 1, there is a 99% probability of the diagnosis pyometra versus CEH. Levels of PG-metabolite 3054 pmol l 1, 2388 pmol l 1 or 1666 pmol l 1 indicates a 95%, 90% or 80% probability of pyometra, respectively. At high PG-metabolite levels (above about 3000 pmol l 1), PG-metabolite alone is enough for differentiation of pyometra versus CEH. The results of the present study showed that PG-metabolite analysis is valuable in the diagnosis and prediction of severity of uterine diseases. # 2005 Elsevier Inc. All rights reserved. Keywords: Dog; Endometritis; Prostaglandins; Uterine inflammation; SIRS
1. Introduction
* Corresponding author. Tel.: +46 18 672918; fax: +46 18 673534. E-mail address: [email protected] (R. Hagman). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.11.002
The diagnosis of pyometra (chronic purulent endometritis post-oestrum) in bitches can be clinically difficult to separate from bitches with cystic endometrial hyperplasia (CEH) in combination with sterile
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seromucous fluid in the uterine lumen (hydrometra or mucometra depending on the water content of the fluid) [1]. Generally pyometra is associated with more and severe systemic signs of illness due to the bacterial infection and following immune response while often the only sign of CEH (defined as proliferation and cystic hyperplasia of endometrial cysts) is failure to conceive [2]. It is however important to be able to differentiate between the two diseases since bitches with pyometra are at risk of a suddenly developing endotoxic shock which may be fatal while bitches with non-infected uteri (CEH/mucometra) not are regarded as potential emergencies [3]. Prostaglandins are derived from arachidonic acid and have many both physiological and pharmacological roles and as important mediators in inflammatory events [4]. The uterine endometrium is known to synthesise and release prostaglandins and mainly prostaglandin F2a (PGF2a) [5]. The systemic release of PGF2a, can be followed by measurement of its more stable main circulating metabolite 15-keto-13,14-dihydro-PGF2a (PG-metabolite) [6]. In dogs the concentrations of PGF2a have been shown to be physiologically elevated during parturition and the post-partum period [7–10]. Increased plasma levels of PG-metabolite have also been demonstrated in pathological inflammatory conditions of the uterus in several species, including canines [5,11–14]. In bitches with pyometra, infection with predominantly Gram-negative bacteria such as Escherichia coli is generally present with inflammation and accumulation of pus in the uterus [15,16]. Gram-negative bacteria release cell wall compounds, endotoxins, into the circulation during vigorous growth or cell disintegration. The endotoxins are biologically active by initiating the release of many inflammatory mediators (cytokine cascade) and are thought to be responsible for many systemic symptoms of bitches with pyometra [17–21]. Endotoxins are very potent compounds also in stimulating prostaglandin release and PGF2a has been demonstrated to be a reliable and sensitive marker of experimentally induced endotoxemia in cattle, goats, pigs and horses [4,21–25]. Endotoxemia has been demonstrated in two out of four studies of endotoxin involvement in bitches with pyometra [15,26–28]. In one of these studies, the plasma endotoxin concentrations were related to outcome (survival or death) due to the disease [28]. Cystic endometrial hyperplasia (CEH) develops as a consequence of an abnormal response of the uterus to repeated progestational stimulation during the luteal phase of the estrous cycle [29]. CEH is considered to be
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the initial phase of a combined CEH–pyometra complex and it is generally believed that CEH predisposes for a bitch for pyometra [29,30]. Both CEH and pyometra can possibly develop independently of each other [1]. De Bosschere and others suggested that CEH and pyometra/endometritis should be divided into two separate entities due to their clinical and histopathological differences [1]. This separation is sometimes difficult since the severity of clinical symptoms does not always correlate with the severity of the uterine pathology [1]. Clinically it would be helpful to accurately predict the diagnosis before surgery. The risk of increased morbidity associated with delaying surgery in bitches with pyometra (potential uterine rupture, peritonitis and sepsis) could then be minimised and facilitate the decision of optimal therapeutic regime in both CEH and pyometra cases. In cattle with endometritis, the possible application of PG-metabolite measurements to predict severity of the uterine inflammation and survival has been shown [13]. It seems likely that analysis of PG-metabolite could facilitate the differentiation between cases without inflammation (CEH/mucometra) and cases of pyometra/endometritis in canines as well, and possibly also predict severity of the uterine disease. Pyometra has been associated with the systemic inflammatory response syndrome (SIRS) and the presence of SIRS has previously been linked with lower survival rates and longer hospitalisation [2,31,32]. In SIRS the initial local inflammatory reaction shifts through the cascade release of the endogenous inflammatory mediators to an uncontrollable systemic reaction where mediators directly stimulate other mediators and continue without the presence of the initiating agent [31]. Various incidents such as severe trauma, infection or endotoxin can initiate the inflammatory process which can be present with bacteremia (sepsis) or without. Diagnostic criteria for dogs with the highest sensitivity in the identification of systemic inflammatory response syndrome (SIRS) have been determined by Hauptman et al. [33]. Assessment of SIRS is important in order to determine severity of the disease, optimise treatments and prevent fatal outcomes, especially in patients at a high risk of developing shock or multiple organ dysfunctions [31,34]. The objectives of this study were (i) to compare PGmetabolite levels in healthy bitches with bitches suffering from pyometra or CEH and to (ii) evaluate if the PG-metabolite analysis can differentiate between pyometra and CEH and further to (iii) determine the predictive value of PG-metabolite levels for severity of clinical status, and to determine outcome.
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2. Materials and methods 2.1. Animals 2.1.1. Pyometra and cystic endometrial hyperplasia (CEH)/mucometra group The study was approved by the Uppsala County Ethical Board, Tierp, Sweden prior to onset of the clinical investigations. Sixty-nine privately owned bitches with the presumptive diagnosis of pyometra or CEH were included in the present study. All bitches were treated by ovariohysterectomy at the Department of Small Animal Clinical Sciences, Swedish University of Agricultural Studies, Uppsala, Sweden. The diagnosis was based on case history, physical examination and diagnostic imaging using ultrasonography and/or radiology to demonstrate an enlarged, fluid-filled uterus. The diagnosis of pyometra or CEH (in ten of these cases, whereof mucometra in four), was verified visually during the ovariohysterectomy and confirmed by histopathological examination. The admitting clinician filled out a form specifying rectal temperature, heart rate, respiratory rate, mucus membrane colour, capillary refill time, location for pain response at abdominal palpation, hydration status and general attitude at the time of admission. None of the pyometra bitches had previously been medically treated for pyometra. After surgery the rectal temperature, heart rate and respiratory rate was noted daily for as long as the patient was admitted on a special form from which also the length of hospitalisation was determined. Additional information on treatment, complications to treatment and mortality were obtained from the medical records. 2.1.2. Control group Healthy adult intact bitches (n = 9) were used as controls. A history questionnaire was filled out, ensuring that the owner considered the bitch healthy for at least 8 weeks prior to the examination. Physical examination was performed and documented by two of the authors. None of the control bitches had previously been medically treated for pyometra. 2.2. Blood collection and analyses 2.2.1. Pyometra and cystic endometrial hyperplasia (CEH/mucometra) group Blood samples for biochemical, hematological and PG-metabolite analysis were obtained immediately before surgery in EDTA, sodium-heparinised and nonadditive vacutainer tubes (Becton-Dickinson, Stock-
holm, Sweden). Biochemical and hematological analyses were performed using routine methods, at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. The following hematology parameters were analysed: packed cell volume (PCV), hemoglobin (Hb), erythrocyte volume fraction (EVF), white blood cell count (WBC), differential count of WBC including total count (BN) and percentage band neutrophils (PBN), nucleated erythrocytes, erythrocyte morphology, red blood cell mean corpuscular volume (MCV), red blood cell mean corpuscular hemoglobin concentration (MCHC) and platelet count (TPK). The following serum biochemical parameters were determined: alanine aminotransferase (ALAT), albumin, alkaline phosphatase (AP), blood urea nitrogen (BUN), cholesterol, glucose, total protein and the electrolytes sodium (Na), potassium (K), chloride (Cl) and calcium (Ca). For analysis of PGmetabolite, sodium heparinised plasma was stored at 70 8C until assayed at the Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden. A radioimmuno assay (RIA) was used to analyse 15-ketodihydro-PGF2a in duplicates [5]. The practical detection limit of the assay was 200 pmol l 1. Where necessary, dilution of the samples was performed to allow for accurate readings on the standard curve. 2.2.2. Control group Blood samples for the control bitches were collected through the same procedure as for the diseased group after the physical examination. 2.3. Histopathological examinations The diagnosis of CEH and pyometra according to the previously proposed definition by Dow was confirmed by gross and histopathological examination of hematoxylin–eosin stained sections of formaldehyde-fixated uteri and ovaries at the Department of Pathology, National Veterinary Institute (SVA), Uppsala, Sweden [29,34]. The definition of pyometra was chronic purulent metritis, acute purulent metritis or purulent endometritis [34,35]. The histopathological diagnosis of CEH was defined by hyperplasia of the endometrium in combination with endometrial cysts without any inflammatory changes [34]. The criteria for the diagnosis mucometra were presence of hyperplastic and cystic or atrophic endometrium in combination with mucus or serous fluid present in the uterine lumen on gross or microscopical examination [34].
