- Email: [email protected]
Cystic endometrial hyperplasia- pyometra complex in the bitch: should the two entities be disconnected?
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ELSEVIER
CYSTIC ENDOMETRIAL HYPERPLASIA- PYOMETRA COMPLEX IN THE BITCH : SHOULD THE TWO ENTITLES BE DISCONNECTED ? H. De Bosschere, la R. Ducatelle, 1 H. Vermeirsch, 2 W. Van Den Broeck 2 and M. Coryn3 1Department of Pathology, Bacteriology and Avian Diseases 2Department of Morphology 3Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine, University of Ghent Salisburylaan 133, B-9820 Merelbeke, Belgium Received for publication: June 23, 2000 Accepted : December 13, 2000 ABSTRACT The uteri of 26 clinically healthy bitches and 42 bitches with a clinical suspicion of pyometra were examined histologically using a computerized image analysis system. Histologic lesions were characterised mainly by thickening or atrophy of the endometrium and by varying degrees of cystic changes of the glands. These lesions were observed in most of the clinically healthy bitches as well as in all of the clinically ill animals. In most of the ill bitches a variable degree of inflammation also was found. Some bitches with clinical signs indicative for pyometra had no inflammatory reaction in the uterus. These bitches were misdiagnosed as suffering from pyometra, confirming the difficulty of diagnosing pyometra by simple clinical examination. Determination of sex hormone serum levels revealed that all dogs in both groups were either in metestrus or in anestrus. Based on the results of this study the cystic endometrial hyperplasia-pyometra complex can be divided in two entities: a cystic endometrial hyperplasia-mucometra complex and an endometritis-pyometra complex. Both entities bear many similarities with each other, except for the inflammatory reaction in the endometritis-pyometra complex. It is concluded from this study that the latter complex probably does not necessarily follow the former, but that both can arise de novo. © 2001 by Elsevier Science Inc.
Keywords: dog, cystic endometrial hyperplasia, mucometra, pyometra, computer image analysis
Acknowledgments The authors wish to thank Ch. Puttevils for his technical support. The Flemish Institute for the Advancement of Scientific Technological research in the Industry (Brussels, Belgium) is gratefully acknowledged for giving a grant to H. De Bosschere. aCorrespondence and reprint address: [email protected] Theriogenology 55:1509-1519, 2001 © 2001 Elsevier Science Inc.
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Theriogenology INTRODUCTION
During the estrous cycle, the canine uterus undergoes a number ofmorphologic changes under the influence of progesterone and estrogen (4). Cystic endometrial hyperplasia (CEH) is an abnormal response of the bitch's uterus to these ovarian hormones (2,17,18,22). Indeed, CEH is assumed to be an exaggerated response of the uterus to chronic or repeated progestational stimulation during the luteal phase of the estrous cycle, with accumulation of fluid within the endomeWial glands and the uterine lumen (13). It has been considered by many authors to be the initial phase in the development of pyometra (11,17,28). The lesions of pyometra in the bitch are considered to result from bacterial and hormonal interaction (2,17,18,22), with hormonal factors playing a predisposing role in the development of the disease (2,10,17,18,21,22). Therefore it is common to refer to these entities as the cystic endometrial hyperplasia-pyometra complex. However, this supposed sequence of events was never actually proven, nor was it ever reproduced fully in experimental studies. The source of the uterine bacteria probably is the urinary tract or anogenital region. The most frequently isolated bacterium in canine pyometra is Escherichia coli (15,27,29). Specific endometrial and myometrial receptors for E. coli are present in the infected uterus, theoretically enhancing the colonization of bacteria in the uterus (17,20,27). Although progesterone stimulation is considered by many authors as the initiating step in the development of the CEH-pyometra complex, serum progesterone concentrations are not significantly different between normal animals and animals with the disorder (18). This led to speculation by our group and others that the manifestations of CEH may be mediated through abnormalities in endometrial receptors for estrogen and/or progesterone (7,8,14,30,31). In CEH the endometrium is thickened due to an increase in the size and number of endometrial glands. The hyperplastic and hypertrophic endometrial glands have an increased secretory activity, and sterile fluid may accumulate in the glands and in the lumen of the uterus, resulting in mucometra or hydrometra, depending on the viscosity of the uterine content (I 3,20). Barton (5) considered mucometra an advanced CEH, with atrophy of the uterine wall and a mucus-filled lumen. By itself, cystic endometrial hyperplasia is not associated with clinical signs (a mucometra sometimes causes abdominal distention), unless the uterine content becomes infected; this is referred to as pyometra (5). The uterine wall in cases of so called CEH-pyometra shows a wide range of lesions. These lesions originally were put into 4 categories by Dow (10), a classification that is still in use, although it obviously causes difficulties in linking lesion classification to the clinical classification. This is mainly due to the lack of lesion category for a number of patients, mainly those cases exhibiting mucometra. Because thorough analysis of lesions in the uterine wall may help us understand the pathology and pathogenetic mechanism, the purpose of the present study was to examine the histological lesions of the uterine wall in detail. We hoped to generate a classification of the lesions that could better match with the clinical observations. The present study was undertaken using a set of samples from healthy bitches and bitches with CEH-pyometra, and analyzing the samples by computerized histomorphometry.
