- Email: [email protected]
Ovarian Abnormalities in the Bitch
J.
CO~IP. PATH.
1960. VOL. 70.
59
OVARIAN ABNORMALITIES IN THE BITCH By
C. Dow Veterinary School, Universi!J of Glasgow INTRODUCTION
Pathological studies of the canine female genital tract have been largely concerned with uterine disease; ovarian abnormalities have received little attention. Mulligan (1949) observed 7 ovarian tumours in a survey of 1000 tumours of the dog. Cotchin (1951, 1954) found that only 24 of 2361 canine tumours were of ovarian origin. Bloom (1954) recorded a variety of ovarian tumours in the bitch but gave no indication of incidence. None of these workers gave any indication of the frequency of ovarian tumours in the dog population, nor did they record the incidence of these tumours with respect to age or parity. There is no adequate description of the histological and histochemical features of these canine neoplasms. This publication is based on the examination of the ovaries of 400 unselected bitches submitted for routine autopsy in the University of Glasgow Veterinary Hospital. MATERIALS AND METHODS
Each ovary was sectioned through the hilus, fixed in formol-sublimate or neutral formol, dehydrated and cleared in an alcohol-amyl acetatebenzene series and double-embedded in celloidin and paraffin. Routine staining was by haemalum and eosin; other methods used in selected cases were picro-Mallory, Gordon and Sweet's reticulin stain, toluidine blue, alcian blue, Southgate's mucicarmine, sudan black B, periodic acid-Schiff before and after hydrolysis with diastase and the methylene blue extinction test. RESULTS
Incidence One hundred and fifteen of the 400 bitches examined showed ovarian lesions, an incidence of 28'75 per cent. The relationship of the various changes to age is shown in Fig. I. As would be expected these are diseases of adult life and increase with age. Of the 400 animals in the survey, 93 had a total of 127 tumours of various types in one or more sites. As twenty-five bitches had primary ovarian tumours, 27 per cent. of all tumour cases and 20 per cent. of all tumours were ovarian in origin. In only one case was the ovarian tumour considered to be the primary cause of disease. All ovarian tumours were in bitches of 5 years and over. The frequency of each ovarian lesion is shown in Table I. Tumours Granulosa Cell Tumours Though tumours of granulosa cell ongm were found m 13
60
OVARIAN ABNORMALITIES IN THE BITCH
Fig.
I
100
r--
r--
90 80 70 r0-
6O
r
Z UJ
r0-
c-
50
I-
r--r--
U 40
a: ~ 30
-
20
IC I
rIl-f
2 345 6
r-
r--
8 9 10 II 12 13 14 15 16
AGE Fig.
I.
The age incidence of ovarian abnormalities in the bitch.
animals, only in 9 were the ovaries considered abnormal at autopsy. The bitches ranged from 5 to 15 years and IO of them were nulliparous. The tumours were commonly round or oval with a smooth or slightly lobulated surface and ranged from 0'4 to IO cm. in diameter. The smaller tumours were solid on section, but some of the larger ones were soft and friable and contained cystic spaces of various sizes. The cut surface had a granular appearance and was cream or faintly yellow with areas of haemorrhage and necrosis in the larger masses. Metastases were not observed in any of these cases though a microscopic tumour nodule was found in the contralateral ovary of one case. The unaffected ovaries contained corpora lutea in 9 animals and were inactive in the other 4 cases. There was an ascites and hydrothorax of undetermined origin in one animal. Histology. The predominant cell type in 9 cases was similar to the granulosa cells present in normal medium-sized follicles. The cells were small and polyhedral with a small amount of palely eosinophilic cytoplasm in which there were a moderate number of argentophil granules. The relatively large round or oval nuclei were central in position. They had an abundant chromatin network and a well-defined nuclear membrane. Mitotic figures were rare in most cases. The histological pattern varied considerably, not only from case to case but also in different fields of individual sections. All were well encapsulated and showed no evidence of local infiltration. The most common arrangement was diffuse fields of granulosa cells in which a variable number of folliculoid structures occurred. These acini were lined by a layer of columnar type cells and contained an eosinophilic fluid. They varied in size from small Call-Exner rosettes to relatively
3
,:
14 15
,6
I
I'
'I
.--I~----
0 I
I
2
6 3
23 18
13
II 12
2
34
10
0 2 2 I
I
0 0
0 0
37
39
I
I I
I
i
I
0 0
0
0 0
0
0 I 0 I
0 0 0
0 0
I
1
I
I
0 0
0
0 0
0
I
0 0: 0 [
0
1
o! 0
0 0
1 _ _1
Adenocarcinoma
0 0
0
0 0
0
0 0 I 0
0 0 0
0 0
Fibroma
:--1---;---- :--1"
0 0
0
2
0 I 2
0 0 0
0 0
Granulosa I Pseudocell I Serous mucinous tumours I cystadenoma' cystadenoma
8 9
7
39 42
23 33 32
3 4 5
6
39 27
No. oj animals
I 2
Age
TABLE
1
I
I
0
2
3
3
4 4
I
I'
I
I, 2
I
1 0 '0 0
0 0,
0 0
I 2 0
0 0 0
0 [
Lutein cysts
Germinal cysts
I
I'
0 0
I
2
3
4
2
2
3 3
0 0 0
I 0 '0
1----
0
I 0
2
3
0 0 0
0 0
Rete cysts
0 0
,: " -~:~I ,: :
2
5 4
5
4 5
4 3
3
2
0 [
Solitary Multiple follicular follicular cysts cysts 1--
AGE INCIDENCE AND LESIONS FOUND
tI
Ol
:E
o
()
OVARIAN ABNORMALITIES IN THE BITCH