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2.4. Determination of a systemic inflammatory response A patient was regarded as SIRS-positive if two or more of the following criteria are met: respiratory rate > 20 min 1; heart rate > 120 beats min 1; WBC < 6 or >16 (109 l 1) or percentage band neutrophils (PBN) > 3%; temperature < 38.1 or >39.2 [29]. 2.5. Statistical analysis Statistical analyses were performed with the programme Statistica (Version 6.0, StatSoft Inc., Tulsa, USA). A one-way ANOVA (Tukey, post hoc) was used to test for differences in PG-metabolite levels by patient group (control, pyometra and CEH/mucometra). Fisher’s exact test, Spearman’s rank correlation coefficient (rS) and Pearson’s product moment correlation coefficient (rP) were used to test for associations between nominal, ordinal and interval-scale variables, respectively. Binary logistic regression analyses were used to evaluate blood parameters in relation to controls and bitches with pyometra or CEH/mucometra by histopathological examinations. The regression models were tested for sensitivity and specificity. Values below detection limit, which in the data set occurred for PGmetabolite, BN, segmented neutrophils, eosinophils, and basophils were set to zero in the calculations. Twosample t-tests were used to test for differences in PGmetabolite levels between bitches with SIRS or without SIRS, as tested for pyometras and CEH/mucometras, pyometra group and CEH/mucometra group, respectively. Significance was accepted at P < 0.05 for all statistical tests used in this study. Equations used to create Figs. 3–5 were as follows: equation used for calculation of the estimated probability of PG-metabolite in combination with PBN for determination of the diagnosis pyometra versus CEH/ mucometra: estimated probability of pyometra = exp ( 1.1966 + (0.000999) PG-metabolite + (0.200471) PBN)/(1 + exp( 1.1966 + (0.000999) PG-metabolite + (0.200471) PBN)). P 0.0001. Equation used for calculation of the estimated probability of PG-metabolite in combination with WBC for determination of the diagnosis pyometra versus CEH/mucometra: estimated probability of pyometra = exp( 2.0962 + (0.001152) PG-metabolite + (0.118737) WBC)/ (1 + exp( 2.0962 + (0.001152) PG-metabolite + (0.118737) WBC)). P 0.0001. Equation used for calculation of the estimated probability of PGmetabolite for determination of the diagnosis pyometra
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versus CEH/mucometra: estimated probability of pyometra = exp( 0.4849 + (0.001123) PG-metabolite/(1 + exp( 0.4849 + (0.001123) PG-metabolite)) (figure not shown), P 0.0001. 3. Results Of the 69 bitches included in disease groups of the present study 59 had the diagnose pyometra and 10 cases CEH/mucometra, confirmed by histopathology. The pyometra patients, CEH/mucometra group and control group consisted of 35, 9 and 6 different breeds, respectively. Clinical findings, blood biochemical and hematological parameters in all three groups are presented in Table 1. Mean values for the controls and the CEH/mucometra and pyometra groups were: MCV (68, 69 and 67 [fl]), MCHC (352, 353 and 355 [g l 1]), Na (155, 153 and 155 [mmol l 1]), K (4.6, 4.4 and 4.4 [mmol l 1]), Cl (122, 124, 123 [mmol l 1]) and Ca (2.6, 2.7, 2.6 [mmol l 1]), respectively. The presence of abnormalities in case history and physical examination and administration of antimicrobial treatment in controls and bitches diagnosed with pyometra or CEH/mucometra are presented in Table 2. Fisher’s exact test was used for the calculations of Pvalue of PG-metabolite levels, age, duration of clinical signs and blood hematological and chemistry parameters (Table 2). The plasma levels of PG-metabolite were significantly increased in bitches with pyometra compared with bitches with CEH/mucometra (P = 0.003) and the controls (P = 0.003) (Fig. 1). The number of bitches in the different patient groups and controls, divided into five different categories of PG-metabolite-levels, are illustrated in Fig. 2. Regarding other parameters, in this study significant differences between bitches with pyometra and CEH/mucometra were demonstrated in WBC (P = 0.001), BN (P = 0.015), PBN (P = 0.001), segmented neutrophils (P = 0.006), monocytes (P = 0.010), cholesterol (P = 0.009) and albumin (P = 0.026). The levels of PG-metabolite were not significantly higher in bitches with CEH/mucometra compared with the controls. When comparing bitches with CEH/mucometra and controls there were significant differences in levels of albumin (P = 0.037), lymphocytes (P = 0.001), monocytes (P = 0.003). PG-metabolite levels were significantly correlated to WBC (rp = 0.351), BN (rp = 0.403), PBN (rp = 0.434), segmented neutrophils (rp = 0.303), lymphocytes (rp = 0.235), monocytes (rp = 0.287), creatinine (rp = 0.306), ALAT (rp = 0.390), glucose (rp = 0.277) and albumin (rp = 0.537). PG-metabolite levels were
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Table 1 Mean, standard deviation and range values for selected hematological and serum biochemistry parameters in different patient groups Control
Age (years) Temperature (8C) Duration of clinical signs (days) PG-metabolite (pmol l 1) Hemoglobin (g l 1) EVF EPC (1012 l 1) WBC (109 l 1) BN (109 l 1) PBN (%) Neutrophils (109 l 1) Eosinophils (109 l 1) Basophils (109 l 1) Lymphocytes (109 l 1) Monocytes (109 l 1) Platelet count (109 l 1) Creatinine (mmol l 1) ALAT (mkat l 1) AP (mkat l 1) BUN (mmol l 1) Glucose (mmol l 1) Protein (g l 1) Albumin (g l 1) Cholesterol (mmol l 1)
CEH/mucometra
Pyometra
Mean S.D. (range)
n
Mean S.D. (range)
n
Mean S.D. (range)
n
6.7 1.7 (4.3–9.7) 38.5 0.4 (37.9–39.0) 0
9 9 9
8.8 3.1 (2.5–12.0) 38.9 0.7 (38.2–40.2) 3 3 (0–7)
10 7 8
9.9 2.4 (2.5–13.6) 39.1 0.6 (37.9–40.6) 7 6 (1–28)
58 56 48
86 338 (0.0–772) 174 20.3 (146–210) 0.49 0.05 (0.41–0.58) 7.38 0.62 (6.70–8.40) 10.1 3.0 (7.0–16.2) 0.01 0.03 (0.00–0.10) 0.11 0.32 (0.00–0.96) 6.1 1.9 (4.1–10.4) 0.8 0.8 (0.0–2.5) 0.01 0.03 (0.0–0.1) 2.6 1.0 (1.6–5.0) 0.5 0.3 (0.1–1.0) 345 101 (148–524) 83 21 (46–112) 0.6 0.3 (0.3–1.3) 1.7 0.7 (1.0–3.2) 7.2 4.0 (3.5–16.3) 5.0 0.4 (4.2–5.5) 76 7 (66–85) 35 3 (32–40) 6.4 1.7 (4.2–8.8)
9 9 9 6 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9
415 998 (0–3200) 137 19.8 (104–173) 0.39 0.06 (0.30–0.49) 5.52 0.91 (4.30–7.30) 9.4 5.2 (3.8–21.4) 0.19 0.43 (0.0–1.3) 1.48 3.81 (0.00–12.0) 7.1 4.6 (2.7–17.3) 0.3 0.3 (0.0–1.0) 0.0 0.0 1.0 0.4 (0.3–1.7) 0.8 0.5 (0.1–1.5) 252 110 (35–414) 79 31 (53–148) 0.6 0.4 (0.2–1.1) 2.4 2.4 (0.6–8.0) 4.6 1.8 (2.0–7.9) 5.4 0.4 (4.8–6.0) 76 7 (65–84) 30 3 (26–35) 7.2 2.8 (5.0–13.3
10 10 10 9 10 10 10 10 10 10 10 10 9 8 9 8 8 8 8 8 8
6278 5757 (0–33500) 136 24.4 (96–207) 0.38 0.07 (0.27–0.58) 5.96 1.76 (3.9–16.0) 22.8 11.4 (4.5–48.4) 2.86 2.96 (0.0–12.7) 11.9 9.97 (0.00–38.9) 15.3 8.1 (2.4–36.4) 0.3 0.6 (0.0–4.1) 0.02 0.08 (0.0–0.5) 1.5 0.9 (0.0–3.9) 2.82.3 (0.0–10.1) 233 118 (22–681) 81 42 (44–257) 0.6 0.8 (0.1–5.6) 6.0 6.0 (0.4–34) 5.1 4.0 (1.6–23.1) 5.0 1.2 (2.4–8.0) 80 13 (43–139) 27 5 (18–37) 9.8 2.5 (4.7–14.1)
58 57 57 51 56 56 56 56 56 56 56 56 55 53 57 57 48 47 49 49 48
Temperature: body temperature; duration: duration of clinical signs; PG-metabolite: prostaglandin F2a metabolite; Hb: hemoglobin; EVF: red blood cell volume fraction; EPC: red blood cell particle concentration; WBC: white blood cell count; BN: band neutrophils; neutrophils: segmented neutrophils; eosinophils: segmented eosinophils; basophils: segmented basophils; ALAT: alanine amino transferase; AP: alkaline phosphatase; BUN: blood urea nitrogen.