Theriogenology
1511 MATERIALS AND METHODS
Animals Ovariohysterectomy was performed in 68 adult female dogs (ranging in age from 9 months to 15 years) of different breeds, either for the purpose of spaying (n = 26) or because of clinical signs indicative ofpyometra (n= 42). Clinical symptoms considered indicative for pyometra were lethargy, depression, anorexia, polyuria and polydipsia, vomiting, fever and in some cases distention of the abdomen. Eighteen animals had been treated with progesterone derivates for estrus suppression. Seventeen of these 18 animals received multiple treatments. Progesterone treated animals were present in all groups (groups are defined below). Tissue Samples Samples of both uterus horns were collected and fixed in a 10% phosphate-buffered formaldehyde solution for 48 hours and thereafter were processed routinely. They were embedded in paraffin, sectioned at 10 ~tm and stained with haematoxylin-eosin (HE). On the basis ofhistologicai criteria, the bitches were divided into 7 groups, as described in the results section and as shown in Table 2. Blood Samples Blood samples were taken immediately before surgery and used to determine the serum levels of sex steroids using radioimmunoassays. The concentration of progesterone was measured after extraction with petroleumether without further purification, using an antiserum against progesterone-11-hemisuccinate-BSA raised in sheep (19). The concentration of estradiol1713 was measured after extraction with diethylether without further purification using an antiserum against 1713-estradiol-3-hemisuccinate-BSA raised in sheep (19). The detection limit for progesterone was 0.005 ng and for estradiol-1713 5pg. The inter- and intra assay variations for progesterone were 7.05% and 8.75%. The inter- and intra assay variations for estradiol-1713 were 5.75% and 8.30%. Feldman and Nelson (13) reported that the plasma progesterone concentration in the anestrus bitch is low (<0.5 ng/mL). Progesterone concentrations remain below 1.0 ng/mL during proestrus and then begin to rise at the onset of estrus, typically being greater than 2.0 ng/mL. Progesterone concentrations continue to increase throughout estrus and the first several weeks of metestrus, followed by a plateau in blood levels and then a slow return toward basal concentrations. The return to concentrations less than 1.0 ng/mL marks the end of metestrus. The interval between 1.0 ng/mL and 0.5 ng/mL is considered late metestrus to early anestrus. Comparable values for progesterone concentrations related to estrus stages were used by Bledinger and others (6) although these authors used a higher concentration of progesterone to outline the different categories (2.0 ng/mL of progesterone instead of 0.5 ng/mL). The latter authors also used the estradiol-1713 concentration to determine the cycle stage. The limit for estradiol-1713 was set at 25 pg/mL. The techniques in these studies however were not the same as
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the ones used in the present study. The concentrations o f estradiol-17[3 and progesterone therefore are not really comparable at this level. In the present study, an adapted scheme of Felman and Nelson (13) and Bledinger and others (6) was used to determine the different stages of the reproductive cycle in the bitch: progesterone <0.5 ng/mL and estradiol-171] <25 pg/mL = anestrus; progesterone <1 ng/mL and estradiol-1713 >25 pg/mL = proestrus; progesterone > 1 ng/mL and estradiol-1713 >25 pg/mL = estrus; progesterone >0.5 ng/mL and estradiol-179 <25 pg/mL = metestrus. MorphometricAnalysis Morphometric analysis was done using a computerized image-analysis system composed of a video camera, a a frame grabberb and specially designed software c. The percentage of endometriurn occupied by endometriat gland lumina was calculated using the parameter "area percentage" (area%) in the software. This parameter was used to grade the cystic changes of the endometrial glands. The thickness of the endometrium and myometrium was measured in the HE-stained sections at 4 randomly selected places and was averaged. An endometrium/myometrium (endo/myo) ratio was calculated. The relative changes in thickness of endometrium and myometrium in pathological samples were compared with normal uterine samples. Pathological samples of progesterone-treated cases were compared to non treated cases within the same group. The endometrial stroma was evaluated for edema, hemorrhages, fibroblast proliferation and inflammatory reaction. StatisticalAnalysis All morphometric results were analysed with the Mann-Whitney test to a level of significance of P<0.05. The variation is expressed as the standard deviation (SD). RESULTS Progesterone-treated animals were represented in all groups. No significant difference was observed in the serum hormone levels between progesterone-treated and non treated bitches. Similar pathologies were observed in progesterone-treated bitches as in non treated bitches and the pathological changes were not more severe in progesterone-treated bitches than in non treated bitches. Therefore, in further analysis no distinction was made between progesteronetreated and non treated animals. The 68 animals represented 26 clinically healthy bitches and 42 bitches with clinical signs of pyometra (see Materials and Methods).
a Kappa type CF 15/4 MCC(RGB), Gleichen Germany b Matrox Meteor - Type RGB PPB c OPTIMAS version 6.5 - Media Cybernetics®, Silver Spring, U.S.A.
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Clinically Healthy Bitches The morphology of the uteri of 26 clinically healthy bitches was histologically very variable (Table t). One constant phenomenon however was that none of the uterine samples showed inflammatory reaction. In these clinically healthy bitches, 6 uterine samples were found with no histologic alterations. These samples were characterized by uniformity in size of the endometrial glands outlined by cylindrical (metestrus) to cuboidal (anestrus) cells. During metestrus the endometrial glands were highly branched and coiled. In anestrus the length and diameter of the endometrial glands was low. The average area% of endometrium occupied by gland lumina was 9.23% + 4.60. No cystic endometrial glands were present and the stroma showed no abnormalities. The endo/myo ratio was 0.74 + 0.30. These bitches were in the metestrous (2) and anestrus stage (4) of the cycle according to their serum sex steroid levels. In most of these clinically healthy bitches (n = 20) uterine pathologies were found. Some bitches (n= 8) had only mild changes. We classified them as mild CEH in Table 1 and Table 2. They had an average glandular luminal area% of 16.07% + 4.86, a few variable sized endometrial cysts outlined with flattened epithelial cells, an endo/myo ratio of 1.27 + 0.44 and no remarkable stromal changes. Four of these bitches were in the metestral stage and 4 in the anestral stage of the cycle. Nine other bitches had severe uterine changes. We classified them as severe CEH in Table 1 and Table 2. They had an area% of endometrium occupied by gland lumina of 42.91% + 15.04, severe cystic endometrial hyperplasia and an endo/myo ratio of 1.03 + 0.71. There was occasional stromal edema, vascular congestion and small hemorrhages. The endometrial glands were increased in number and size, and were distributed haphazardly and irregularly throughout the stroma. In some cases with extreme cyst formation the propria of the endometrium was atrophic. On the basis of progesterone and estradiol-1713 concentrations, 7 of these 9 bitches were in metestrus and 2 were in anestrus. In 3 bitches histologic lesions characteristic ofmucometra were found (Table 2). The endometrial gland lumina in these bitches occupied only 0.60% + 1.04 of the endometrium. They had severe endometrial atrophy with almost no endometrial glands. The endometrium was reduced to a narrow annular stromal tissue band surrounding the uterine lumen, which was filled with mucus. The endo/myo ratio was 0.07 + 0.06. Stromal tissue in some cases was almost absent. Hormonally one bitch was in metestrus, the other two in anestrus. Bitches with Clinical Signs Indicative of Pyometra Forty-two bitches were ill, showing clinical symptoms indicative of pyometra (see Materials and Methods). These symptoms varied in intensity. The severity of clinical symptoms was not related to the severity of uterine pathologies. In this category, one group (n= 12) histologically had no inflammatory lesions in the uterus. Thus they were misdiagnosed as pyometra cases. Most (n= 30) however had histological signs of inflammation in the uterus wall or pyometra.
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Ill Bitches with No Uterine Inflammation In this group uterine findings were similar to those described above for clinically healthy bitches. This included 5 normal uterine samples (3 in metestrus and 2 in anestms) with a glandular area% of I 1.01 + 6.45 and an endo/myo ratio of 0.83 + 0.21. Four animals with mild CEH (3 in metestrus and 1 in anestrus) had a glandular area% of 20.74 + 2.66 and an endo/myo ratio of 0.70 + 0.69. Two bitches had severe CEH (1 in metestrus and 1 in anestrus) with a glandular area% of 49.05 + 5.73 and an endo/myo ratio of 1.40 + 0.21. One animal in anestrus had a mucometra with the glandular lumina occupying 0.70 % and an endo/myo ratio of 0.03. These animals were classified into corresponding groups (Table I, cells with *). Table 1. Number of bitches in each group Group
Healthy bitches
Clinically ill bitches Total number of cases
Normal 6 Mild CEH 8 Severe CEH 9 Mucometra 3 Endometritis 0 Pyometra (hyperplastic) 0 Pyometra (atrophic) 0 Total: 26 Cells with *: Ill bitches with no uterine inflammation Cells with °: Ill bitches with uterine inflammation
5* 4* 2* 1* 4° 23 ° 3o 42
11 12 11 4 4 23 3 68
Ill Bitches with Uterine Inflammation In 4 bitches the endometrium showed a mild infiltration of inflammatory cells, neutrophils, lymphocytes and plasma cells. The area% of endometrium occupied by gland lumina was 15.60% + 4.09. The endometrial glands formed either few large cysts or several smaller cysts. The endo/myo ratio was 1.26 + 0.29. A mild stromal proliferation of fibroblasts and edema was observed. These animals were classified in the endometritis group as shown in Table 2. Hormonally one bitch was in metestrus and three were in anestrus. In 23 bitches severe inflammatory cell infiltrates were found, consisting of neutrophils, macrophages, lymphocytes and plasma cells. These inflammatory cells were present in variable proportions (depending on the sample) and were found in the uterine lumen, in the endometrial glands, in the stroma of the endometrium and in the myometrium. In the latter they were especially concentrated around the stratum vasculare between the inner and outer layers of the tunica muscularis. Severe endometrial cyst formation occupied 39.2% + 15.60 of the endometrium with the exception of two cases that had an area% of glandular lumina below 25%. The endo/myo ratio was 1.20 + 0.51. Moderate to severe stromal proliferation of fibroblasts, occasional ulceration, necrosis, edema, hemorrhages, abcess formation in blocked glands and squamous metaplasia of the endometrial surface epithelium were observed. These findings correspond with the criteria of (hyperplastic) pyometra (Table 2). Hormonally 16 bitches were in metestrus and 7 in anestrus.
