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Reprod Dom Anim 39, 136–140 (2004)Ó 2004 Blackwell Verlag, BerlinISSN 0936-6768 Ultrasonography and Cystic Hyperplasia–Pyometra Complex in the Bitch E Bigliardi1, E Parmigiani1, S Cavirani2, A Luppi3, L Bonati4 and A Corradi3 Units of 1Obstetrics and Reproduction, 2Infectious Diseases, 3Pathology, and 4Internal Medicine, Department of Animal Health, Faculty ofVeterinary Medicine, University of Parma, Parma, Italy be one of the most important growth factors with a Cystic endometrial hyperplasia–pyometra complex is the most strong mitogenic effect on the uterus (De Cock et al.
frequent and important endometrial disorder encountered in 2002). In many cases of CEH secondary bacterial bitches. The pathogenesis of the disease is related to the activity infections occur causing pyometra. However, in other of progesterone [Feldman and Nelson, Canine and Feline cases the uterine contents are sterile.
Endocrinology and Reproduction (1996) W.B. Saunders, Phil- A general classification of CEH–pyometra complex adelphia]. Cystic endometrial hyperplasia (CEH) is an abnor- has been proposed according to Dow’s and De Bossc- mal response of the bitch’s uterus to ovarian hormones [De here’s criteria (Dow 1958; De Bosschere et al. 2001).
Bosschere et al. Theriogenology (2001) 55, 1509]. CEH is The average age of animals undergoing elective ovarian considered by many authors to be an exaggerated response ofthe uterus to chronic progestational stimulation during the hysterectomy (OHE) in Italy is higher compared with the luteal phase of the oestrous cycle, causing an abnormal USA or UK. Therefore, in Italy, bitches over the age of accumulation of fluid within the endometrial glands and 5 years are more frequently diagnosed with CEH.
uterine lumen (De Bosschere et al. 2001). The resulting lesions The aim of this study was to evaluate if transabdom- of pyometra are due to the interaction between bacteria and inal uterine ultrasonography can be a useful and reliable hormones. The aim of this study was to evaluate if trans- diagnostic method to confirm Dow’s and De Bosschere’s abdominal uterine ultrasonography can be a useful and reliable histopathological classification of CEH–pyometra com- diagnostic method to confirm Dow’s [Veterinary Record (1958) 70, 1102] and De Bosschere’s histopathological classification ofCEH–pyometra complex. The study was carried out on 45bitches with pyometra, 10 purebreeds and 35 crossbreeds, 1– 15 years old, 20% of which had whelped at least once. None ofthese animals had received exogenous oestrogen or progester- The study was performed on 45 ovario-hysterectomized one treatment. On admission the 45 animals were in the luteal bitches with clinical diagnosis of pyometra. The age of phase of the oestrus cycle. Clinical signs, blood parameters, bitches ranged from 1 to 15 years. Ten were purebreeds uterine ultrasonography, bacterial swabs and uterine histopa- and 35 were crossbreeds, and 20% had whelped. None logical results were recorded. Results suggest that ultrasono- of the dogs studied had received exogenous oestrogen or graphic examination is a useful and reliable tool for the progesterone treatment. All animals were in the luteal diagnosis of cystic endometrial hyperplasia.
In each case, clinical examination, haematological analysis, bacterial culture ultrasound and histopathologywere carried out. The main clinical symptoms consid- Excessive or prolonged oestrogenic or progestinic stimu- ered were hyperthermia, polydipsia, vomit, diarrhoea, lation, whether natural or synthetic, induces endometrial changes in the bitch (Hardy and Osborne 1994; De Cock Peripheral blood samples were collected from the et al. 1997). The most important endometrial hormone- radial vein into Vacutainer tubes (BD Franklin Lakes, induced changes in bitches may be classified in four NJ, USA). The blood samples [ethylenediaminetetra- hyperplastic conditions: (i) cystic hyperplasia–pyometra acetic acid (EDTA)] were processed within 3–4 h after complex; (ii) endometrial hyperplasia associated with collection: blood cell counts were performed using an pseudopregnancy; (iii) oestrogen-induced hyperplasia; Automated Cell Count (Medonic CA 570-Delcon, and (iv) endometrial polyps (McEntee 1990). Exogenous Stockholm, Sweden). The following parameters were oestrogens and progesterone administration predispose evaluated: erythrocytes (RGB), mean corpuscular vol- bitches to endometrial disease, the severity of which is ume (MCV), packed cell volume (HCT), platelets (PLT), dose dependent (Nelson and Kelly 1976).