Table 2 The presence of selected case history and physical examination findings in control bitches, bitches with cystic endometrial hyperplasia/mucometra (CEH/M) and bitches with pyometra Case history and physical examination findings Polydipsia and/or polyuria Gastrointestinal signs Lethargy Abdominal pain on palpation Purulent-like vaginal discharge Mucous membranes hyperemic, discoloured or pale Capillary refill time 3s Temperature > 39.2 8C Heart rate > 120 beats/min Respiratory rate > 20 breaths/min Attitude moderately depressed at admission Antimicrobial treatment Presence of two or more SIRS-criteria
Control, % (n = 9)
Pyometra and CEH, % (n = 69)
P-value
CEH/mucometra, % (n = 10)
Pyometra, % (n = 59)
P-value
0 0 0 0 0 0
(9) (9) (9) (9) (9) (9)
65 61 56 72 58 20
(66) (66) (66) (65) (66) (65)
<0.001 <0.001 <0.001 <0.001 <0.001 0.032
55 11 11 62 55 0
(9) (9) (9) (8) (9) (8)
68 70 64 75 57 23
(56) (56) (56) (56) (56) (56)
0.47 0.001 0.004 0.43 1.0 0.10
0 0 0 0 0
(9) (9) (9) (9) (9)
9 30 26 49 78
(66) (64) (61) (43) (65)
0.33 0.13 0.76 0.19 <0.001
0 14 0 83 33
(9) (7) (7) (6) (9)
11 30 28 44 85
(56) (56) (53) (36) (55)
0.58 0.66 0.17 0.18 0.002
29 (62) 49 (68)
0.004 0.17
0 (9) 22 (9)
33 (9) 20 (10)
27 (52) 53 (58)
0.70 0.08
Fisher’s exact test was used for the calculations of P-value for the level of statistical significance of the differences between the groups.
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within the pyometra group (P = 0.045) but not significant within the CEH/mucometra group (twosample t-tests) (Fig. 5). 4. Discussion 4.1. Differentiation of pyometra versus CEH/ mucometra
Fig. 1. Prostaglandin F2a metabolite (PG-metabolite) levels in controls and bitches with cystic endometrial hyperplasia/mucometra (CEH) or pyometra. Bars indicate 1 standard error of the mean (S.E.) (Anova and Tukey post hoc test).
also significantly correlated to the age of the patient (rp = 0.264), length of the hospitalisation (rp = 0.314), body temperature (rp = 0.313) and heart rate (rp = 0.317). The number and percentage of bitches with two or more criteria of SIRS in each respective group (control, CEH/ mucometra, pyometra) are demonstrated in Table 2. When PG-metabolite levels in bitches with and without SIRS were compared, the differences were significant in the group consisting of both pyometra and CEH/mucometra cases (P = 0.01), just significant
Fig. 2. Percentage of the controls, cystic endometrial hyperplasia/ mucometra (CEH) and pyometra bitches as divided into five groups according to their prostaglandin F2a metabolite (PG-metabolite) plasma levels.
This is the first study of PG-metabolite analysis in the differentiation between pyometra and CEH/mucometra in bitches. Pyometra is in general considered a surgical emergency, unlike CEH/mucometra, since the risk of uterine rupture, endotoxemia and following inflammatory reactions may transform a clinically stable pyometra to an emergency case in a matter of hours [2,30]. PG-metabolite analysis alone is the single parameter with highest sensitivity (98.3%) and specificity (80.0%), in the present study, for the differentiation of pyometra versus CEH/mucometra in bitches here a fluid-filled uterus is diagnosed. This sensitivity is higher than what has been reported for a single parameter previously in this differentiation [34]. To obtain the highest sensitivity for differentiation between pyometra and CEH/mucometra in this study the analysis of PG-metabolite was combined with analysis of PBN which yielded a sensitivity of 100.0% and a specificity of 90.0%. Compared with analysis of PGmetabolite alone, the combination of PG-metabolite and PBN had both higher sensitivity and specificity. In clinical work the preference of a test is to have high sensitivity (ability to correctly diagnose a pyometra) since the surgery on a ‘‘false positive pyometra’’ (CEH/ mucometra) patient has no negative consequences for the patient (except for breeding bitches) compared with a test with high specificity which in this case could mean that a pyometra could be falsely diagnosed as CEH/mucometra and perhaps not treated as an emergency. The combination of PG-metabolite with any other blood parameter than PBN led to decreased sensitivity and specificity compared with PG-metabolite alone (albumin 87.5; BN 80.0%; monocytes 80.0%) (Fig. 3). If the PG-metabolite level in a bitch is 4524 pmol l 1, there is a 99% probability of the diagnosis pyometra versus CEH/mucometra. PG-metabolite levels of 3054 pmol l 1, 2388 pmol l 1 or 1666 pmol l 1 indicates a 95%, 90% or 80% probability of pyometra, respectively. The analysis of PG-metabolite concentrations therefore has high diagnostic value. Routine blood parameters such as WBC, BN, segmented neutrophils, monocytes, albumin, and AP have
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Fig. 3. Contour plot of estimated 80, 90, 95, and 99% probability of the diagnose pyometra vs. cystic endometrial hyperplasia/mucometra (CEH) in a bitch, based on percentage band neutrophils (PBN) and plasma prostaglandin F2a metabolite (PG-metabolite) concentrations. Bisection of the values for PBN and PG-metabolite to the right of each respective graph indicates 80%, 90%, 95% or 99% probability of the diagnosis pyometra.
Fig. 4. Contour plot of estimated 80, 90, 95, and 99% probability of the diagnose pyometra vs. cystic endometrial hyperplasia/mucometra (CEH) in a bitch, based on white blood cell counts (WBC) and plasma prostaglandin F2a metabolite (PG-metabolite) concentrations. Bisection of the values for WBC and PG-metabolite to the right of each respective graph indicates 80%, 90%, 95% or 99% probability of the diagnosis pyometra.
sensitivities of 98.2, 94.6, 100, 98.2, 100, and 100%, respectively, but the diagnostic value in the differentiation between pyometra and CEH/mucometra is low because of their low specificity (20.0%) with the exception of BN (specificity 80.0). At high PGmetabolite levels (above about 3000 pmol l 1), PGmetabolite alone is enough for differentiation of pyometra versus CEH/mucometra, but at lower PGmetabolite levels (about 2000 pmol l 1) the combination of PG-metabolite with PBN, WBC or BN (if sufficiently high) will increase the probability of pyometra (Figs. 4 and 5). An advantage with the analysis of PG-metabolite alone or in combination with WBC in the differentiation of pyometra versus CEH/mucometra is that it allows for diagnosis without the differential count of WBC. An inexpensive test for rapid detection of PG-metabolite would be clinically very useful in the diagnosis of pyometra versus CEH/mucometra. Other clinical parameters useful to differentiate between pyometra and CEH/mucometra are the general attitude of the bitch at the time of admission, lethargy and gastrointestinal signs (Table 2). In the present study significant differences between pyometra and CEH/ mucometra were also demonstrated in WBC, BN, PBN, segmented neutrophils, lymphocytes, monocytes, cholesterol and albumin (data not shown). These parameters have previously been shown to be elevated (WBC, BN, segmented neutrophils, monocytes, cholesterol) or decreased (albumin) in bitches with pyometra reflecting the chronic bacterial infection, endotoxemia, following intrahepatic cholestasis and acute phase reaction [18,19].
4.2. PG-metabolite levels in pyometras versus controls PG-metabolite concentrations were significantly (P = 0.003) increased in bitches with pyometra compared with the controls (Fig. 1). These results are in accordance with the study by Vandeplassche et al. [12]. The synthesis of prostaglandins is stimulated by the bacterial infection and inflammation of the uterus as is observed both in canines and many other domestic
Fig. 5. Prostaglandin F2a metabolite (PG-metabolite) concentrations in controls and bitches with cystic endometrial hyperplasia/mucometra (CEH) or pyometra, with and without a systemic inflammatory response (SIRS). Bars indicate standard error of the mean (two-sample t-tests).