large cysts (Fig. 2). In one of the tumours there were several large cystic cavities lined by many layers of granulosa cells which were surrounded by a distinct connective tissue capsule. These structures resembled the folliculoma malignum of von Kahlden. The fluid within the cysts was weakly P.A.S. positive, but was not metachromatic and did not stain with alcian blue or with Southgate's mucicarmine. Methylene blue extinction occurred at pH 5. The connective tissue stroma in the majority of these cases was not conspicuous and reticulin fibres did not penetrate the granulosa cell masses. In 2 cases there were numerous tissue trabeculae extending in from the capsule and splitting up the granulosa cells into round or oval columns. This connective tissue proliferation was very prominent in one case and granulosa cells appeared as small isolated islands or columns. In this cylindromatous pattern, the cells immediately adjacent to the trabeculae were columnar in type and lay on a thin layer of argyrophil fibres. In one specimen of the diffuse cellular type there were areas of transition from typical granulosa cells to plump, fusiform cells resembling those of the theca interna. These thecal cells were arranged in an irregular interlacing manner and were separated by distinct bands of fibrous tissue. Unlike the granulosa cells, they had a well-developed reticulin network and did not have intra-cytoplasmic argentophil granules. Both the granulosa and thecal cells in this tumour contained small numbers of lipid granules. There was transformation of the cells into typical granulosa lutein cells in 3 of these tumours. In 2 cases this change was focal and occurred mainly in the centre of diffuse cellular masses, but in the third case luteinisation had occurred in the majority of the cells. The transitional stages between granulosa and lutein cells were similar to those observed in the normal follicle during the later stages of maturation. There was a gradual increase in the size and in the lipid content of the cells as the degree of luteinisation increased. Though hormone assays were not performed on body fluids or on tumour tissue some evidence of ovarian functional activity could be gained from histological examination of the uterus, vagina and skin. The vaginal epithelium was hyperplastic and exhibited a degree of cornification indicative of oestrogenic activity in 3 cases in which the contralateral ovary was atrophic. In these, the endometrium showed slight glandular dilatation and stromal fibrosis similar to that observed following prolonged oestrogen treatment. One of these animals showed atrophy of the epidermis and hair follicles suggestive of prolonged oestrogenic activity. There were 9 cases in which the vaginal and endometrial histology was compatible with metoestrus. In 7 of them the contralateral ovary contained corpora lutea and the other 2 were associated with luteinised tumours. There was no evidence of progestational activity in the endometrium or vagina of the third luteinised tumour.
Serous Cystadenoma Tumours of this type were found in 8 animals ranging from 6 to 13 years old. Five of them were nulliparous and only one had whelped more than one litter. Six of the tumours were small unilocular structures of 0·8 to 1·5 cm. in diameter and 2 were multilocular masses of 6 to 8 cm. in diameter. Intraluminal papillary
C. DOW
growth was prominent in most specimens. The contralateral ovary contained follicular cysts in 3 cases and corpora lutea in 2 cases. Histology. There was evidence of multicentric origin in all the small tumours with the formation of microscopic papillary cysts in other parts of the ovarian cortex. Papillary cysts were present in the contralateral ovary in 2 cases. The walls of the cysts were composed of dense vascular connective tissue which was hyaline in some of the larger specimens. Arising from this capsule were a number of intracystic papilligerous processes which almost occluded the lumen in some of the smaller cysts. These processes varied greatly in size and configuration. They were commonly slender and villiform with fine connective tissue cores in the smaller cysts. In the larger cysts, the processes were thicker and more irregular in shape. Some were simple papillae with broad cores of oedematous connective tissue, whilst others exhibited a more complex arborescent pattern (Fig. 3). In the latter, numerous acini were included in the substance of the processes, producing an adenomatous appearance. Extracystic growth was not prominent but small solid papillomata were present in 3 cases. Small calcified granules, the so-called psammoma bodies, were present in the stroma of 2 of the tumours. The lining epithelium was composed of a single layer of cells which varied considerably in type, even in individual cysts. The predominant type of epithelium was composed of cuboidal or low columnar cells with central vesicular nuclei and homogeneous cytoplasm. The epithelium in 2 cases showed certain similarities to that of the oviduct and was composed of tall ciliated columnar cells and intercalary cells with deeply basophilic cytoplasm. A few Schiff-positive granules were present in these tubal-type cells but not elsewhere. Lipid was not present in the epithelium of any of the tumours. The cyst lumina contained pale eosinophilic fluid with an admixture of cell debris and blood in some cases This fluid was Schiff-positive, but was not metachromatic and did not stain with alcian blue or Southgate's mucicarmine. It did not contain lipid. There was no histological evidence of derangement of the oestrus cycle due to these tumours. The endometrial and vaginal histology was compatible with the functional activity shown by the contralateral ovary.