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Three bitches showed severe inflammatory reaction of the endometrium and myometrium (neutrophils, lymphocytes, plasma cells and macrophages). They were different from the previous group of 23 dogs because the area% of endometrium occupied by the gland lumina was 3.60% + 3.34. There was histologically severe endometrial atrophy with almost no endometrial cysts, stroma or glands, and myometrial hyperplasia was observed. The endo/myo ratio was 0.12 + 0. l 7. The stroma of one bitch was completely ulcerated. The other two bitches showed some edema and congestion. These three animals were listed in the (atrophic) pyometra group (Table 2). The three bitches were hormonally in metestrus. On the basis of the observed histological alterations the different cases could be allocated to 7 different classes (Table 2). The number of cases represented in each group can be found in Table 1. The groups mild CEH, severe CEH and mucometra contain clinically healthy bitches and ill bitches with no uterine inflammation. The groups endometritis, (hyperplastic) pyometra and (atrophic) pyometra are represented by the clinically ill bitches with uterine inflammation (Table 1). Table 2. Criteria to catalogue pathological changes in the uterus Group Normal
Inflammatory Reaction Negative
Area Histology of %* cysts < 25% No cysts
Mild CEH
Negative
<25%
Severe CEH
Negative
>25%
Mucometra
Negative
< 10%
Endometritis
Mild
<25%
Pyometra (hyperplastic
Severe
>25%
Few/small cysts Many/large cysts No cysts No/few small cysts Many/large cysts
Endo/myo Ratio Reference value = 0.78 Increased
Fibroblast Proliferation Negative
Increased
Negative
Much decreased Increased
Negative
Increased
Moderate
Negative
Mild
) Pyometra Severe < 10% No cysts Much Negative (atrophic) decreased * Percentage of endometfium occupied by gland lumina (limits arbitrary choosen) DISCUSSION The present study bears a discrepancy between the clinical and histologic findings. Mild to severe lesions of CEH were found in clinically healthy bitches, which also were described by Allen (1) and Feldman and Nelson (13). Barton (5) associated CEH with infertility. In contrast, clinical symptoms always were observed in cases where an inflammatory reaction was found in the uterine wall. Endometritis-pyometra is obviously a clinical disease entity, whereas CEH-mucometra is not. The bacterial species most frequently isolated from bitches with pyometra is E. coli (27,29). Endotoxin from an E. coli infected uterus may produce
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Theriogenology
some of the clinical signs of pyometra (9,16). Allen (1) and Barton (5) associated inflammatory reaction with infection, and considered the inflammatory reaction in endometritis/pyometra to be the result of an uterine infection. In the present study, 12 of 42 bitches with clinical signs indicative for pyometra were observed without inflammatory reaction in the uterus. These bitches were misdiagnosed as suffering from pyometra and they were ovariohysterectomized. This confirms the difficulty of diagnosing pyometra by simple clinical examination. The clinical symptoms of pyometra are variable. Pyometra commonly is accompanied by lethargy, depression, anorexia, polyuria and polydipsia, vomiting and progression to shock (5,18). Similar symptoms also can be observed in other disorders, such as renal failure, diabetes mellitus, hyperadrenocorticism and generalized hepatic disease (18). In the present study, more cases of mild and severe CEH were found in dogs in metestrus than in anestrus. This agrees with the findings of Feldman and Nelson (13) that CEH develops during metestrus. In the present study also more clinical cases of pyometra were in metestrus than in anestrus. This supports the hypothesis that CEH does not always precede pyometra (13,25). Pyometra is not necessarily the consequence of an infected CEH uterus as suggested by Feldman and Nelson (13) and Nomura and Funahashi (25). Another argument which substantiate this claim is that the average age of bitches with CEH and of bitches with pyometra was similar, so that pyometra appeared in bitches not older than those with CEH. It is extremely rare for pyometra to occur in a bitch that was not under the influence of progesterone (metestrus) at the time that the infection began. But in some bitches the syndrome progresses slowly, presumably because the infection is mild. By the time the disease creates clinical signs in these bitches, the ovarian cycle of these bitches may have continued into anestrus, although the disease began during metestrus (13). Nomura (24) was able to reproduce CEH with artificial intra-uterine stimuli, and he also reproduced pyometra independently from CEH (23,25). To our knowledge, our study is the first report on naturally occurring CEH and pyometra that provides evidence in support of the two diseases being separate entities. This means that the CEH-mucometra entity and the endometritis-pyometra entity probably can originate independently from each other. Both entities bear many similarities with each other, except for the inflammatory reaction in the endometritis-pyometra complex. The histologic picture of CEH is very similar to that of early normal placentation in the bitch and to deciduoma in rodents (24). Normally fertilized ova are the trigger for deciduoma formation, but the initial trigger in natural cases of CEH is still unclear (25). In the pathogenesis of CEH and pyometra, each of these entities may have its own trigger in the development of lesions. Changes in sex hormone receptor concentrations as reported previously (8) may at least increase the susceptibility of the uterus to CEH. When bacteria constitute the trigger, the result is pyometra (25). The CEH-mucometra entity and the endometritis-pyometra entity have a similar histological appearance. Grading of the lesions therefore can be done similarly except for the inflammatory reaction in the second entity (Table 2): mild CEIl, severe CEH and mucometra of
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the CEH-mucometra entity correspond respectively with endometritis, (hyperplastic) pyometra and (atrophic) pyometra of the endometritis-pyometra entity. Another argument for considering CEH-mucometra and endometritis-pyometra as two separate entities is that in both entities more bitches are in the metestrus stage of the ovarian cycle than in anestrus, supporting a metestral onset of the pathology in both entities. It can not be excluded however that bitches with the CEH-mucometra entity are predisposed to develop the endomeWitis-pyometra entity. Normal development of the endometrium during the estrous cycle in the bitch is well described by Barrau and others (4). The thickness of the uterus wall increases from pro-estrus to early metestrus, and then decreases. The endometrium and myometrium both follow the same evolution (3,26). During pro-estrus the endo/myo ratio is high, due to edema and hyperplasia of the endometrium (26). During estrus the ratio decreases strongly, and then during metestrus and anestrus a slight increase is observed. From estrus to early metestrus, the uterine glands undergo mitosis and hypertrophy, forming the highly branched and coiled uterine glands. The uterine gland cells increase in size. During late metestrus the size of the uterine gland ceils reduces more rapidly than does the size of the uterine gland lumen, resulting in the cystic aspect of the gland. In this study the average endo/myo ratio of the mild CEH, severe CEH, endometritis and pyometra cases is higher than in normal cases, mainly due to endometrial hyperplasia. However, there is considerable individual variation. Endometrial hyperplasia is mainly the result of cystic deformation of glands in CEH cases and of cystic deformation of glands, stromal proliferations of fibroblasts and inflammatory reaction in endometritis-pyometra cases. In mild CEH cases it could be questioned whether the cystic deformation of uterine glands is physiological or pathological. In the latter case it could be the initiating phase of more advanced CEH, i.e. severe CEH. The large cysts in severe CEH definitely are pathological. Fayrer-Hosken and others (12) described a c a s e of CEH-pyometra with fibrosis of the endometrium. In the present study, stromal proliferation of fibroblasts was observed in all endomctfitis and (hyperplastic) pyometra cases. Stromal fibroblasts were suggested to play a role in the pathogenesis of endometritis-pyometra on the basis of their sex hormone receptor pattern (31). Because no difference was observed between progesterone-treated and non treated animals in the same group, progesterone treatment may predispose, but does not seem to enhance the pathology. LITERATURE I. Allen WE. Fertility and Obstetrics in the Dog. Oxford: Blackwell Scientific Publications,
1992;82-86. 2. Arthur GH, Noakes DE, Pearson H. Infertility in the dog and the cat. In: Arthur GH (ed), Veterinary Reproduction and Obstetrics (Theriogenology). London: Bailli~re Tindall, 1989;496-500. 3. Banks WJ. Female reproductive system. In: Applied Veterinary Histology, London: William & Wilkins, 1981 ;494-515. 4. Barrau MD, Abel JH Jr., Verhage HG, Tietz WJ. Development of the endometrium during the estrous cycle in the bitch. Am J Anat 1975;142(1):47-66.