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animals [3,4,10,11,21,23]. The highest PG-metabolite value in the pyometra bitches was 33,500 pmol l 1. This can be compared with the maximum concentrations measured at parturition of 74,900 31,900 pmol l 1 [8,9]. The reference range for non-pregnant healthy bitches in all stages of the oestrous cycle is 0.05– 2.1 nmol l 1 and the levels are generally low during the first weeks of pregnancy [8,9,11]. The levels of PGmetabolite for the controls in this study were 0.29 0.05 nmol l 1 which is in the same order of magnitude as in the other studies [8,9,11]. The levels of PG-metabolite were not significantly higher in bitches with CEH/mucometra compared with the controls likely because of the absence of bacterial infection, endotoxemia and uterine inflammation according to histopathological examinations. 4.3. PG-metabolite correlations PG-metabolite levels were correlated to the following hematological and blood chemistry parameters: WBC, BN, PBN, segmented neutrophils, lymphocytes, monocytes, creatinine, ALAT, glucose, and albumin. PG-metabolite levels were also correlated to duration of clinical illness prior to admission, hospitalisation length and measurements of temperature, heart rate, and dehydration at the time of admission. It seems logical that the PG-metabolite concentrations are correlated to parameters normally responsive to endotoxemia since endotoxins are potent inducers of the release and synthesis of prostaglandins [17,22,23]. 4.4. Prognostic value of PG-metabolite analysis As for prognostic value, the PG-metabolite concentrations were correlated to morbidity as measured in this study by length of the hospitalisation stay. Bitches operated for pyometra are dismissed 1–2 days after surgery, with few exceptions. Additional complications with severely depressed general condition will result in longer hospitalisation stays. The pre-operative measurement of PG-metabolite can thus help the clinician in determining the severity of the pyometra roughly judged by how long time the patient will need to be hospitalised. To study PG-metabolite concentrations in relation to degree of uterine pathological changes would be a logical continuation of the present study. To evaluate any possible predictive values of PG-metabolite for survival of an increased patient number is needed since only two bitches died of causes related to pyometra in this study. Two other bitches died, 2 and 14 days after surgery, respectively, but it is uncertain
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whether the deaths were consequences of the pyometra since no autopsies were performed. The bitch with the highest observed level of PGmetabolite, 33,500 pmol l 1, died the day after surgery as a consequence of myocarditis and other sequela of the uterine bacterial infection. The other confirmed death was a bitch with a thin-walled uterus which ruptured during the operation and she died 1 h after the surgery was completed. That bitch had low (400 pmol l 1) levels of PG-metabolite before surgery and was perhaps an example of severely damaged endometrium which reduces the endogenous production of prostaglandins, as has been described in the mare [10]. The levels of PG-metabolite were correlated to rectal temperature, heart rate, WBC and PBN, four out of five clinical parameters used in determining whether systemic inflammatory response syndrome (SIRS) is present. The presence of SIRS has clinical importance since it is linked with higher mortality rates, in spite of the fact that mortality was very low in the present study [2]. PG-metabolite analysis could possibly be used as an additional SIRS indicator if a rapid test for veterinary clinics is made readily available in the future. Analysis of PG-metabolite can be useful in the clinical work where it sometimes can be hard to differentiate pyometra from CEH/mucometra when the general condition of the bitch is relatively unaffected. A sensitive, rapid and inexpensive PG-metabolite test for the prediction of pyometra would enable clinicians to decide whether it is possible to postpone surgery (CEH/ mucometra) until optimal resources are available or whether the bitch needs to be monitored as an emergency and ovariohysterectomised as soon as the clinical condition allows (pyometra). The results of this study indicate the possibility to use PG-metabolite analysis to diagnose endometrial pathologies, predict the severity of the pyometra (determined by prolonged hospitalisation stays and systemic inflammation) and differentiate between pyometra and CEH/mucometra. Acknowledgements We express our sincere gratitude to Pauli Snoeijs for help with the statistical analysis of the data, Lillemor Abersten and Anna Gerentz Bohlin for collection of the samples, Erika Karlstam for the histopathological examinations and Susanna Sternberg for comments on the manuscript. The present study was financially supported by Agria Insurance Co. Research Foundation and The Swedish Kennel Club Research Foundation.
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References [1] De Bosschere H, Ducatelle R, Vermeirsch H, Van Den Broeck W, Coryn M. Cystic endometrial hyperplasia–pyometra complex in the bitch: should the two entities be disconnected. Theriogenology 2001;55:1509–19. [2] Dow C. The cystic endometrial hyperplasia–pyomtra complex in the bitch. J Comp Path 1959;69:237–50. [3] Hardie EM. Life-threatening bacterial infection. Comp Cont Educ Pract Vet 1995;17:763–77. [4] Fredriksson G. Some reproductive and clinical aspects of endotoxins in cows with special emphasis on the role of prostaglandins. Acta Vet Scand 1984;25:365–77. [5] Heap RB, Poyser NL. Prostaglandins in pyometrial fluid from the cow, bitch and ferret. Brit J Pharm 1975;55:515–8. [6] Granstro¨m E, Kindahl H. Radioimmunoassay of the major plasma metabolite of PGF2a, 15-keto-13,14-dihydro-PGF2a. Meth Enzymol 1982;86:320–39. [7] Concannon PW, Isaman L, Frank DA, Michel FJ, Currie WB. Elevated concentrations of 13,14-dihydro-15-keto-prostaglandin F2a in maternal plasma during prepartum luteolysis and parturition in dogs (Canis familiaris). J Reprod Fertil 1988;84:71–7. [8] Williams BJ, Watts JR, Wright PJ, Shaw G, Rebfree MB. Effect of sodium cloprostenol and flunixin meglumine on luteolysis and the timing of birth in bitches. J Reprod Fertil 1999;116: 103–11. [9] Olsson K, Bergstro¨m A, Kindahl H, Lagerstedt A-S. Increased plasma concentrations of vasopressin, oxytocin, cortisol and the prostaglandin F2a metabolite during labour in the dog. Acta Physiol Scand 2003;179:281–7. [10] Veronesi MC, Battocchio M, Marinelli L, Faustini M, Kindahl H, Cairoli F. Correlations among body temperature, plasma progesterone, cortisol and prostaglandin F2a of the periparturient bitch. J Vet Med A 2002;49:264–8. [11] Hughes JP, Stabenfelt GH, Kindahl H, Kennedy PC, Edqvist LE, Neely DP, et al. Pyometra in the mare. J Reprod Fertil 1979; 27:321–9. [12] Vandeplassche M, Coryn M, De Schepper J. Pyometra in the bitch: cytological, bacterial, histological and endocrinological characteristics. Vlaams Diergen Tijds 1991;60:207–11. [13] Mateus L, Lopes da Costa L, Diniz P, Ziecik AJ. Relationship between endotoxin and prostaglandin (PGE2 and PGFM) concentrations and ovarian function in dairy cows with puerperal endometritis. Anim Reprod Sci 2003;76:143–54. [14] Kindahl H, Odensvik K, Aiumlamai S, Fredriksson G. Uteroovarian relationships during the bovine post-partum period. Anim Reprod Sci 1992;28:363–9. [15] Fransson B, Lagerstedt A-S, Hellme´n E, Jonsson P. Bacteriological findings, blood chemistry profile and plasma endotoxin levels in bitches with pyometra or other uterine disease. J Vet Med 1997;44:417–26. [16] Dhaliwal GK, England GC, Noakes DE. Uterine bacterial flora and uterine lesions in bitches with cystic endometrial hyperplasia (pyometra). Vet Rec 1998;143:659–61. ˚ sheim A ˚ . Pathogenesis of renal damage and polydipsia in dogs [17] A with pyometra. J Am Vet Med Assoc 1965;147:736–45.