Pseudomucinous Cystadenoma Two tumours of this type were found in maiden bitches of 7 and 9 years old. The older animal had shown some evidence of oestral abnormality for almost 2 years. The tumours formed unilateral lobulated masses of 7 to IO cm. in diameter. On section, each was composed of a number of lobules containing a viscid milky fluid. The cyst walls were thin and translucent with only occasional papillary growths. Histology. The tumours were composed of a number of relatively large cysts surrounded by a honeycombed mass of microscopic acini (Fig. 4). The cysts had a well-defined connective tissue capsule containing coarse collagen fibres and dense bundles of elastin. Smooth muscle fibres were not evident in the capsule. The few papillae which projected into the cyst lumina were long and slender. The cyst epithelium rested on an
ov ARrAN
ABNORMALITIES IN THE BITCH
argyrophil basement membrane which was particularly prominent In the small locules. The cysts were lined by a single layer of very tall columnar cells with clear refractile cytoplasm. The oval nuclei which were deeply basophilic were situated close to the basement membrane. Small nucleoli were evident in many nuclei. The cell membranes were prominent and there were no cilia on the free border. Large goblet cells were numerous in the epithelium of some cysts and absent in others. The cytoplasm of all the epithelial cells contained aggregated masses of Schiff-positive material. This material also stained with mucicarmine and alcian blue and showed B-metachromasia with toluidine blue. The cyst lumina also contained fluid of this nature. The cytoplasm of some of the cells also contained a number of minute, finely dispersed lipid droplets. Adena-carcinoma Autopsy of a 9 year old maiden bitch revealed a nodular mass 9 X 7 cm. in the left ovary. On section, the bulk of the tumour was firm and solid, but a cystic cavity of 2 cm. diameter was present in the centre. There was widespread peritoneal dissemination of the tumour with the formation oflarge solid masses in the omentum and mesenteries. Small discrete metastatic nodules were present in the sublumbar lymph glands, liver and lungs. There was a moderate degree of ascites and hydrothorax. Histology. The structure of the primary tumour varied in different areas. Long branching papillary processes extended into the lumina of the central cystic portion (Fig. 5) and were covered by a tall columnar epithelium with central deeply staining nuclei similar to those of a serous cystadenoma. The epithelium ranged in height from one to many layers and had a moderate mitotic rate. Some of the central cvsts had welldefined connective tissue capsules, but elsewhere the papillary processes sprung from broad fields of solid adenocarcinoma. The major portion of the tumour was less well differentiated and was composed of solid sheets of cells with only occasional acini. The connective tissue stroma was fine and diffusely arranged throughout the tumour. The metastases were of a similar solid pattern with only a few scattered acini. There was a small amount of Schiff-positive fluid in some of the acini and in a few of the surrounding epithelial cells. This material was not metachromatic and did not stain with mucicarmine or alcian blue. The contralateral ovary contained a number of corpora lutea which were regressing, The endometrial and vaginal histology was consistent with the later stages of metoestrus. 12 X
Fibroma A single rounded mass of 9 cm. diameter was an incidental finding at autopsy in an 8 year old bitch which had borne 3 litters, the last 6 months prior to death. The tumour was solid and hard to cut and had a whitish, homogeneous surface. There were no corpora lutea or follicles in the other ovary and the uterus appeared normal.
c.
DOW
Histology. The tumour was composed of interlacing bands of fibrous tissue. The majority of the connective tissue bundles were moderately cellular with a fine collagenous stroma. The cells were fusiform with plump, deeply basophilic nuclei resembling those of the theca interna. Unlike the latter they did not contain lipid or P.A.S.-positive material. In other bundles, the connective tissue was relatively acellular and the collagen fibres were coarse and frequently hyalinised. In such bundles, the cells were small with scanty cytoplasm and elongated dense nuclei. There were no mitotic figures in any part of the tumour and smooth muscle cells were present only in the walls of vessels. Though normal ovarian structures were present in the hilar portion of the capsule, no epithelial elements were present within the tumour. The other ovary contained only primary follicles and remnants of corpora lutea. The endometrial histology was consistent with the terminal stages of metoestrus.