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5. Barton C. Diseases of the uterus - Cystic endometrial hyperplasia/pyometra complex. In: Morgan RV (ed) Handbook of Small Animal Practice, 2no ed., New York: Churchill Livingstone, 1992;655-658. 6. Bledinger K, Bostedt H, Hoffmann B. Hormonal state and effects of the use of an antiprogestin in bitches with pyometra. J Reprod Fertil 1997;Supplement 51:317-325. 7. De Cock H, Ducatelle R, Logghe JP. Immunohistochemical localization of estrogen receptor in the normal canine female genital tract. Domest Anita Endocrinol 1997;14(3):133-147. 8. De Cock H, Vermeirsch H, Ducatelle R, De Schepper J. Immunohistochemical analysis of estrogen receptors in cystic-endometritis-pyometra complex in the bitch. Theriogenology 1997;48:1035-1047. 9. De Schepper J, Van Der Stock J, Capiau E. The characteristic pattern of aspartate aminotransferase and alanine aminotransferase in the bitch with the cystic hyperplasiapyometra complex: effect of medical or surgical treatment. Vet Res Commun 1987;11:6575. 10. Dow C. The cystic hyperplasia-pyometra complex in the bitch. Vet Rec 1958;70(49): 11021108. 11. Dow C. Experimental reproduction of the cystic hyperplasia-pyometra complex in the bitch. J Pathol Bacteriol 1959;78:267-278. 12. Fayrer-Hoskens R, Durham DH, Allen S, Miller-Liebl DM, Caudle AB. Follicular cystic ovaries and cystic endometrial hyperplasia in a bitch. JAMA 1992;201(1): 107-108. 13. Feldman EC, Nelson RW. Chapter 21: Cystic endometrial hyperplasia/pyometra complex. In: Canine and Feline Endocrinology and Reproduction, 2"a ed., Philadelphia: W.B. Saunders, 1996;605-618. 14. Fernandes PA, Bowen RA, Sawyer HR, Nett TM, Gorell TA. Concentration of receptors for estradiol and progesterone in canine endometrium during estrus and diestrus. Am J Vet Res 1989;50(1):64-67. 15. Fransson B, Lagerstedt A-S, Hellmen E, Jonsson P. Bacteriological findings, blood chemistry profile and plasma endotoxin levels in bitches with pyometra or other uterine diseases. J Vet Med 1997;44:417-426. 16. Gilbert RO. Diagnosis and treatment ofpyometra in bitches and queens. Compend Continuing Educ Pract 1992; 14(6):777-785. 17. Hardy RM. Cystic endometrial hyperplasia-pyometra complex. In: Shille VM (ed), Current Therapy in Theriogenology: diagnosis, treatment and prevention of reproductive diseases in animals. W.B. Saunders, Philadelphia, 1980;624-630. 18. Hardy RM, Osborne CA. Canine Pyometra: pathophysiology, diagnosis and treatment of uterine and extra-uterine lesions. J Am Anim Hosp Assoc 1974;10:245-268. 19. Henry M, Figueiredo AEF, Palhares MS, Coryn M. Clinical and endocrine aspects of the oestrous cycle in donkeys (Equus asinus). J Reprod Fertil 1987; 35(Supplement):297-303. 20. Johnson CA. Chapter 125: Cystic Endometrial Hyperplasia, Pyometra, and Infertility. In: Ettinger SJ, Feldman EC (ed): Textbook of Veterinary Internal Medicine. 4th ed. Philadelphia: W.B. Saunders, 1995; 1636-1642. 21. McEntee M. The uterus: atrophic, metaplastic, and proliferative lesions. In: MeEntee M (ed): Reproductive pathology of domestic mammals. New York: Academic Press Inc., 1990;170-176. 22. MiUer-Liebl D, Fayer-Hosken R, Caudle A, Downs M. Reproductive tract diseases that cause infertility in the bitch. Vet Med 1994;89:1047-1054.
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23. Nomura K. Canine pyometra with cystic endometrial hyperplasia experimentally induced by E. coli inoculation. Jpn J Vet Sci 1983;45(2):237-240. 24. Nomura K. Induction ofdeciduoma in the dog. J Vet Med Sci 1994;56(2):365-369. 25. Nomura K, Funahashi H. Histological characteristics of canine deciduoma induced by intrauterine inoculation of E. coli suspension. J Vet Med Sci 1999;61 (4):433-438. 26. Pineda MH. Reproductive patterns of dogs. In: Veterinary Endocrinology and Reproduction, 3rd edition, Philadelphia: Lea & Febiger, 1989;460-486. 27. Sandholm M, Vasenius H, Kivist6 A-K. Pathogenesis of canine pyometra. J Am Vet Med Assoc 1975;167:1006-1010. 28. Sevelius E, Tidholm A, Thoren-Tolling K. Pyometra in the dog. J Am Anim Hosp Assoc 1990;36:33-38. 29. Vandeplassche M, Coryn M, De Schepper J. Pyometra in the bitch: cytological, bacterial, histological and endocrinological characteristics. Vlaams Diergeneeskundig Tijdschrift 1991 ;60:207-211. 30. Vermeirsch H Simoens P, Hellemans A, Coryn M, Lauwers H. Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 2000;53:773-788. 31. Vermeirsch H, Simoens P, Lauwers H, Coryn M. Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 1999;51 (4):729-743.