[18] Fletcher JR, Ramwell PW. Modification by aspirin and indomethacin of the hemodynamic and prostaglandin releasing effects of E. coli endotoxin in the dog. Brit J Pharm 1977;61: 175–81. [19] Børresen B. Pyometra in the dog — a pathophysiological investigation. Part IV. Functional derangement of extragenital organs. Nord Vet Med 1980;32:255–68. [20] Børresen B, Skrede S. Pyometra in the dog — a pathophysiological investigation. Part V. The presence of intrahepatic cholestasis and ‘‘acute phase reaction’’. Nord Vet Med 1980;32: 378–86. [21] Fox ES, Thomas P, Broitman SA. Hepatic mechanisms for clearance and detoxification of bacterial endotoxins. J Nutr Biochem 1990;1:620–8. [22] Fredriksson G, Kindahl H, Edqvist L-E. Endotoxin induced prostaglandin release and corpus luteum function in goats. Anim Reprod Sci 1985;8:109–21. [23] Aiumlamai S, Kindahl H. Clinical and blood biochemical changes during induction of endotoxemia in heifers. Acta Vet Scand 1990;31:501–4. [24] Holst H, Edqvist L-E, Kindahl H, Rylander R. Effects of oral and intravenous administration of endotoxin in prepubertal gilts. J Vet Med A 1993;40:33–44. [25] Daels PF, Starr M, Kindahl H, Fredriksson G, Hughes JP, Stabenfeldt GH. Effect of Salmonella typhimurium endotoxin on PGF-2 alpha release and fetal death in the mare. J Reprod Fertil Suppl 1987;35:485–92. [26] Børresen B, Naess B. Microbial, immunological and toxicological aspects of canine pyometra. Acta Vet Scand 1977;18: 569–71. [27] Wessels BC, Wells MT. Antiendotoxin immunotherapy for canine pyometra endotoxemia. J Am Anim Hosp Assoc 1989; 25:455–60. [28] Okano S, Tagawa M, Takase K. Relationship of the blood endotoxin concentration and prognosis in dogs with pyometra. J Vet Med Sci 1998;60:1265–7. [29] Dow C. The cystic endometrial hyperplasia–pyometra complex in the bitch. Vet Rec 1958;70(49):1102–8. [30] Sandholm M, Vasenius H, Kivisto¨ AK. Pathogenesis of canine pyometra. J Am Vet Med Assoc 1975;167:1006–10. [31] Purvis D, Kirby R. Systemic inflammatory response syndrome. Septic shock. Vet Clin N Am Small Anim Pract 1994;24: 1225–47. [32] Brady CA, Otto CM. Systemic inflammatory response syndrome, sepsis and multiple organ dysfunctions. Vet Clin N Am Small Anim Pract 2001;31:1147–62. [33] Hauptmann JG, Walshaw R, Olivier NB. Evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs. Vet Surg 1997;26:393–7. [34] Fransson BA, Karlstam E, Bergstro¨m A, Park JS, Evans MA, Ragle CA. C-reactive protein in the differentiation of pyometra from cystic endometrial hyperplasia/mucometra in dogs. J Am Anim Hosp Assoc 2004;40:391–9. [35] Børresen B. Pyometra in the dog — a pathophysiological investigation. Part I. The pyometra syndrome, a review. Nord Vet Med 1975;27:508–17.
Differentiation between pyometra and cystic endometrial hyperplasia/mucometra in bitches by prostaglandin F2a metabolite analysis R. Hagman a,*, H. Kindahl b, B.A. Fransson c, A. Bergstro¨m a, B. Stro¨m Holst d, A.-S. Lagerstedt a a Department of Small Animal Clinical Sciences, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 7037, SE-75007 Uppsala, Sweden b Department of Clinical Sciences, Division of Comparative Reproduction, Obstetrics and Udder Health, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 7037, SE-75007 Uppsala, Sweden c Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164-7060, USA d Department of Small Animals, National Veterinary Institute, SE-75189 Uppsala, Sweden
Received 1 October 2004; received in revised form 2 November 2005; accepted 4 November 2005
Abstract Bitches with pyometra are potential emergency cases which may be clinically difficult to differentiate from cases of cystic endometrial hyperplasia (CEH) in combination with mucometra. In the present study plasma prostaglandin F2a, as measured by its main metabolite 15-keto-13,14-dihydro-PGF2a (PG-metabolite) concentrations, blood biochemical and hematological parameters were measured in 59 bitches with pyometra, 10 bitches with CEH and nine controls to determine if PG-metabolite could differentiate between the three uterine conditions. Bitches with pyometra had significantly higher plasma levels of PG-metabolite than bitches with CEH (P = 0.002) and the controls (P = 0.002). PG-metabolite analysis alone had a high sensitivity (98.3%) and a high specificity (80.0%) for the differentiation of pyometra versus CEH in bitches where fluid in the uterus was diagnosed. When a combination of PG-metabolite and percentage band neutrophils (PBN) was used for differentiation of the two diagnoses, a sensitivity of 100% and specificity of 90.0% was obtained. This means that the combination of PG-metabolite and PBN analysis allows for differentiation between cases of pyometra and CEH. If the PG-metabolite level in a bitch is 4524 pmol l 1, there is a 99% probability of the diagnosis pyometra versus CEH. Levels of PG-metabolite 3054 pmol l 1, 2388 pmol l 1 or 1666 pmol l 1 indicates a 95%, 90% or 80% probability of pyometra, respectively. At high PG-metabolite levels (above about 3000 pmol l 1), PG-metabolite alone is enough for differentiation of pyometra versus CEH. The results of the present study showed that PG-metabolite analysis is valuable in the diagnosis and prediction of severity of uterine diseases. # 2005 Elsevier Inc. All rights reserved. Keywords: Dog; Endometritis; Prostaglandins; Uterine inflammation; SIRS
1. Introduction
* Corresponding author. Tel.: +46 18 672918; fax: +46 18 673534. E-mail address: [email protected] (R. Hagman). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.11.002
The diagnosis of pyometra (chronic purulent endometritis post-oestrum) in bitches can be clinically difficult to separate from bitches with cystic endometrial hyperplasia (CEH) in combination with sterile
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seromucous fluid in the uterine lumen (hydrometra or mucometra depending on the water content of the fluid) [1]. Generally pyometra is associated with more and severe systemic signs of illness due to the bacterial infection and following immune response while often the only sign of CEH (defined as proliferation and cystic hyperplasia of endometrial cysts) is failure to conceive [2]. It is however important to be able to differentiate between the two diseases since bitches with pyometra are at risk of a suddenly developing endotoxic shock which may be fatal while bitches with non-infected uteri (CEH/mucometra) not are regarded as potential emergencies [3]. Prostaglandins are derived from arachidonic acid and have many both physiological and pharmacological roles and as important mediators in inflammatory events [4]. The uterine endometrium is known to synthesise and release prostaglandins and mainly prostaglandin F2a (PGF2a) [5]. The systemic release of PGF2a, can be followed by measurement of its more stable main circulating metabolite 15-keto-13,14-dihydro-PGF2a (PG-metabolite) [6]. In dogs the concentrations of PGF2a have been shown to be physiologically elevated during parturition and the post-partum period [7–10]. Increased plasma levels of PG-metabolite have also been demonstrated in pathological inflammatory conditions of the uterus in several species, including canines [5,11–14]. In bitches with pyometra, infection with predominantly Gram-negative bacteria such as Escherichia coli is generally present with inflammation and accumulation of pus in the uterus [15,16]. Gram-negative bacteria release cell wall compounds, endotoxins, into the circulation during vigorous growth or cell disintegration. The endotoxins are biologically active by initiating the release of many inflammatory mediators (cytokine cascade) and are thought to be responsible for many systemic symptoms of bitches with pyometra [17–21]. Endotoxins are very potent compounds also in stimulating prostaglandin release and PGF2a has been demonstrated to be a reliable and sensitive marker of experimentally induced endotoxemia in cattle, goats, pigs and horses [4,21–25]. Endotoxemia has been demonstrated in two out of four studies of endotoxin involvement in bitches with pyometra [15,26–28]. In one of these studies, the plasma endotoxin concentrations were related to outcome (survival or death) due to the disease [28]. Cystic endometrial hyperplasia (CEH) develops as a consequence of an abnormal response of the uterus to repeated progestational stimulation during the luteal phase of the estrous cycle [29]. CEH is considered to be
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the initial phase of a combined CEH–pyometra complex and it is generally believed that CEH predisposes for a bitch for pyometra [29,30]. Both CEH and pyometra can possibly develop independently of each other [1]. De Bosschere and others suggested that CEH and pyometra/endometritis should be divided into two separate entities due to their clinical and histopathological differences [1]. This separation is sometimes difficult since the severity of clinical symptoms does not always correlate with the severity of the uterine pathology [1]. Clinically it would be helpful to accurately predict the diagnosis before surgery. The risk of increased morbidity associated with delaying surgery in bitches with pyometra (potential uterine rupture, peritonitis and sepsis) could then be minimised and facilitate the decision of optimal therapeutic regime in both CEH and pyometra cases. In cattle with endometritis, the possible application of PG-metabolite measurements to predict severity of the uterine inflammation and survival has been shown [13]. It seems likely that analysis of PG-metabolite could facilitate the differentiation between cases without inflammation (CEH/mucometra) and cases of pyometra/endometritis in canines as well, and possibly also predict severity of the uterine disease. Pyometra has been associated with the systemic inflammatory response syndrome (SIRS) and the presence of SIRS has previously been linked with lower survival rates and longer hospitalisation [2,31,32]. In SIRS the initial local inflammatory reaction shifts through the cascade release of the endogenous inflammatory mediators to an uncontrollable systemic reaction where mediators directly stimulate other mediators and continue without the presence of the initiating agent [31]. Various incidents such as severe trauma, infection or endotoxin can initiate the inflammatory process which can be present with bacteremia (sepsis) or without. Diagnostic criteria for dogs with the highest sensitivity in the identification of systemic inflammatory response syndrome (SIRS) have been determined by Hauptman et al. [33]. Assessment of SIRS is important in order to determine severity of the disease, optimise treatments and prevent fatal outcomes, especially in patients at a high risk of developing shock or multiple organ dysfunctions [31,34]. The objectives of this study were (i) to compare PGmetabolite levels in healthy bitches with bitches suffering from pyometra or CEH and to (ii) evaluate if the PG-metabolite analysis can differentiate between pyometra and CEH and further to (iii) determine the predictive value of PG-metabolite levels for severity of clinical status, and to determine outcome.