Secondary Tumours
Though 74 of the bitches submitted to autopsy had metastases from tumours arising in a variety of sites, only in 3 cases were secondary deposits found in the ovaries. Gross involvement of both ovaries was present at autopsy in 2 cases with widespread metastases from lymphoid tumours of the small intestine. A microscopic secondary was found in one ovary of an animal with a widely metastasising mammary carcinoma. Lymphosarcoma and mammary carcinoma were the commonest malignant tumours found at autopsy. Ovarian Cysts Follicular Cysts
Solitary cysts were found in 41 animals of 2 to 15 years of age and at various stages of the oestrus cycle. Twenty-nine of the bitches were nulliparous and only 7 were multiparous. The majority of the cysts measured I to 1·5 cm. in diameter but a few ranged up to 5 cm. On section they were thin-walled, monolocular and contained a clear watery fluid. Corpora lutea at various stages of growth and regression were present in the affected and normal ovaries of 3 I cases. There was cystic hyperplasia of the endometrium in I I cases. Multiple ovarian cysts were present in a further 22 animals of 5 years and over. Eighteen of these were maiden bitches. The cysts formed masses up to 10 cm. in diameter. The component cysts were commonly small and appeared to be monolocular. There was bilateral involvement in 7 cases. In 5 bitches, there was a concomitant cystic glandular hyperplasia of the endometrium. Histology. Evidence of follicular origin was readily apparent in the smaller solitary cysts. They were lined by several layers of granulosa cells within a capsule composed of regressing theca and compressed ovarian stroma. The nuclei of the granulosa cells were less regularly shaped than those of the normal follicle and showed complete loss of polarity. Degenerate remnants of the cumulus 06phorus could be seen in many of the cysts. The nuclei became compressed and orientated parallel to the capsule as the size of the cysts increased. The lining of the largest cysts consisted E
66
OVARIAN ABNORMALITIES IN THE BITCH
of a single layer of cuboidal or flattened cells. The capsule was dense, fibrous and often hyalinised. Where multiple cysts were present, the smaller ones were lined by a typical granulosa cell layer whilst the larger ones were lined by a low cuboidal epithelium. The epithelium in some of the larger cysts had completely disappeared and they were lined by dense hyalinised connective tissue. All the cysts presented a simple cavity with no evidence of intracystic growth or of communication between individual cysts. All stages of follicular aad luteal growth and regression were present in the cases with single cysts and in those with unilateral multicystic change but were absent in cases with bilateral involvement. There was no histological evidence of oestrogenic activity in the endometrium or vagina of any of these cases.
Lutein Cysts Solitary cysts lined by luteal tissue were found in the ovaries of 9 bitches of 2 to 13 years. Six of the animals were nulliparous. The cysts ranged from 1·5 to 3 cm. in diameter and had thicker and more opaque walls than those of the follicular type. The ovaries in all cases also contained normal corpora lutea. Histology. The normal corpus luteum in the dog shows central cavitation for a short time after ovulation but this rapidly disappears. None of the normal corpora lutea in these cases showed such cavitation. Six of the cysts were completely lined by several layers of granulosa lutein cells arranged on a capsule of compressed ovarian stroma. The lutein cells in the smaller cysts were similar to those of neighbouring normal corpora lutea and were supported by vascular connective tissue trabeculae extending in from the capsule. A thin layer of fibrin was deposited on the inner surface of the layer of lutein cells. The cells lining the larger cysts were less well preserved; the cells on the luminal surface were compressed and many contained pyknotic nuclei. A layer of fine connective tissue separated the lutein cells from the lumen in several of the larger cysts. Only a portion of the wall was lined by lutein cells in 3 cases, the remainder being lined by granulosa cells like those in follicular cysts (Fig. 6). Luteinisation of the granulosa cells was focal in 2 cases and a gradual transition from one cell type to another could be traced. Similar foci of luteinisation were present in the walls of the cyst in the third case, but the change appeared to be present in the theca interna since the granulosa cell layer continued over the lutein cells without interruption. All these lutein cysts contained a pale eosinophilic fluid which did not stain with sudan black B or with P.A.S. The morphology of the uterus and vagina in these cases was consistent with the progesterone phase of the cycle and this, in the presence of normal corpora lutea, gave no indication of the hormonal activity of the cysts.