leucocytes (WBC), haemoglobin (HGB), mean corpus- Cystic hyperplasia pyometra (CEH) complex is the cular haemoglobin concentration (MCHC).
most frequent and important endometrial disorder in Serum samples were analysed for glucose, urea, bitches, the exact pathogenesis of which is still creatinine, total protein, cholesterol, Asparate Amino unknown. Sex steroid hormones and their receptors Transferase (AST), Alanine Amino Transferase (ALT), play an important role and exogenous administration of and Alkaline Phosphatase (ALP). Enzyme activity was progesterone is often associated with CEH. However, determined at 25°C, using an automated filter photom- CEH is often present also in bitches in the luteal phase eter (Cobas Mira Plus; Roche Diagnostic System, Basel, which have not received any hormonal treatment.
Switzerland). Plasma oestrogen and progesterone con- Furthermore, there is an exceptionally high, progestin- centrations were determined by radioimmunoassay induced production of insulin-like growth factor-I (IGF-I) in the dog. IGF-I is now generally accepted to U.S. Copyright Clearance Centre Code Statement: 0936–6768/2004/3903–0136$15.000/0 Cystic Hyperplasia–Pyometra Complex in the Bitch Ultrasound was performed using Caris (Esaote Bio- paraffin (56–58°C). Five micrometre microtome sections medica, Florence, Italy) with 2.5–3.5–5–7.5–10 MHz were stained with haematoxylin–eosin (H&E), Van sectorial probes. The uterus was examined to evaluate Gieson, and periodic acid-Schiff (PAS). H&E sections the integrity of endometrium, presence of exudates and were viewed using a Nikon E-800 light microscope cystic hyperplasia of endometrial glands. The widest (Tokyo, Japan) and the images were captured with a cross-sectional diameter of uterine horns was measured digital camera (SV Micro; Sound Vision, Waltham, by electronic calipers. The ovaries were examined to MA, USA). Morphometric analysis was performed on 5 evaluate the presence of pathological changes such as serial slides from each uterine sample, using Image Pro- cysts, neoplasia, etc (Shille et al. 1984). The following Plus software (Media Cybernetics, Silver Spring, MD, USA) to determine morphological parameters accordingto De Bosschere’s classification. The De Bosschere’s and Group A: No cysts, normal endometrial surface and Dow’s classifications were compared with ultrasound results. Statistical analysis was performend by SPSS Group B: Few and small cysts, normal endometrial Group C: Many and large cysts, irregular surface and Group D: Many and large cysts in all the uterus, The interval between the onset of pro-oestrus to the diagnosis of CEH–pyometra complex ranged from 20 to atrophic endometrium, hyperechoic uterine 70 days (mean 35 days). Thirty-six bitches (80%) showed vaginal discharge, 47% hyperthermia, polydip- Swab specimens (Copan, Italy) were collected asepti- sia, polyuria, and in several cases there was vomiting.
cally from each uterine horn after hysterectomy. The Uterine exudate was present in all bitches (65% pus, swabs (in transport medium and at 5°C) were sent to the laboratory within 2 h after sampling. Each sample was Neutrophilia ranging from 15 000 to 60 000/ml plated on blood agar and McConkey agar. They were (means value 23 000/ml) was detected in 75% of bitches.