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2. Materials and methods 2.1. Animals 2.1.1. Pyometra and cystic endometrial hyperplasia (CEH)/mucometra group The study was approved by the Uppsala County Ethical Board, Tierp, Sweden prior to onset of the clinical investigations. Sixty-nine privately owned bitches with the presumptive diagnosis of pyometra or CEH were included in the present study. All bitches were treated by ovariohysterectomy at the Department of Small Animal Clinical Sciences, Swedish University of Agricultural Studies, Uppsala, Sweden. The diagnosis was based on case history, physical examination and diagnostic imaging using ultrasonography and/or radiology to demonstrate an enlarged, fluid-filled uterus. The diagnosis of pyometra or CEH (in ten of these cases, whereof mucometra in four), was verified visually during the ovariohysterectomy and confirmed by histopathological examination. The admitting clinician filled out a form specifying rectal temperature, heart rate, respiratory rate, mucus membrane colour, capillary refill time, location for pain response at abdominal palpation, hydration status and general attitude at the time of admission. None of the pyometra bitches had previously been medically treated for pyometra. After surgery the rectal temperature, heart rate and respiratory rate was noted daily for as long as the patient was admitted on a special form from which also the length of hospitalisation was determined. Additional information on treatment, complications to treatment and mortality were obtained from the medical records. 2.1.2. Control group Healthy adult intact bitches (n = 9) were used as controls. A history questionnaire was filled out, ensuring that the owner considered the bitch healthy for at least 8 weeks prior to the examination. Physical examination was performed and documented by two of the authors. None of the control bitches had previously been medically treated for pyometra. 2.2. Blood collection and analyses 2.2.1. Pyometra and cystic endometrial hyperplasia (CEH/mucometra) group Blood samples for biochemical, hematological and PG-metabolite analysis were obtained immediately before surgery in EDTA, sodium-heparinised and nonadditive vacutainer tubes (Becton-Dickinson, Stock-
holm, Sweden). Biochemical and hematological analyses were performed using routine methods, at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. The following hematology parameters were analysed: packed cell volume (PCV), hemoglobin (Hb), erythrocyte volume fraction (EVF), white blood cell count (WBC), differential count of WBC including total count (BN) and percentage band neutrophils (PBN), nucleated erythrocytes, erythrocyte morphology, red blood cell mean corpuscular volume (MCV), red blood cell mean corpuscular hemoglobin concentration (MCHC) and platelet count (TPK). The following serum biochemical parameters were determined: alanine aminotransferase (ALAT), albumin, alkaline phosphatase (AP), blood urea nitrogen (BUN), cholesterol, glucose, total protein and the electrolytes sodium (Na), potassium (K), chloride (Cl) and calcium (Ca). For analysis of PGmetabolite, sodium heparinised plasma was stored at 70 8C until assayed at the Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden. A radioimmuno assay (RIA) was used to analyse 15-ketodihydro-PGF2a in duplicates [5]. The practical detection limit of the assay was 200 pmol l 1. Where necessary, dilution of the samples was performed to allow for accurate readings on the standard curve. 2.2.2. Control group Blood samples for the control bitches were collected through the same procedure as for the diseased group after the physical examination. 2.3. Histopathological examinations The diagnosis of CEH and pyometra according to the previously proposed definition by Dow was confirmed by gross and histopathological examination of hematoxylin–eosin stained sections of formaldehyde-fixated uteri and ovaries at the Department of Pathology, National Veterinary Institute (SVA), Uppsala, Sweden [29,34]. The definition of pyometra was chronic purulent metritis, acute purulent metritis or purulent endometritis [34,35]. The histopathological diagnosis of CEH was defined by hyperplasia of the endometrium in combination with endometrial cysts without any inflammatory changes [34]. The criteria for the diagnosis mucometra were presence of hyperplastic and cystic or atrophic endometrium in combination with mucus or serous fluid present in the uterine lumen on gross or microscopical examination [34].
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2.4. Determination of a systemic inflammatory response A patient was regarded as SIRS-positive if two or more of the following criteria are met: respiratory rate > 20 min 1; heart rate > 120 beats min 1; WBC < 6 or >16 (109 l 1) or percentage band neutrophils (PBN) > 3%; temperature < 38.1 or >39.2 [29]. 2.5. Statistical analysis Statistical analyses were performed with the programme Statistica (Version 6.0, StatSoft Inc., Tulsa, USA). A one-way ANOVA (Tukey, post hoc) was used to test for differences in PG-metabolite levels by patient group (control, pyometra and CEH/mucometra). Fisher’s exact test, Spearman’s rank correlation coefficient (rS) and Pearson’s product moment correlation coefficient (rP) were used to test for associations between nominal, ordinal and interval-scale variables, respectively. Binary logistic regression analyses were used to evaluate blood parameters in relation to controls and bitches with pyometra or CEH/mucometra by histopathological examinations. The regression models were tested for sensitivity and specificity. Values below detection limit, which in the data set occurred for PGmetabolite, BN, segmented neutrophils, eosinophils, and basophils were set to zero in the calculations. Twosample t-tests were used to test for differences in PGmetabolite levels between bitches with SIRS or without SIRS, as tested for pyometras and CEH/mucometras, pyometra group and CEH/mucometra group, respectively. Significance was accepted at P < 0.05 for all statistical tests used in this study. Equations used to create Figs. 3–5 were as follows: equation used for calculation of the estimated probability of PG-metabolite in combination with PBN for determination of the diagnosis pyometra versus CEH/ mucometra: estimated probability of pyometra = exp ( 1.1966 + (0.000999) PG-metabolite + (0.200471) PBN)/(1 + exp( 1.1966 + (0.000999) PG-metabolite + (0.200471) PBN)). P 0.0001. Equation used for calculation of the estimated probability of PG-metabolite in combination with WBC for determination of the diagnosis pyometra versus CEH/mucometra: estimated probability of pyometra = exp( 2.0962 + (0.001152) PG-metabolite + (0.118737) WBC)/ (1 + exp( 2.0962 + (0.001152) PG-metabolite + (0.118737) WBC)). P 0.0001. Equation used for calculation of the estimated probability of PGmetabolite for determination of the diagnosis pyometra