Germinal Cysts These cysts were found on microscopic examination of the ovaries of 20 bitches of 6 to 13 years. Eleven of the animals were nulliparous. They were found in otherwise normal ovaries at all stages of the
C. DOW
oestrus cycle and also in association with other ovarian abnormalities. The cysts were microscopic in size, multiple and commonly bilateral. They were present as irregularly round or oval structures in the cortices of ovaries of the senile type in which there were numerous indentations of the germinal epithelium (Fig. 7). They were lined by a non-secretory cuboidal or low columnar epithelium of the germinal type. There was no evidence of cilia or of metaplastic change. The epithelium lay directly on the undifferentiated ovarian stroma with only slight condensation of argyrophil fibres without the formation of a definite basement membrane. The cysts contained a pale eosinophilic fluid which did not stain with sudan black B or P.A.S. Rete Cysts
Rete tubules were present as a small mass of irregular, anastomosing tubules in the hilar region of over 90 per cent of the ovaries examined. They were lined by a single layer of closely packed columnar cells with large, pale basal nuclei and ragged free borders. Single microscopic cysts were present near the hilus of thc ovary in close association with groups of rete tubules in 9 animals of 4 to I I years of age. The cysts were unilateral. The smaller cysts were irregular in outline and were lined by an epithelium similar to that of the normal rete tubules. The larger cysts were more rounded and were lined by an epithelium in which the cells showed loss of polarity. The cells were cuboidal in type and the free edge was more even in outline than in the normal tubules. Intra-cystic papillary processes were present in the smaller cysts but the larger ones were essentially monolocular. The fluid in the cysts was eosinophilic and stained pink with P.A.S. The stroma underlying the cysts was composed of dense fibrous tissue and did not contain muscle fibres. DISCUSSION
Twenty-five primary ovarian tumours were found in a histological study of the genital tract of 400 bitches submitted to routine post-mortem examination. Ovarian tumours represented 20 per cent of all tumours found in the animals under consideration. Both Mulligan (1949) and Cotchin (1951, 1954) observed that I per cent of all canine tumours examined by them were of ovarian origin. This disparity in incidence may possibly be explained by the fact that many of the tumours included in the present study were found only on histological examination. The affected animals ranged from 5 to 15 years of age and 18 out of the 25 were nulliparous which suggests an increased incidence of ovarian neoplasms with age and with absence of pregnancy. The survey suggests that there is a close similarity between the common ovarian tumours in the bitch and in women. Only in the
68
OVARIAN ABNORMALITIES IN THE BITCH
granulosa cell tumours are there obvious structural differences and, as Willis (1953) points out, the canine tumours show greater variations in morphology and some show distinct resemblances to the papillary adenomas. The granulosa cell tumours in the dog show no evidence of malignancy. Histological evidence of oestrogenic activity was found in the endometrium and vagina in only 3 of the 13 tumours of this type examined. Traut and Marchetti (1940) found that 39 out of 54 human granulosa cell tumours showed evidence of hormonal activity. Another major difference from a comparable series of human ovarian neoplasms is the absence of teratomata from the present series though both Storm (1947) and Cotchin (1956) have observed single specimens. It is noteworthy that tumours of the canine testis have not been recorded. It is interesting to note the low incidence of secondary tumours of the ovary in the bitch. Novak (1952) states that the commonest metastatic tumours of the human ovary arise from primary carcinomas of the pelvic organs and the intestines. Primary carcimoma of these regions is rare in the dog and though there were 18 cases of intestinal lymphosarcoma in the animals examined, only in 2 cases were there ovarian secondaries. Twenty-nine of the animals had carcinomata of the mammary glands with pulmonary metastases, yet ovarian involvement was present in only one case. Willis (1953) found a similarly low incidence of ovarian metastases from widely disseminated mammary carcinomas in women. Non-neoplastic cysts of the ovaries were found in 90 animals. Follicular cysts were the commonest and were found in 16 per cent of the ovaries examined. There was no histological evidence of oestrogen activity in any of the cases of follicular cysts. The incidence of all types of ovarian cysts was higher in nulliparous animals and in those over 5 years of age. CONCLUSIONS
A histological examination of the ovaries of 400 bitches, submitted to routine post-mortem, revealed primary tumours in 25 cases, secondary tumours in 3 cases and non-neoplastic cysts in a further 90 animals. A detailed histopathological description of the individual lesions is given in addition to an examination of their secretory activity with regard to mucin and lipid. Analysis of age incidence suggests that both tumours and cysts of the bitch are more common in animals over 5 years of age and in nulliparous animals. REFERENCES
Bloom, F. (1954). Pathology of the Dog and Cat. American Veterinary Publications; Evanston. Cotchin, E. (1951). Vet. Rec., 63, 67; (1954). Ibid., 66, 879; (1956). Neoplasms qf the Domesticated Animals. Commonwealth Agricultural Bureaux.
c.
DOW
[0 face /Jagc 68
C . DOW
69
Mulligan, R. M. (1949). Neoplasms oj the Dog. Williams and Wilkins Co.; Baltimore. Novak, E. (1952). Gynecologic and Obstetric Pathology. W. B. Saunders Co.; London. Storm, R. E. (1947). N . arner. Vet., 28,30. Traut, H. F ., and Marchetti, A. A. (1940). Surg. Gynec. & Obst., 70, 632. Willis, R. A. ( 1953) . Pathology of Tumours. Butterworth & Co.; London. [Received for publication, February 13th, 19591 LEGENDS
Fig.