•
L:t.. ,')
ELSEVIER
CYSTIC ENDOMETRIAL HYPERPLASIA- PYOMETRA COMPLEX IN THE BITCH : SHOULD THE TWO ENTITLES BE DISCONNECTED ? H. De Bosschere, la R. Ducatelle, 1 H. Vermeirsch, 2 W. Van Den Broeck 2 and M. Coryn3 1Department of Pathology, Bacteriology and Avian Diseases 2Department of Morphology 3Department of Reproduction, Obstetrics and Herd Health Faculty of Veterinary Medicine, University of Ghent Salisburylaan 133, B-9820 Merelbeke, Belgium Received for publication: June 23, 2000 Accepted : December 13, 2000 ABSTRACT The uteri of 26 clinically healthy bitches and 42 bitches with a clinical suspicion of pyometra were examined histologically using a computerized image analysis system. Histologic lesions were characterised mainly by thickening or atrophy of the endometrium and by varying degrees of cystic changes of the glands. These lesions were observed in most of the clinically healthy bitches as well as in all of the clinically ill animals. In most of the ill bitches a variable degree of inflammation also was found. Some bitches with clinical signs indicative for pyometra had no inflammatory reaction in the uterus. These bitches were misdiagnosed as suffering from pyometra, confirming the difficulty of diagnosing pyometra by simple clinical examination. Determination of sex hormone serum levels revealed that all dogs in both groups were either in metestrus or in anestrus. Based on the results of this study the cystic endometrial hyperplasia-pyometra complex can be divided in two entities: a cystic endometrial hyperplasia-mucometra complex and an endometritis-pyometra complex. Both entities bear many similarities with each other, except for the inflammatory reaction in the endometritis-pyometra complex. It is concluded from this study that the latter complex probably does not necessarily follow the former, but that both can arise de novo. © 2001 by Elsevier Science Inc.
Keywords: dog, cystic endometrial hyperplasia, mucometra, pyometra, computer image analysis
Acknowledgments The authors wish to thank Ch. Puttevils for his technical support. The Flemish Institute for the Advancement of Scientific Technological research in the Industry (Brussels, Belgium) is gratefully acknowledged for giving a grant to H. De Bosschere. aCorrespondence and reprint address: [email protected] Theriogenology 55:1509-1519, 2001 © 2001 Elsevier Science Inc.
O093-691X/O1/$-see front matter PII: S0093-691X(01 )00498-8
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Theriogenology INTRODUCTION
During the estrous cycle, the canine uterus undergoes a number ofmorphologic changes under the influence of progesterone and estrogen (4). Cystic endometrial hyperplasia (CEH) is an abnormal response of the bitch's uterus to these ovarian hormones (2,17,18,22). Indeed, CEH is assumed to be an exaggerated response of the uterus to chronic or repeated progestational stimulation during the luteal phase of the estrous cycle, with accumulation of fluid within the endomeWial glands and the uterine lumen (13). It has been considered by many authors to be the initial phase in the development of pyometra (11,17,28). The lesions of pyometra in the bitch are considered to result from bacterial and hormonal interaction (2,17,18,22), with hormonal factors playing a predisposing role in the development of the disease (2,10,17,18,21,22). Therefore it is common to refer to these entities as the cystic endometrial hyperplasia-pyometra complex. However, this supposed sequence of events was never actually proven, nor was it ever reproduced fully in experimental studies. The source of the uterine bacteria probably is the urinary tract or anogenital region. The most frequently isolated bacterium in canine pyometra is Escherichia coli (15,27,29). Specific endometrial and myometrial receptors for E. coli are present in the infected uterus, theoretically enhancing the colonization of bacteria in the uterus (17,20,27). Although progesterone stimulation is considered by many authors as the initiating step in the development of the CEH-pyometra complex, serum progesterone concentrations are not significantly different between normal animals and animals with the disorder (18). This led to speculation by our group and others that the manifestations of CEH may be mediated through abnormalities in endometrial receptors for estrogen and/or progesterone (7,8,14,30,31). In CEH the endometrium is thickened due to an increase in the size and number of endometrial glands. The hyperplastic and hypertrophic endometrial glands have an increased secretory activity, and sterile fluid may accumulate in the glands and in the lumen of the uterus, resulting in mucometra or hydrometra, depending on the viscosity of the uterine content (I 3,20). Barton (5) considered mucometra an advanced CEH, with atrophy of the uterine wall and a mucus-filled lumen. By itself, cystic endometrial hyperplasia is not associated with clinical signs (a mucometra sometimes causes abdominal distention), unless the uterine content becomes infected; this is referred to as pyometra (5). The uterine wall in cases of so called CEH-pyometra shows a wide range of lesions. These lesions originally were put into 4 categories by Dow (10), a classification that is still in use, although it obviously causes difficulties in linking lesion classification to the clinical classification. This is mainly due to the lack of lesion category for a number of patients, mainly those cases exhibiting mucometra. Because thorough analysis of lesions in the uterine wall may help us understand the pathology and pathogenetic mechanism, the purpose of the present study was to examine the histological lesions of the uterine wall in detail. We hoped to generate a classification of the lesions that could better match with the clinical observations. The present study was undertaken using a set of samples from healthy bitches and bitches with CEH-pyometra, and analyzing the samples by computerized histomorphometry.