cultured aerobically in a controlled atmosphere of 5% All other haemotological parameters (RGB, MCV, CO2 at 37°C. Isolates were re-plated and identified by HCT, PLT, HGB, MCHC) were within normal range.
biochemical test (Api test; Biomeriaux, Marcy L’etoile, Blood levels of AST and creatinine were elevated while France). Escherichia coli cultures were serotyped using the other serum enzyme levels were normal. There was a test tube agglutination method (Orskov et al. 1977).
significant increase of the AST (p < 0.01) and a positive Presence of cytotoxic necrotizing factor (CNF) was correlation between AST and WBC (r ¼ 0.4; p < 0.01).
checked on VERO cells (Caprioli et al. 1983). Kirby- Progesterone levels ranged from 2 to 25 ng/ml, while Bauer method was applied for antibiotic sensitivity.
oestrogen values were at the basal levels (< 8 pg/ml).
Dow’s gross morphology criteria, histological lesion Escherichia coli was isolated from 28 of 45 cases and De Bosschere’s histomorphological classification (62.2%), all from bitches in Dow’s groups III and IV are given (Tables 1 and 2). Tissue samples (1 cm2), were (Table 3). Thirteen different serotypes were identified; collected from the middle portion of each uterine horn.
04 K) and 032 K+ strains were isolated from six and Biopsies were immediatly fixed in calcium-buffered five bitches, respectively. CNFwas detected in 14 of 28 formalin solution 10%, with pH 7.4, then embedded in (50%) E. coli-infected uteri. CNFwas identified in 50% Table 1. Dow’s gross pathology criteria and histological lesions Endometrial hyperplasia without endometritis Hyperplastic endometrium with irregular cysts Enlargement with thinning of uterine wall Endometrium and myometrium atrophic cysts Table 2. De Bosschere’s histomorphometrical classification E Bigliardi, E Parmigiani, S Cavirani, A Luppi, L Bonati and A Corradi Table 3. Bacteria isolated from uterine swabs in relationship withDow’s groups Fig. 1. Ultrasound image of uterine horn: the uterine content is iperechoic and the endometrial glands are many and large (group C) Fig. 2. Ultrasound of uterine horn with endometrial hyperplasia: severe endometrial surface lesions and endometrial glands distension high degree of correlation with Dow’s groups III and IV of E. coli serotypes in groups III–IV. In the presence of (Table 4). In several cases, an irregular hyperplastic CFN, the integrity of the endometrial epithelium was endometrial surface was also present (Figs 1 and 2).
reduced and the degree of inflammatory cell infiltration Mean values of uterine diameter were 2.9 cm (groups A–B), 5.5 cm (group C) and 4.7 cm (group D) according In addition, Streptococcus canis was isolated in five with Dow’s gross criteria (Table 5).
animals (11.1%); other bacteria (Enterobacter cloacae, The histopathology showed that the endometrial Proteus spp., Klebsiella spp. and Pseudomonas spp.) glands of bitches in groups A and B (9%) had a were detected in eight bitches (17.8%). In four cases tendency to be hyperplastic more than cystic in nature, (8.9%) uterine cultures were sterile (Table 3).