G ranulosa cell tumour showing follicular structures of various sizes. Hand E x 65. Fig. 3· Serous cystadenoma showing thick branching intracystic papillary processes' Hand E x 2 0. Fig. 4· Pseudomucinous cystadenoma composed of numerous cysts of varying size. The cysts a re lined by a characteristic tall columnar epithelium with clear refractile cytoplasm. Hand E x 65. Fig. 5· Adeno-carcinoma showing long branching papillary processes extending into the lumen of the central cystic portion. Hand E x 65. Fig. 6. Simple ovaria n cyst lined partly by granulosa cells a nd partly by luteal cells. Hand E x 65 . Fig. 7. Group of germinal cysts in the cortex of the ovary. They are lined by a ow l cuboidal epithelium. Hand E x 2 0. 2.
CO~IP. PATH.
1960. VOL. 70.
59
OVARIAN ABNORMALITIES IN THE BITCH By
C. Dow Veterinary School, Universi!J of Glasgow INTRODUCTION
Pathological studies of the canine female genital tract have been largely concerned with uterine disease; ovarian abnormalities have received little attention. Mulligan (1949) observed 7 ovarian tumours in a survey of 1000 tumours of the dog. Cotchin (1951, 1954) found that only 24 of 2361 canine tumours were of ovarian origin. Bloom (1954) recorded a variety of ovarian tumours in the bitch but gave no indication of incidence. None of these workers gave any indication of the frequency of ovarian tumours in the dog population, nor did they record the incidence of these tumours with respect to age or parity. There is no adequate description of the histological and histochemical features of these canine neoplasms. This publication is based on the examination of the ovaries of 400 unselected bitches submitted for routine autopsy in the University of Glasgow Veterinary Hospital. MATERIALS AND METHODS
Each ovary was sectioned through the hilus, fixed in formol-sublimate or neutral formol, dehydrated and cleared in an alcohol-amyl acetatebenzene series and double-embedded in celloidin and paraffin. Routine staining was by haemalum and eosin; other methods used in selected cases were picro-Mallory, Gordon and Sweet's reticulin stain, toluidine blue, alcian blue, Southgate's mucicarmine, sudan black B, periodic acid-Schiff before and after hydrolysis with diastase and the methylene blue extinction test. RESULTS
Incidence One hundred and fifteen of the 400 bitches examined showed ovarian lesions, an incidence of 28'75 per cent. The relationship of the various changes to age is shown in Fig. I. As would be expected these are diseases of adult life and increase with age. Of the 400 animals in the survey, 93 had a total of 127 tumours of various types in one or more sites. As twenty-five bitches had primary ovarian tumours, 27 per cent. of all tumour cases and 20 per cent. of all tumours were ovarian in origin. In only one case was the ovarian tumour considered to be the primary cause of disease. All ovarian tumours were in bitches of 5 years and over. The frequency of each ovarian lesion is shown in Table I. Tumours Granulosa Cell Tumours Though tumours of granulosa cell ongm were found m 13
60
OVARIAN ABNORMALITIES IN THE BITCH
Fig.
I
100
r--
r--
90 80 70 r0-
6O
r
Z UJ
r0-
c-
50
I-
r--r--
U 40
a: ~ 30
-
20
IC I
rIl-f
2 345 6
r-
r--
8 9 10 II 12 13 14 15 16
AGE Fig.
I.
The age incidence of ovarian abnormalities in the bitch.
animals, only in 9 were the ovaries considered abnormal at autopsy. The bitches ranged from 5 to 15 years and IO of them were nulliparous. The tumours were commonly round or oval with a smooth or slightly lobulated surface and ranged from 0'4 to IO cm. in diameter. The smaller tumours were solid on section, but some of the larger ones were soft and friable and contained cystic spaces of various sizes. The cut surface had a granular appearance and was cream or faintly yellow with areas of haemorrhage and necrosis in the larger masses. Metastases were not observed in any of these cases though a microscopic tumour nodule was found in the contralateral ovary of one case. The unaffected ovaries contained corpora lutea in 9 animals and were inactive in the other 4 cases. There was an ascites and hydrothorax of undetermined origin in one animal. Histology. The predominant cell type in 9 cases was similar to the granulosa cells present in normal medium-sized follicles. The cells were small and polyhedral with a small amount of palely eosinophilic cytoplasm in which there were a moderate number of argentophil granules. The relatively large round or oval nuclei were central in position. They had an abundant chromatin network and a well-defined nuclear membrane. Mitotic figures were rare in most cases. The histological pattern varied considerably, not only from case to case but also in different fields of individual sections. All were well encapsulated and showed no evidence of local infiltration. The most common arrangement was diffuse fields of granulosa cells in which a variable number of folliculoid structures occurred. These acini were lined by a layer of columnar type cells and contained an eosinophilic fluid. They varied in size from small Call-Exner rosettes to relatively