Theriogenology
1511 MATERIALS AND METHODS
Animals Ovariohysterectomy was performed in 68 adult female dogs (ranging in age from 9 months to 15 years) of different breeds, either for the purpose of spaying (n = 26) or because of clinical signs indicative ofpyometra (n= 42). Clinical symptoms considered indicative for pyometra were lethargy, depression, anorexia, polyuria and polydipsia, vomiting, fever and in some cases distention of the abdomen. Eighteen animals had been treated with progesterone derivates for estrus suppression. Seventeen of these 18 animals received multiple treatments. Progesterone treated animals were present in all groups (groups are defined below). Tissue Samples Samples of both uterus horns were collected and fixed in a 10% phosphate-buffered formaldehyde solution for 48 hours and thereafter were processed routinely. They were embedded in paraffin, sectioned at 10 ~tm and stained with haematoxylin-eosin (HE). On the basis ofhistologicai criteria, the bitches were divided into 7 groups, as described in the results section and as shown in Table 2. Blood Samples Blood samples were taken immediately before surgery and used to determine the serum levels of sex steroids using radioimmunoassays. The concentration of progesterone was measured after extraction with petroleumether without further purification, using an antiserum against progesterone-11-hemisuccinate-BSA raised in sheep (19). The concentration of estradiol1713 was measured after extraction with diethylether without further purification using an antiserum against 1713-estradiol-3-hemisuccinate-BSA raised in sheep (19). The detection limit for progesterone was 0.005 ng and for estradiol-1713 5pg. The inter- and intra assay variations for progesterone were 7.05% and 8.75%. The inter- and intra assay variations for estradiol-1713 were 5.75% and 8.30%. Feldman and Nelson (13) reported that the plasma progesterone concentration in the anestrus bitch is low (<0.5 ng/mL). Progesterone concentrations remain below 1.0 ng/mL during proestrus and then begin to rise at the onset of estrus, typically being greater than 2.0 ng/mL. Progesterone concentrations continue to increase throughout estrus and the first several weeks of metestrus, followed by a plateau in blood levels and then a slow return toward basal concentrations. The return to concentrations less than 1.0 ng/mL marks the end of metestrus. The interval between 1.0 ng/mL and 0.5 ng/mL is considered late metestrus to early anestrus. Comparable values for progesterone concentrations related to estrus stages were used by Bledinger and others (6) although these authors used a higher concentration of progesterone to outline the different categories (2.0 ng/mL of progesterone instead of 0.5 ng/mL). The latter authors also used the estradiol-1713 concentration to determine the cycle stage. The limit for estradiol-1713 was set at 25 pg/mL. The techniques in these studies however were not the same as
Theriogenology
1512
the ones used in the present study. The concentrations o f estradiol-17[3 and progesterone therefore are not really comparable at this level. In the present study, an adapted scheme of Felman and Nelson (13) and Bledinger and others (6) was used to determine the different stages of the reproductive cycle in the bitch: progesterone <0.5 ng/mL and estradiol-171] <25 pg/mL = anestrus; progesterone <1 ng/mL and estradiol-1713 >25 pg/mL = proestrus; progesterone > 1 ng/mL and estradiol-1713 >25 pg/mL = estrus; progesterone >0.5 ng/mL and estradiol-179 <25 pg/mL = metestrus. MorphometricAnalysis Morphometric analysis was done using a computerized image-analysis system composed of a video camera, a a frame grabberb and specially designed software c. The percentage of endometriurn occupied by endometriat gland lumina was calculated using the parameter "area percentage" (area%) in the software. This parameter was used to grade the cystic changes of the endometrial glands. The thickness of the endometrium and myometrium was measured in the HE-stained sections at 4 randomly selected places and was averaged. An endometrium/myometrium (endo/myo) ratio was calculated. The relative changes in thickness of endometrium and myometrium in pathological samples were compared with normal uterine samples. Pathological samples of progesterone-treated cases were compared to non treated cases within the same group. The endometrial stroma was evaluated for edema, hemorrhages, fibroblast proliferation and inflammatory reaction. StatisticalAnalysis All morphometric results were analysed with the Mann-Whitney test to a level of significance of P<0.05. The variation is expressed as the standard deviation (SD). RESULTS Progesterone-treated animals were represented in all groups. No significant difference was observed in the serum hormone levels between progesterone-treated and non treated bitches. Similar pathologies were observed in progesterone-treated bitches as in non treated bitches and the pathological changes were not more severe in progesterone-treated bitches than in non treated bitches. Therefore, in further analysis no distinction was made between progesteronetreated and non treated animals. The 68 animals represented 26 clinically healthy bitches and 42 bitches with clinical signs of pyometra (see Materials and Methods).
a Kappa type CF 15/4 MCC(RGB), Gleichen Germany b Matrox Meteor - Type RGB PPB c OPTIMAS version 6.5 - Media Cybernetics®, Silver Spring, U.S.A.
Theriogenology
1513
Clinically Healthy Bitches The morphology of the uteri of 26 clinically healthy bitches was histologically very variable (Table t). One constant phenomenon however was that none of the uterine samples showed inflammatory reaction. In these clinically healthy bitches, 6 uterine samples were found with no histologic alterations. These samples were characterized by uniformity in size of the endometrial glands outlined by cylindrical (metestrus) to cuboidal (anestrus) cells. During metestrus the endometrial glands were highly branched and coiled. In anestrus the length and diameter of the endometrial glands was low. The average area% of endometrium occupied by gland lumina was 9.23% + 4.60. No cystic endometrial glands were present and the stroma showed no abnormalities. The endo/myo ratio was 0.74 + 0.30. These bitches were in the metestrous (2) and anestrus stage (4) of the cycle according to their serum sex steroid levels. In most of these clinically healthy bitches (n = 20) uterine pathologies were found. Some bitches (n= 8) had only mild changes. We classified them as mild CEH in Table 1 and Table 2. They had an average glandular luminal area% of 16.07% + 4.86, a few variable sized endometrial cysts outlined with flattened epithelial cells, an endo/myo ratio of 1.27 + 0.44 and no remarkable stromal changes. Four of these bitches were in the metestral stage and 4 in the anestral stage of the cycle. Nine other bitches had severe uterine changes. We classified them as severe CEH in Table 1 and Table 2. They had an area% of endometrium occupied by gland lumina of 42.91% + 15.04, severe cystic endometrial hyperplasia and an endo/myo ratio of 1.03 + 0.71. There was occasional stromal edema, vascular congestion and small hemorrhages. The endometrial glands were increased in number and size, and were distributed haphazardly and irregularly throughout the stroma. In some cases with extreme cyst formation the propria of the endometrium was atrophic. On the basis of progesterone and estradiol-1713 concentrations, 7 of these 9 bitches were in metestrus and 2 were in anestrus. In 3 bitches histologic lesions characteristic ofmucometra were found (Table 2). The endometrial gland lumina in these bitches occupied only 0.60% + 1.04 of the endometrium. They had severe endometrial atrophy with almost no endometrial glands. The endometrium was reduced to a narrow annular stromal tissue band surrounding the uterine lumen, which was filled with mucus. The endo/myo ratio was 0.07 + 0.06. Stromal tissue in some cases was almost absent. Hormonally one bitch was in metestrus, the other two in anestrus. Bitches with Clinical Signs Indicative of Pyometra Forty-two bitches were ill, showing clinical symptoms indicative of pyometra (see Materials and Methods). These symptoms varied in intensity. The severity of clinical symptoms was not related to the severity of uterine pathologies. In this category, one group (n= 12) histologically had no inflammatory lesions in the uterus. Thus they were misdiagnosed as pyometra cases. Most (n= 30) however had histological signs of inflammation in the uterus wall or pyometra.
1514
Theriogenology
Ill Bitches with No Uterine Inflammation In this group uterine findings were similar to those described above for clinically healthy bitches. This included 5 normal uterine samples (3 in metestrus and 2 in anestms) with a glandular area% of I 1.01 + 6.45 and an endo/myo ratio of 0.83 + 0.21. Four animals with mild CEH (3 in metestrus and 1 in anestrus) had a glandular area% of 20.74 + 2.66 and an endo/myo ratio of 0.70 + 0.69. Two bitches had severe CEH (1 in metestrus and 1 in anestrus) with a glandular area% of 49.05 + 5.73 and an endo/myo ratio of 1.40 + 0.21. One animal in anestrus had a mucometra with the glandular lumina occupying 0.70 % and an endo/myo ratio of 0.03. These animals were classified into corresponding groups (Table I, cells with *). Table 1. Number of bitches in each group Group
Healthy bitches
Clinically ill bitches Total number of cases
Normal 6 Mild CEH 8 Severe CEH 9 Mucometra 3 Endometritis 0 Pyometra (hyperplastic) 0 Pyometra (atrophic) 0 Total: 26 Cells with *: Ill bitches with no uterine inflammation Cells with °: Ill bitches with uterine inflammation
5* 4* 2* 1* 4° 23 ° 3o 42
11 12 11 4 4 23 3 68
Ill Bitches with Uterine Inflammation In 4 bitches the endometrium showed a mild infiltration of inflammatory cells, neutrophils, lymphocytes and plasma cells. The area% of endometrium occupied by gland lumina was 15.60% + 4.09. The endometrial glands formed either few large cysts or several smaller cysts. The endo/myo ratio was 1.26 + 0.29. A mild stromal proliferation of fibroblasts and edema was observed. These animals were classified in the endometritis group as shown in Table 2. Hormonally one bitch was in metestrus and three were in anestrus. In 23 bitches severe inflammatory cell infiltrates were found, consisting of neutrophils, macrophages, lymphocytes and plasma cells. These inflammatory cells were present in variable proportions (depending on the sample) and were found in the uterine lumen, in the endometrial glands, in the stroma of the endometrium and in the myometrium. In the latter they were especially concentrated around the stratum vasculare between the inner and outer layers of the tunica muscularis. Severe endometrial cyst formation occupied 39.2% + 15.60 of the endometrium with the exception of two cases that had an area% of glandular lumina below 25%. The endo/myo ratio was 1.20 + 0.51. Moderate to severe stromal proliferation of fibroblasts, occasional ulceration, necrosis, edema, hemorrhages, abcess formation in blocked glands and squamous metaplasia of the endometrial surface epithelium were observed. These findings correspond with the criteria of (hyperplastic) pyometra (Table 2). Hormonally 16 bitches were in metestrus and 7 in anestrus.