which corresponds to mucometra (De Bosschere’s clas- In all cases, ultrasound examination revealed the sification) (Table 5). The difference between groups A presence of uterine exudates such as blood, mucus, pus and B was the presence of plasma cell endometrial and cystic endometrial hyperplasia (Figs 1 and 2). These results were confirmed histopathologically (Figs 3 and 4; The dominant pathology observed (91%) was char- Table 4). Ultrasound examination was able to clearly acterized by severe endometrial gland hyperplasia with evaluate endometrial integrity, variation of uterine wall the presence of large cystic glands. These lesions are thickness, uterine distension and cystic endometrial compatible with Dow’s groups III and IV and severe glands. The glands had increased in size and number CEH and hyperplastic pyometra (De Bosschere’s clas- as endometrial anechoic areas (1–2 mm). The ultra- sification) (Table 5). On pathology, samples from bit- sound diagnosis of cystic endometrial hyperplasia has ches in group C showed a large population of shown to be mainly correct for all the cases. Indeed has inflammatory cells in the endometrium, miometrium Cystic Hyperplasia–Pyometra Complex in the Bitch Table 5. Dow’s criteria and De Bossechere’s classification of uterinesamples Fig. 3. Uterine horn section: abnormal distension of endometrial Fig. 4. Uterine horn section: presence of exudates and cystic endome- Mu, mucometra; MCEH, mild cystic endometrial hyperplasia; SCEH, cysticendometrial hyperplasia; EHP, endometritis and hyperplastic pyometra; AP, Table 4. Ultrasonography and Dow’s classification of CEH (number thinness) were classified as atrophic pyometra (AP)(Tables 2 and 5).
DiscussionProgesterone plays an important role in triggering the and also in in the endometrial glands from several development of CEH (Nelson and Kelly 1976; Hardy samples. In samples from group D, cystic endometrial and Osborne 1994) and it is logical to suppose that hyperplasia was associated with a small number treatment with exogenous progestins may increase the probability of pyometra (Blendinger et al. 1997; Koois- According to De Bosschere’s classification (Table 2), tra et al. 1997). In addition, oestrogens have stimulatory the bitches in group D with cystic endometrium, effects on uterine progesterone receptors. A recent study hypertrophic and fibrotic miometrium were classified demostrated that in case of cystic endometrial hyperpl- as hyperplastic pyometra, whereas those with atrophic asia, oestrogen and progesterone receptors are modified endometrium and miometrium (decreased uterine wall (Niskanen and Thrusfield 1988). Furthermore, certain E Bigliardi, E Parmigiani, S Cavirani, A Luppi, L Bonati and A Corradi factors associated with endometrial trauma or irritation interact with sex steroid hormones, stimulating the Blendinger K, Bostedt H, Hoffman B, 1997: Proceedings of the development of CEH (De Cook et al. 2002). Inflamma- 3rd International Symposium on Canine and Feline Repro- tion is closely associated with growth factors such as duction. J Reprod Fertil 51 (suppl.), 317.
IGF-I that have a potential mitogenic effect on the Caprioli A, Falbo V, Roda LG, Ruggeri FM, Zona C, 1983: uterus. In our study, bitches had not received exogenous Partial purification and characterization of Escherichia coli treatment of progesterone and therefore endogenously toxic factor that induces morphological cell alterations.
produced progesterone during the oestrus cycle may have induced overproduction of GH a major regulator De Cock H, Vermeirsch H, Ducatelle R, De Schepper J, 1997: of circulating IGF-I (De Cock et al. 2002). In our study Immunohistochemical analysis of estrogen receptors incystic-endometritis-pyometra complex in the bitch. Therio- 90% of the bitches had clinical signs of pyometra (groups B, C and D). We belive that when clinical signs De Cock H, Ducatelle R, Tilmant K, De Schepper J, 2002: are severe it is necessary to perform an ultrasonographic Possible role for insuline-like factor-I in the pathogenesis of examination to diagnose the degree of the lesions. Our cystic endometrial hyperplasia pyometra complex in the unpubblished data demonstred that medical treatment bitch. Theriogenology 57, 2271–2287.
of patients with severe CEH (groups C and D) is never De Cook H, Ducatelle R, Tilman K, De Schepper J, 2002: succesful. Therefore the medical treatment with Pgf2al- Possible role for insuline-like factor-I in the pathogenesis of pha and antibiotics of mild CEH cases (groups A and B) cystic endometrial hyperplasia piometra complex in the bitch. Theriogenology 57, 2271–2287.