3
,:
14 15
,6
I
I'
'I
.--I~----
0 I
I
2
6 3
23 18
13
II 12
2
34
10
0 2 2 I
I
0 0
0 0
37
39
I
I I
I
i
I
0 0
0
0 0
0
0 I 0 I
0 0 0
0 0
I
1
I
I
0 0
0
0 0
0
I
0 0: 0 [
0
1
o! 0
0 0
1 _ _1
Adenocarcinoma
0 0
0
0 0
0
0 0 I 0
0 0 0
0 0
Fibroma
:--1---;---- :--1"
0 0
0
2
0 I 2
0 0 0
0 0
Granulosa I Pseudocell I Serous mucinous tumours I cystadenoma' cystadenoma
8 9
7
39 42
23 33 32
3 4 5
6
39 27
No. oj animals
I 2
Age
TABLE
1
I
I
0
2
3
3
4 4
I
I'
I
I, 2
I
1 0 '0 0
0 0,
0 0
I 2 0
0 0 0
0 [
Lutein cysts
Germinal cysts
I
I'
0 0
I
2
3
4
2
2
3 3
0 0 0
I 0 '0
1----
0
I 0
2
3
0 0 0
0 0
Rete cysts
0 0
,: " -~:~I ,: :
2
5 4
5
4 5
4 3
3
2
0 [
Solitary Multiple follicular follicular cysts cysts 1--
AGE INCIDENCE AND LESIONS FOUND
tI
Ol
:E
o
()
OVARIAN ABNORMALITIES IN THE BITCH
large cysts (Fig. 2). In one of the tumours there were several large cystic cavities lined by many layers of granulosa cells which were surrounded by a distinct connective tissue capsule. These structures resembled the folliculoma malignum of von Kahlden. The fluid within the cysts was weakly P.A.S. positive, but was not metachromatic and did not stain with alcian blue or with Southgate's mucicarmine. Methylene blue extinction occurred at pH 5. The connective tissue stroma in the majority of these cases was not conspicuous and reticulin fibres did not penetrate the granulosa cell masses. In 2 cases there were numerous tissue trabeculae extending in from the capsule and splitting up the granulosa cells into round or oval columns. This connective tissue proliferation was very prominent in one case and granulosa cells appeared as small isolated islands or columns. In this cylindromatous pattern, the cells immediately adjacent to the trabeculae were columnar in type and lay on a thin layer of argyrophil fibres. In one specimen of the diffuse cellular type there were areas of transition from typical granulosa cells to plump, fusiform cells resembling those of the theca interna. These thecal cells were arranged in an irregular interlacing manner and were separated by distinct bands of fibrous tissue. Unlike the granulosa cells, they had a well-developed reticulin network and did not have intra-cytoplasmic argentophil granules. Both the granulosa and thecal cells in this tumour contained small numbers of lipid granules. There was transformation of the cells into typical granulosa lutein cells in 3 of these tumours. In 2 cases this change was focal and occurred mainly in the centre of diffuse cellular masses, but in the third case luteinisation had occurred in the majority of the cells. The transitional stages between granulosa and lutein cells were similar to those observed in the normal follicle during the later stages of maturation. There was a gradual increase in the size and in the lipid content of the cells as the degree of luteinisation increased. Though hormone assays were not performed on body fluids or on tumour tissue some evidence of ovarian functional activity could be gained from histological examination of the uterus, vagina and skin. The vaginal epithelium was hyperplastic and exhibited a degree of cornification indicative of oestrogenic activity in 3 cases in which the contralateral ovary was atrophic. In these, the endometrium showed slight glandular dilatation and stromal fibrosis similar to that observed following prolonged oestrogen treatment. One of these animals showed atrophy of the epidermis and hair follicles suggestive of prolonged oestrogenic activity. There were 9 cases in which the vaginal and endometrial histology was compatible with metoestrus. In 7 of them the contralateral ovary contained corpora lutea and the other 2 were associated with luteinised tumours. There was no evidence of progestational activity in the endometrium or vagina of the third luteinised tumour.