Theriogenology
1515
Three bitches showed severe inflammatory reaction of the endometrium and myometrium (neutrophils, lymphocytes, plasma cells and macrophages). They were different from the previous group of 23 dogs because the area% of endometrium occupied by the gland lumina was 3.60% + 3.34. There was histologically severe endometrial atrophy with almost no endometrial cysts, stroma or glands, and myometrial hyperplasia was observed. The endo/myo ratio was 0.12 + 0. l 7. The stroma of one bitch was completely ulcerated. The other two bitches showed some edema and congestion. These three animals were listed in the (atrophic) pyometra group (Table 2). The three bitches were hormonally in metestrus. On the basis of the observed histological alterations the different cases could be allocated to 7 different classes (Table 2). The number of cases represented in each group can be found in Table 1. The groups mild CEH, severe CEH and mucometra contain clinically healthy bitches and ill bitches with no uterine inflammation. The groups endometritis, (hyperplastic) pyometra and (atrophic) pyometra are represented by the clinically ill bitches with uterine inflammation (Table 1). Table 2. Criteria to catalogue pathological changes in the uterus Group Normal
Inflammatory Reaction Negative
Area Histology of %* cysts < 25% No cysts
Mild CEH
Negative
<25%
Severe CEH
Negative
>25%
Mucometra
Negative
< 10%
Endometritis
Mild
<25%
Pyometra (hyperplastic
Severe
>25%
Few/small cysts Many/large cysts No cysts No/few small cysts Many/large cysts
Endo/myo Ratio Reference value = 0.78 Increased
Fibroblast Proliferation Negative
Increased
Negative
Much decreased Increased
Negative
Increased
Moderate
Negative
Mild
) Pyometra Severe < 10% No cysts Much Negative (atrophic) decreased * Percentage of endometfium occupied by gland lumina (limits arbitrary choosen) DISCUSSION The present study bears a discrepancy between the clinical and histologic findings. Mild to severe lesions of CEH were found in clinically healthy bitches, which also were described by Allen (1) and Feldman and Nelson (13). Barton (5) associated CEH with infertility. In contrast, clinical symptoms always were observed in cases where an inflammatory reaction was found in the uterine wall. Endometritis-pyometra is obviously a clinical disease entity, whereas CEH-mucometra is not. The bacterial species most frequently isolated from bitches with pyometra is E. coli (27,29). Endotoxin from an E. coli infected uterus may produce
1516
Theriogenology
some of the clinical signs of pyometra (9,16). Allen (1) and Barton (5) associated inflammatory reaction with infection, and considered the inflammatory reaction in endometritis/pyometra to be the result of an uterine infection. In the present study, 12 of 42 bitches with clinical signs indicative for pyometra were observed without inflammatory reaction in the uterus. These bitches were misdiagnosed as suffering from pyometra and they were ovariohysterectomized. This confirms the difficulty of diagnosing pyometra by simple clinical examination. The clinical symptoms of pyometra are variable. Pyometra commonly is accompanied by lethargy, depression, anorexia, polyuria and polydipsia, vomiting and progression to shock (5,18). Similar symptoms also can be observed in other disorders, such as renal failure, diabetes mellitus, hyperadrenocorticism and generalized hepatic disease (18). In the present study, more cases of mild and severe CEH were found in dogs in metestrus than in anestrus. This agrees with the findings of Feldman and Nelson (13) that CEH develops during metestrus. In the present study also more clinical cases of pyometra were in metestrus than in anestrus. This supports the hypothesis that CEH does not always precede pyometra (13,25). Pyometra is not necessarily the consequence of an infected CEH uterus as suggested by Feldman and Nelson (13) and Nomura and Funahashi (25). Another argument which substantiate this claim is that the average age of bitches with CEH and of bitches with pyometra was similar, so that pyometra appeared in bitches not older than those with CEH. It is extremely rare for pyometra to occur in a bitch that was not under the influence of progesterone (metestrus) at the time that the infection began. But in some bitches the syndrome progresses slowly, presumably because the infection is mild. By the time the disease creates clinical signs in these bitches, the ovarian cycle of these bitches may have continued into anestrus, although the disease began during metestrus (13). Nomura (24) was able to reproduce CEH with artificial intra-uterine stimuli, and he also reproduced pyometra independently from CEH (23,25). To our knowledge, our study is the first report on naturally occurring CEH and pyometra that provides evidence in support of the two diseases being separate entities. This means that the CEH-mucometra entity and the endometritis-pyometra entity probably can originate independently from each other. Both entities bear many similarities with each other, except for the inflammatory reaction in the endometritis-pyometra complex. The histologic picture of CEH is very similar to that of early normal placentation in the bitch and to deciduoma in rodents (24). Normally fertilized ova are the trigger for deciduoma formation, but the initial trigger in natural cases of CEH is still unclear (25). In the pathogenesis of CEH and pyometra, each of these entities may have its own trigger in the development of lesions. Changes in sex hormone receptor concentrations as reported previously (8) may at least increase the susceptibility of the uterus to CEH. When bacteria constitute the trigger, the result is pyometra (25). The CEH-mucometra entity and the endometritis-pyometra entity have a similar histological appearance. Grading of the lesions therefore can be done similarly except for the inflammatory reaction in the second entity (Table 2): mild CEIl, severe CEH and mucometra of
Theriogenology
1517
the CEH-mucometra entity correspond respectively with endometritis, (hyperplastic) pyometra and (atrophic) pyometra of the endometritis-pyometra entity. Another argument for considering CEH-mucometra and endometritis-pyometra as two separate entities is that in both entities more bitches are in the metestrus stage of the ovarian cycle than in anestrus, supporting a metestral onset of the pathology in both entities. It can not be excluded however that bitches with the CEH-mucometra entity are predisposed to develop the endomeWitis-pyometra entity. Normal development of the endometrium during the estrous cycle in the bitch is well described by Barrau and others (4). The thickness of the uterus wall increases from pro-estrus to early metestrus, and then decreases. The endometrium and myometrium both follow the same evolution (3,26). During pro-estrus the endo/myo ratio is high, due to edema and hyperplasia of the endometrium (26). During estrus the ratio decreases strongly, and then during metestrus and anestrus a slight increase is observed. From estrus to early metestrus, the uterine glands undergo mitosis and hypertrophy, forming the highly branched and coiled uterine glands. The uterine gland cells increase in size. During late metestrus the size of the uterine gland ceils reduces more rapidly than does the size of the uterine gland lumen, resulting in the cystic aspect of the gland. In this study the average endo/myo ratio of the mild CEH, severe CEH, endometritis and pyometra cases is higher than in normal cases, mainly due to endometrial hyperplasia. However, there is considerable individual variation. Endometrial hyperplasia is mainly the result of cystic deformation of glands in CEH cases and of cystic deformation of glands, stromal proliferations of fibroblasts and inflammatory reaction in endometritis-pyometra cases. In mild CEH cases it could be questioned whether the cystic deformation of uterine glands is physiological or pathological. In the latter case it could be the initiating phase of more advanced CEH, i.e. severe CEH. The large cysts in severe CEH definitely are pathological. Fayrer-Hosken and others (12) described a c a s e of CEH-pyometra with fibrosis of the endometrium. In the present study, stromal proliferation of fibroblasts was observed in all endomctfitis and (hyperplastic) pyometra cases. Stromal fibroblasts were suggested to play a role in the pathogenesis of endometritis-pyometra on the basis of their sex hormone receptor pattern (31). Because no difference was observed between progesterone-treated and non treated animals in the same group, progesterone treatment may predispose, but does not seem to enhance the pathology. LITERATURE I. Allen WE. Fertility and Obstetrics in the Dog. Oxford: Blackwell Scientific Publications,
1992;82-86. 2. Arthur GH, Noakes DE, Pearson H. Infertility in the dog and the cat. In: Arthur GH (ed), Veterinary Reproduction and Obstetrics (Theriogenology). London: Bailli~re Tindall, 1989;496-500. 3. Banks WJ. Female reproductive system. In: Applied Veterinary Histology, London: William & Wilkins, 1981 ;494-515. 4. Barrau MD, Abel JH Jr., Verhage HG, Tietz WJ. Development of the endometrium during the estrous cycle in the bitch. Am J Anat 1975;142(1):47-66.