Uterine samples from four bitches in our study were De Schepper J, Van Der Stock J, Capiau E, 1987: The characteristic pattern of aspartate aminotrasferase and bacteriologically sterile. In these cases, bacteria involved alanine aminotrasferase in the bitch with the cystic hyper- in the pathogenesis of CEH, may have been killed either plasia pyometra complex. Effect of medical or surgical by uterine defence mechanisms or by antibiotic therapy treatment. Vet Res Commun 11, 65–75.
(Dhaliwal et al. 1998). The change in AST–ALT serum De Bosschere H, Ducatelle R, Vermeirsch H, Van Den Broeck activity could be caused by the effects of E. coli W, Coryn M, 2001: Cystic endometrial hyperplasia-piome- endotoxins on liver function (De Schepper et al. 1987).
tra complex in the bitch: Should the two entities disconnec- In our study the presence of E. coli Cytotoxin ted? Theriogenology 55, 1509–1519.
Necrotising Factor (CNF) + in uterus was associated Dhaliwal GK, Wray C, Noakes DE, 1998: Uterine bacterial with clinical signs of disease (increased AST, ALT and flora and uterine lesion in bitches with cystic endometrial hyperplasia (pyometra). Veterinay Record 143, 659–661.
Dow C, 1958: The cystic hyperplasia –pyometra complex in Endometrial hyperplasia is the result of cystic defor- the bitch. Veterinary Record 70, 1102–1108.
mation of endometrial glands and stromal proliferation Hardy RM, Osborne CA, 1994: Canine pyometra: phatophys- of fibroblasts with inflammatory reaction (DeBosschere iology, diagnosis and treatment of uterine and extra-uterine et al. 2001). Ultrasonographic examination is always lesion. J Am Anim Hosp Ass 10, 245–268.
useful in detecting uterine exudate. Ultrasonographic Kooistra HS, Okkens AC, Mol JA, Van Garderen E, differential diagnosis of CEH in bitches of groups A and Kirpensteijn J, Runberk A, 1997: Proceedings of 3rd B was not always possible because the lesions were of International Symposium on Canine and Feline Reproduc- moderate degree and not identifiable. Cystic endometri- tion. J Reprod Fertil 51 (suppl.), 355.
al hyperplasia was well identified by ultrasound for McEntee K, 1990: Reproductive Pathology of Domestic bitches in groups C and D because of the extent of Mammals. San Diego, CA: Academic Press, pp. 171–176.
Nelson LW, Kelly WA, 1976: Progesteron related gross and changes in the tissue. In this study CEH involved all microscopic changes in female beagles. Vet Pathol 13, 143– bitches in various degrees. DeBosschere et al. (2001) has suggested that although CEH–mucometra and endome- Niskanen M, Thrusfield MV, 1988: Association between, tritis–pyometra may be two separate entities, it cannot parity, hormonal therapy and breed, and pyometra in be excluded that bitches with the CEH–mucometra Finnish dogs. Vet Rec 143, 493–498.
could be predisposed to endometritis–pyometra.
Orskov I, Orskov F, Jann K, 1977: Serology, chemistry, and We can say that when the clinicians can utilize high- genetics of O and K antigens of Escherichia coli. Bacteriol definition equipment, and are well-trained with good experience, the diagnostic results improve to make a Shille V, Caldrewood-Mays MB, Thatcher M, 1984: Infertility sufficiently good diagnosis and choose the best therapy.
in a bitch associated with short interestrous intervals andcyst follicles: a case report. J Am Anim Hosp Ass 20, 171– In conclusion our study has demonstrated that: (1) Ultrasound investigation is a useful and reliable method to detect pathological uterine alterations.
(2) Ultrasound is an efficient tool to classify CEH in III Authors’ address (for correspondence): Bigliardi Enrico, Unit of and IV Dow’s group and in mucometra, endome- Obstetrics and Reproduction, University of Parma Via del Taglio 8, tritis, hyperplastic pyometra (EHP) and atrophic 43100 Parma, Italy. E-mail: biglio@unipr.it pyometra (AP) of De Bosschere classification, whileit is not always reliable in I and II Dow’s groups.

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