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versus CEH/mucometra: estimated probability of pyometra = exp( 0.4849 + (0.001123) PG-metabolite/(1 + exp( 0.4849 + (0.001123) PG-metabolite)) (figure not shown), P 0.0001. 3. Results Of the 69 bitches included in disease groups of the present study 59 had the diagnose pyometra and 10 cases CEH/mucometra, confirmed by histopathology. The pyometra patients, CEH/mucometra group and control group consisted of 35, 9 and 6 different breeds, respectively. Clinical findings, blood biochemical and hematological parameters in all three groups are presented in Table 1. Mean values for the controls and the CEH/mucometra and pyometra groups were: MCV (68, 69 and 67 [fl]), MCHC (352, 353 and 355 [g l 1]), Na (155, 153 and 155 [mmol l 1]), K (4.6, 4.4 and 4.4 [mmol l 1]), Cl (122, 124, 123 [mmol l 1]) and Ca (2.6, 2.7, 2.6 [mmol l 1]), respectively. The presence of abnormalities in case history and physical examination and administration of antimicrobial treatment in controls and bitches diagnosed with pyometra or CEH/mucometra are presented in Table 2. Fisher’s exact test was used for the calculations of Pvalue of PG-metabolite levels, age, duration of clinical signs and blood hematological and chemistry parameters (Table 2). The plasma levels of PG-metabolite were significantly increased in bitches with pyometra compared with bitches with CEH/mucometra (P = 0.003) and the controls (P = 0.003) (Fig. 1). The number of bitches in the different patient groups and controls, divided into five different categories of PG-metabolite-levels, are illustrated in Fig. 2. Regarding other parameters, in this study significant differences between bitches with pyometra and CEH/mucometra were demonstrated in WBC (P = 0.001), BN (P = 0.015), PBN (P = 0.001), segmented neutrophils (P = 0.006), monocytes (P = 0.010), cholesterol (P = 0.009) and albumin (P = 0.026). The levels of PG-metabolite were not significantly higher in bitches with CEH/mucometra compared with the controls. When comparing bitches with CEH/mucometra and controls there were significant differences in levels of albumin (P = 0.037), lymphocytes (P = 0.001), monocytes (P = 0.003). PG-metabolite levels were significantly correlated to WBC (rp = 0.351), BN (rp = 0.403), PBN (rp = 0.434), segmented neutrophils (rp = 0.303), lymphocytes (rp = 0.235), monocytes (rp = 0.287), creatinine (rp = 0.306), ALAT (rp = 0.390), glucose (rp = 0.277) and albumin (rp = 0.537). PG-metabolite levels were
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Table 1 Mean, standard deviation and range values for selected hematological and serum biochemistry parameters in different patient groups Control
Age (years) Temperature (8C) Duration of clinical signs (days) PG-metabolite (pmol l 1) Hemoglobin (g l 1) EVF EPC (1012 l 1) WBC (109 l 1) BN (109 l 1) PBN (%) Neutrophils (109 l 1) Eosinophils (109 l 1) Basophils (109 l 1) Lymphocytes (109 l 1) Monocytes (109 l 1) Platelet count (109 l 1) Creatinine (mmol l 1) ALAT (mkat l 1) AP (mkat l 1) BUN (mmol l 1) Glucose (mmol l 1) Protein (g l 1) Albumin (g l 1) Cholesterol (mmol l 1)
CEH/mucometra
Pyometra
Mean S.D. (range)
n
Mean S.D. (range)
n
Mean S.D. (range)
n
6.7 1.7 (4.3–9.7) 38.5 0.4 (37.9–39.0) 0
9 9 9
8.8 3.1 (2.5–12.0) 38.9 0.7 (38.2–40.2) 3 3 (0–7)
10 7 8
9.9 2.4 (2.5–13.6) 39.1 0.6 (37.9–40.6) 7 6 (1–28)
58 56 48
86 338 (0.0–772) 174 20.3 (146–210) 0.49 0.05 (0.41–0.58) 7.38 0.62 (6.70–8.40) 10.1 3.0 (7.0–16.2) 0.01 0.03 (0.00–0.10) 0.11 0.32 (0.00–0.96) 6.1 1.9 (4.1–10.4) 0.8 0.8 (0.0–2.5) 0.01 0.03 (0.0–0.1) 2.6 1.0 (1.6–5.0) 0.5 0.3 (0.1–1.0) 345 101 (148–524) 83 21 (46–112) 0.6 0.3 (0.3–1.3) 1.7 0.7 (1.0–3.2) 7.2 4.0 (3.5–16.3) 5.0 0.4 (4.2–5.5) 76 7 (66–85) 35 3 (32–40) 6.4 1.7 (4.2–8.8)
9 9 9 6 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9
415 998 (0–3200) 137 19.8 (104–173) 0.39 0.06 (0.30–0.49) 5.52 0.91 (4.30–7.30) 9.4 5.2 (3.8–21.4) 0.19 0.43 (0.0–1.3) 1.48 3.81 (0.00–12.0) 7.1 4.6 (2.7–17.3) 0.3 0.3 (0.0–1.0) 0.0 0.0 1.0 0.4 (0.3–1.7) 0.8 0.5 (0.1–1.5) 252 110 (35–414) 79 31 (53–148) 0.6 0.4 (0.2–1.1) 2.4 2.4 (0.6–8.0) 4.6 1.8 (2.0–7.9) 5.4 0.4 (4.8–6.0) 76 7 (65–84) 30 3 (26–35) 7.2 2.8 (5.0–13.3
10 10 10 9 10 10 10 10 10 10 10 10 9 8 9 8 8 8 8 8 8
6278 5757 (0–33500) 136 24.4 (96–207) 0.38 0.07 (0.27–0.58) 5.96 1.76 (3.9–16.0) 22.8 11.4 (4.5–48.4) 2.86 2.96 (0.0–12.7) 11.9 9.97 (0.00–38.9) 15.3 8.1 (2.4–36.4) 0.3 0.6 (0.0–4.1) 0.02 0.08 (0.0–0.5) 1.5 0.9 (0.0–3.9) 2.82.3 (0.0–10.1) 233 118 (22–681) 81 42 (44–257) 0.6 0.8 (0.1–5.6) 6.0 6.0 (0.4–34) 5.1 4.0 (1.6–23.1) 5.0 1.2 (2.4–8.0) 80 13 (43–139) 27 5 (18–37) 9.8 2.5 (4.7–14.1)
58 57 57 51 56 56 56 56 56 56 56 56 55 53 57 57 48 47 49 49 48
Temperature: body temperature; duration: duration of clinical signs; PG-metabolite: prostaglandin F2a metabolite; Hb: hemoglobin; EVF: red blood cell volume fraction; EPC: red blood cell particle concentration; WBC: white blood cell count; BN: band neutrophils; neutrophils: segmented neutrophils; eosinophils: segmented eosinophils; basophils: segmented basophils; ALAT: alanine amino transferase; AP: alkaline phosphatase; BUN: blood urea nitrogen.
Table 2 The presence of selected case history and physical examination findings in control bitches, bitches with cystic endometrial hyperplasia/mucometra (CEH/M) and bitches with pyometra Case history and physical examination findings Polydipsia and/or polyuria Gastrointestinal signs Lethargy Abdominal pain on palpation Purulent-like vaginal discharge Mucous membranes hyperemic, discoloured or pale Capillary refill time 3s Temperature > 39.2 8C Heart rate > 120 beats/min Respiratory rate > 20 breaths/min Attitude moderately depressed at admission Antimicrobial treatment Presence of two or more SIRS-criteria
Control, % (n = 9)
Pyometra and CEH, % (n = 69)
P-value
CEH/mucometra, % (n = 10)
Pyometra, % (n = 59)
P-value
0 0 0 0 0 0
(9) (9) (9) (9) (9) (9)
65 61 56 72 58 20
(66) (66) (66) (65) (66) (65)
<0.001 <0.001 <0.001 <0.001 <0.001 0.032
55 11 11 62 55 0
(9) (9) (9) (8) (9) (8)
68 70 64 75 57 23
(56) (56) (56) (56) (56) (56)
0.47 0.001 0.004 0.43 1.0 0.10
0 0 0 0 0
(9) (9) (9) (9) (9)
9 30 26 49 78
(66) (64) (61) (43) (65)
0.33 0.13 0.76 0.19 <0.001
0 14 0 83 33
(9) (7) (7) (6) (9)
11 30 28 44 85
(56) (56) (53) (36) (55)
0.58 0.66 0.17 0.18 0.002
29 (62) 49 (68)
0.004 0.17
0 (9) 22 (9)
33 (9) 20 (10)
27 (52) 53 (58)
0.70 0.08
Fisher’s exact test was used for the calculations of P-value for the level of statistical significance of the differences between the groups.
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within the pyometra group (P = 0.045) but not significant within the CEH/mucometra group (twosample t-tests) (Fig. 5). 4. Discussion 4.1. Differentiation of pyometra versus CEH/ mucometra
Fig. 1. Prostaglandin F2a metabolite (PG-metabolite) levels in controls and bitches with cystic endometrial hyperplasia/mucometra (CEH) or pyometra. Bars indicate 1 standard error of the mean (S.E.) (Anova and Tukey post hoc test).
also significantly correlated to the age of the patient (rp = 0.264), length of the hospitalisation (rp = 0.314), body temperature (rp = 0.313) and heart rate (rp = 0.317). The number and percentage of bitches with two or more criteria of SIRS in each respective group (control, CEH/ mucometra, pyometra) are demonstrated in Table 2. When PG-metabolite levels in bitches with and without SIRS were compared, the differences were significant in the group consisting of both pyometra and CEH/mucometra cases (P = 0.01), just significant
Fig. 2. Percentage of the controls, cystic endometrial hyperplasia/ mucometra (CEH) and pyometra bitches as divided into five groups according to their prostaglandin F2a metabolite (PG-metabolite) plasma levels.