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5. Barton C. Diseases of the uterus - Cystic endometrial hyperplasia/pyometra complex. In: Morgan RV (ed) Handbook of Small Animal Practice, 2no ed., New York: Churchill Livingstone, 1992;655-658. 6. Bledinger K, Bostedt H, Hoffmann B. Hormonal state and effects of the use of an antiprogestin in bitches with pyometra. J Reprod Fertil 1997;Supplement 51:317-325. 7. De Cock H, Ducatelle R, Logghe JP. Immunohistochemical localization of estrogen receptor in the normal canine female genital tract. Domest Anita Endocrinol 1997;14(3):133-147. 8. De Cock H, Vermeirsch H, Ducatelle R, De Schepper J. Immunohistochemical analysis of estrogen receptors in cystic-endometritis-pyometra complex in the bitch. Theriogenology 1997;48:1035-1047. 9. De Schepper J, Van Der Stock J, Capiau E. The characteristic pattern of aspartate aminotransferase and alanine aminotransferase in the bitch with the cystic hyperplasiapyometra complex: effect of medical or surgical treatment. Vet Res Commun 1987;11:6575. 10. Dow C. The cystic hyperplasia-pyometra complex in the bitch. Vet Rec 1958;70(49): 11021108. 11. Dow C. Experimental reproduction of the cystic hyperplasia-pyometra complex in the bitch. J Pathol Bacteriol 1959;78:267-278. 12. Fayrer-Hoskens R, Durham DH, Allen S, Miller-Liebl DM, Caudle AB. Follicular cystic ovaries and cystic endometrial hyperplasia in a bitch. JAMA 1992;201(1): 107-108. 13. Feldman EC, Nelson RW. Chapter 21: Cystic endometrial hyperplasia/pyometra complex. In: Canine and Feline Endocrinology and Reproduction, 2"a ed., Philadelphia: W.B. Saunders, 1996;605-618. 14. Fernandes PA, Bowen RA, Sawyer HR, Nett TM, Gorell TA. Concentration of receptors for estradiol and progesterone in canine endometrium during estrus and diestrus. Am J Vet Res 1989;50(1):64-67. 15. Fransson B, Lagerstedt A-S, Hellmen E, Jonsson P. Bacteriological findings, blood chemistry profile and plasma endotoxin levels in bitches with pyometra or other uterine diseases. J Vet Med 1997;44:417-426. 16. Gilbert RO. Diagnosis and treatment ofpyometra in bitches and queens. Compend Continuing Educ Pract 1992; 14(6):777-785. 17. Hardy RM. Cystic endometrial hyperplasia-pyometra complex. In: Shille VM (ed), Current Therapy in Theriogenology: diagnosis, treatment and prevention of reproductive diseases in animals. W.B. Saunders, Philadelphia, 1980;624-630. 18. Hardy RM, Osborne CA. Canine Pyometra: pathophysiology, diagnosis and treatment of uterine and extra-uterine lesions. J Am Anim Hosp Assoc 1974;10:245-268. 19. Henry M, Figueiredo AEF, Palhares MS, Coryn M. Clinical and endocrine aspects of the oestrous cycle in donkeys (Equus asinus). J Reprod Fertil 1987; 35(Supplement):297-303. 20. Johnson CA. Chapter 125: Cystic Endometrial Hyperplasia, Pyometra, and Infertility. In: Ettinger SJ, Feldman EC (ed): Textbook of Veterinary Internal Medicine. 4th ed. Philadelphia: W.B. Saunders, 1995; 1636-1642. 21. McEntee M. The uterus: atrophic, metaplastic, and proliferative lesions. In: MeEntee M (ed): Reproductive pathology of domestic mammals. New York: Academic Press Inc., 1990;170-176. 22. MiUer-Liebl D, Fayer-Hosken R, Caudle A, Downs M. Reproductive tract diseases that cause infertility in the bitch. Vet Med 1994;89:1047-1054.
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23. Nomura K. Canine pyometra with cystic endometrial hyperplasia experimentally induced by E. coli inoculation. Jpn J Vet Sci 1983;45(2):237-240. 24. Nomura K. Induction ofdeciduoma in the dog. J Vet Med Sci 1994;56(2):365-369. 25. Nomura K, Funahashi H. Histological characteristics of canine deciduoma induced by intrauterine inoculation of E. coli suspension. J Vet Med Sci 1999;61 (4):433-438. 26. Pineda MH. Reproductive patterns of dogs. In: Veterinary Endocrinology and Reproduction, 3rd edition, Philadelphia: Lea & Febiger, 1989;460-486. 27. Sandholm M, Vasenius H, Kivist6 A-K. Pathogenesis of canine pyometra. J Am Vet Med Assoc 1975;167:1006-1010. 28. Sevelius E, Tidholm A, Thoren-Tolling K. Pyometra in the dog. J Am Anim Hosp Assoc 1990;36:33-38. 29. Vandeplassche M, Coryn M, De Schepper J. Pyometra in the bitch: cytological, bacterial, histological and endocrinological characteristics. Vlaams Diergeneeskundig Tijdschrift 1991 ;60:207-211. 30. Vermeirsch H Simoens P, Hellemans A, Coryn M, Lauwers H. Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 2000;53:773-788. 31. Vermeirsch H, Simoens P, Lauwers H, Coryn M. Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 1999;51 (4):729-743.