This is the first study of PG-metabolite analysis in the differentiation between pyometra and CEH/mucometra in bitches. Pyometra is in general considered a surgical emergency, unlike CEH/mucometra, since the risk of uterine rupture, endotoxemia and following inflammatory reactions may transform a clinically stable pyometra to an emergency case in a matter of hours [2,30]. PG-metabolite analysis alone is the single parameter with highest sensitivity (98.3%) and specificity (80.0%), in the present study, for the differentiation of pyometra versus CEH/mucometra in bitches here a fluid-filled uterus is diagnosed. This sensitivity is higher than what has been reported for a single parameter previously in this differentiation [34]. To obtain the highest sensitivity for differentiation between pyometra and CEH/mucometra in this study the analysis of PG-metabolite was combined with analysis of PBN which yielded a sensitivity of 100.0% and a specificity of 90.0%. Compared with analysis of PGmetabolite alone, the combination of PG-metabolite and PBN had both higher sensitivity and specificity. In clinical work the preference of a test is to have high sensitivity (ability to correctly diagnose a pyometra) since the surgery on a ‘‘false positive pyometra’’ (CEH/ mucometra) patient has no negative consequences for the patient (except for breeding bitches) compared with a test with high specificity which in this case could mean that a pyometra could be falsely diagnosed as CEH/mucometra and perhaps not treated as an emergency. The combination of PG-metabolite with any other blood parameter than PBN led to decreased sensitivity and specificity compared with PG-metabolite alone (albumin 87.5; BN 80.0%; monocytes 80.0%) (Fig. 3). If the PG-metabolite level in a bitch is 4524 pmol l 1, there is a 99% probability of the diagnosis pyometra versus CEH/mucometra. PG-metabolite levels of 3054 pmol l 1, 2388 pmol l 1 or 1666 pmol l 1 indicates a 95%, 90% or 80% probability of pyometra, respectively. The analysis of PG-metabolite concentrations therefore has high diagnostic value. Routine blood parameters such as WBC, BN, segmented neutrophils, monocytes, albumin, and AP have
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Fig. 3. Contour plot of estimated 80, 90, 95, and 99% probability of the diagnose pyometra vs. cystic endometrial hyperplasia/mucometra (CEH) in a bitch, based on percentage band neutrophils (PBN) and plasma prostaglandin F2a metabolite (PG-metabolite) concentrations. Bisection of the values for PBN and PG-metabolite to the right of each respective graph indicates 80%, 90%, 95% or 99% probability of the diagnosis pyometra.
Fig. 4. Contour plot of estimated 80, 90, 95, and 99% probability of the diagnose pyometra vs. cystic endometrial hyperplasia/mucometra (CEH) in a bitch, based on white blood cell counts (WBC) and plasma prostaglandin F2a metabolite (PG-metabolite) concentrations. Bisection of the values for WBC and PG-metabolite to the right of each respective graph indicates 80%, 90%, 95% or 99% probability of the diagnosis pyometra.
sensitivities of 98.2, 94.6, 100, 98.2, 100, and 100%, respectively, but the diagnostic value in the differentiation between pyometra and CEH/mucometra is low because of their low specificity (20.0%) with the exception of BN (specificity 80.0). At high PGmetabolite levels (above about 3000 pmol l 1), PGmetabolite alone is enough for differentiation of pyometra versus CEH/mucometra, but at lower PGmetabolite levels (about 2000 pmol l 1) the combination of PG-metabolite with PBN, WBC or BN (if sufficiently high) will increase the probability of pyometra (Figs. 4 and 5). An advantage with the analysis of PG-metabolite alone or in combination with WBC in the differentiation of pyometra versus CEH/mucometra is that it allows for diagnosis without the differential count of WBC. An inexpensive test for rapid detection of PG-metabolite would be clinically very useful in the diagnosis of pyometra versus CEH/mucometra. Other clinical parameters useful to differentiate between pyometra and CEH/mucometra are the general attitude of the bitch at the time of admission, lethargy and gastrointestinal signs (Table 2). In the present study significant differences between pyometra and CEH/ mucometra were also demonstrated in WBC, BN, PBN, segmented neutrophils, lymphocytes, monocytes, cholesterol and albumin (data not shown). These parameters have previously been shown to be elevated (WBC, BN, segmented neutrophils, monocytes, cholesterol) or decreased (albumin) in bitches with pyometra reflecting the chronic bacterial infection, endotoxemia, following intrahepatic cholestasis and acute phase reaction [18,19].
4.2. PG-metabolite levels in pyometras versus controls PG-metabolite concentrations were significantly (P = 0.003) increased in bitches with pyometra compared with the controls (Fig. 1). These results are in accordance with the study by Vandeplassche et al. [12]. The synthesis of prostaglandins is stimulated by the bacterial infection and inflammation of the uterus as is observed both in canines and many other domestic
Fig. 5. Prostaglandin F2a metabolite (PG-metabolite) concentrations in controls and bitches with cystic endometrial hyperplasia/mucometra (CEH) or pyometra, with and without a systemic inflammatory response (SIRS). Bars indicate standard error of the mean (two-sample t-tests).
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animals [3,4,10,11,21,23]. The highest PG-metabolite value in the pyometra bitches was 33,500 pmol l 1. This can be compared with the maximum concentrations measured at parturition of 74,900 31,900 pmol l 1 [8,9]. The reference range for non-pregnant healthy bitches in all stages of the oestrous cycle is 0.05– 2.1 nmol l 1 and the levels are generally low during the first weeks of pregnancy [8,9,11]. The levels of PGmetabolite for the controls in this study were 0.29 0.05 nmol l 1 which is in the same order of magnitude as in the other studies [8,9,11]. The levels of PG-metabolite were not significantly higher in bitches with CEH/mucometra compared with the controls likely because of the absence of bacterial infection, endotoxemia and uterine inflammation according to histopathological examinations. 4.3. PG-metabolite correlations PG-metabolite levels were correlated to the following hematological and blood chemistry parameters: WBC, BN, PBN, segmented neutrophils, lymphocytes, monocytes, creatinine, ALAT, glucose, and albumin. PG-metabolite levels were also correlated to duration of clinical illness prior to admission, hospitalisation length and measurements of temperature, heart rate, and dehydration at the time of admission. It seems logical that the PG-metabolite concentrations are correlated to parameters normally responsive to endotoxemia since endotoxins are potent inducers of the release and synthesis of prostaglandins [17,22,23]. 4.4. Prognostic value of PG-metabolite analysis As for prognostic value, the PG-metabolite concentrations were correlated to morbidity as measured in this study by length of the hospitalisation stay. Bitches operated for pyometra are dismissed 1–2 days after surgery, with few exceptions. Additional complications with severely depressed general condition will result in longer hospitalisation stays. The pre-operative measurement of PG-metabolite can thus help the clinician in determining the severity of the pyometra roughly judged by how long time the patient will need to be hospitalised. To study PG-metabolite concentrations in relation to degree of uterine pathological changes would be a logical continuation of the present study. To evaluate any possible predictive values of PG-metabolite for survival of an increased patient number is needed since only two bitches died of causes related to pyometra in this study. Two other bitches died, 2 and 14 days after surgery, respectively, but it is uncertain
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whether the deaths were consequences of the pyometra since no autopsies were performed. The bitch with the highest observed level of PGmetabolite, 33,500 pmol l 1, died the day after surgery as a consequence of myocarditis and other sequela of the uterine bacterial infection. The other confirmed death was a bitch with a thin-walled uterus which ruptured during the operation and she died 1 h after the surgery was completed. That bitch had low (400 pmol l 1) levels of PG-metabolite before surgery and was perhaps an example of severely damaged endometrium which reduces the endogenous production of prostaglandins, as has been described in the mare [10]. The levels of PG-metabolite were correlated to rectal temperature, heart rate, WBC and PBN, four out of five clinical parameters used in determining whether systemic inflammatory response syndrome (SIRS) is present. The presence of SIRS has clinical importance since it is linked with higher mortality rates, in spite of the fact that mortality was very low in the present study [2]. PG-metabolite analysis could possibly be used as an additional SIRS indicator if a rapid test for veterinary clinics is made readily available in the future. Analysis of PG-metabolite can be useful in the clinical work where it sometimes can be hard to differentiate pyometra from CEH/mucometra when the general condition of the bitch is relatively unaffected. A sensitive, rapid and inexpensive PG-metabolite test for the prediction of pyometra would enable clinicians to decide whether it is possible to postpone surgery (CEH/ mucometra) until optimal resources are available or whether the bitch needs to be monitored as an emergency and ovariohysterectomised as soon as the clinical condition allows (pyometra). The results of this study indicate the possibility to use PG-metabolite analysis to diagnose endometrial pathologies, predict the severity of the pyometra (determined by prolonged hospitalisation stays and systemic inflammation) and differentiate between pyometra and CEH/mucometra. Acknowledgements We express our sincere gratitude to Pauli Snoeijs for help with the statistical analysis of the data, Lillemor Abersten and Anna Gerentz Bohlin for collection of the samples, Erika Karlstam for the histopathological examinations and Susanna Sternberg for comments on the manuscript. The present study was financially supported by Agria Insurance Co. Research Foundation and The Swedish Kennel Club Research Foundation.
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