Serous Cystadenoma Tumours of this type were found in 8 animals ranging from 6 to 13 years old. Five of them were nulliparous and only one had whelped more than one litter. Six of the tumours were small unilocular structures of 0·8 to 1·5 cm. in diameter and 2 were multilocular masses of 6 to 8 cm. in diameter. Intraluminal papillary
C. DOW
growth was prominent in most specimens. The contralateral ovary contained follicular cysts in 3 cases and corpora lutea in 2 cases. Histology. There was evidence of multicentric origin in all the small tumours with the formation of microscopic papillary cysts in other parts of the ovarian cortex. Papillary cysts were present in the contralateral ovary in 2 cases. The walls of the cysts were composed of dense vascular connective tissue which was hyaline in some of the larger specimens. Arising from this capsule were a number of intracystic papilligerous processes which almost occluded the lumen in some of the smaller cysts. These processes varied greatly in size and configuration. They were commonly slender and villiform with fine connective tissue cores in the smaller cysts. In the larger cysts, the processes were thicker and more irregular in shape. Some were simple papillae with broad cores of oedematous connective tissue, whilst others exhibited a more complex arborescent pattern (Fig. 3). In the latter, numerous acini were included in the substance of the processes, producing an adenomatous appearance. Extracystic growth was not prominent but small solid papillomata were present in 3 cases. Small calcified granules, the so-called psammoma bodies, were present in the stroma of 2 of the tumours. The lining epithelium was composed of a single layer of cells which varied considerably in type, even in individual cysts. The predominant type of epithelium was composed of cuboidal or low columnar cells with central vesicular nuclei and homogeneous cytoplasm. The epithelium in 2 cases showed certain similarities to that of the oviduct and was composed of tall ciliated columnar cells and intercalary cells with deeply basophilic cytoplasm. A few Schiff-positive granules were present in these tubal-type cells but not elsewhere. Lipid was not present in the epithelium of any of the tumours. The cyst lumina contained pale eosinophilic fluid with an admixture of cell debris and blood in some cases This fluid was Schiff-positive, but was not metachromatic and did not stain with alcian blue or Southgate's mucicarmine. It did not contain lipid. There was no histological evidence of derangement of the oestrus cycle due to these tumours. The endometrial and vaginal histology was compatible with the functional activity shown by the contralateral ovary.
Pseudomucinous Cystadenoma Two tumours of this type were found in maiden bitches of 7 and 9 years old. The older animal had shown some evidence of oestral abnormality for almost 2 years. The tumours formed unilateral lobulated masses of 7 to IO cm. in diameter. On section, each was composed of a number of lobules containing a viscid milky fluid. The cyst walls were thin and translucent with only occasional papillary growths. Histology. The tumours were composed of a number of relatively large cysts surrounded by a honeycombed mass of microscopic acini (Fig. 4). The cysts had a well-defined connective tissue capsule containing coarse collagen fibres and dense bundles of elastin. Smooth muscle fibres were not evident in the capsule. The few papillae which projected into the cyst lumina were long and slender. The cyst epithelium rested on an
ov ARrAN
ABNORMALITIES IN THE BITCH
argyrophil basement membrane which was particularly prominent In the small locules. The cysts were lined by a single layer of very tall columnar cells with clear refractile cytoplasm. The oval nuclei which were deeply basophilic were situated close to the basement membrane. Small nucleoli were evident in many nuclei. The cell membranes were prominent and there were no cilia on the free border. Large goblet cells were numerous in the epithelium of some cysts and absent in others. The cytoplasm of all the epithelial cells contained aggregated masses of Schiff-positive material. This material also stained with mucicarmine and alcian blue and showed B-metachromasia with toluidine blue. The cyst lumina also contained fluid of this nature. The cytoplasm of some of the cells also contained a number of minute, finely dispersed lipid droplets. Adena-carcinoma Autopsy of a 9 year old maiden bitch revealed a nodular mass 9 X 7 cm. in the left ovary. On section, the bulk of the tumour was firm and solid, but a cystic cavity of 2 cm. diameter was present in the centre. There was widespread peritoneal dissemination of the tumour with the formation oflarge solid masses in the omentum and mesenteries. Small discrete metastatic nodules were present in the sublumbar lymph glands, liver and lungs. There was a moderate degree of ascites and hydrothorax. Histology. The structure of the primary tumour varied in different areas. Long branching papillary processes extended into the lumina of the central cystic portion (Fig. 5) and were covered by a tall columnar epithelium with central deeply staining nuclei similar to those of a serous cystadenoma. The epithelium ranged in height from one to many layers and had a moderate mitotic rate. Some of the central cvsts had welldefined connective tissue capsules, but elsewhere the papillary processes sprung from broad fields of solid adenocarcinoma. The major portion of the tumour was less well differentiated and was composed of solid sheets of cells with only occasional acini. The connective tissue stroma was fine and diffusely arranged throughout the tumour. The metastases were of a similar solid pattern with only a few scattered acini. There was a small amount of Schiff-positive fluid in some of the acini and in a few of the surrounding epithelial cells. This material was not metachromatic and did not stain with mucicarmine or alcian blue. The contralateral ovary contained a number of corpora lutea which were regressing, The endometrial and vaginal histology was consistent with the later stages of metoestrus. 12 X
Fibroma A single rounded mass of 9 cm. diameter was an incidental finding at autopsy in an 8 year old bitch which had borne 3 litters, the last 6 months prior to death. The tumour was solid and hard to cut and had a whitish, homogeneous surface. There were no corpora lutea or follicles in the other ovary and the uterus appeared normal.
c.
DOW
Histology. The tumour was composed of interlacing bands of fibrous tissue. The majority of the connective tissue bundles were moderately cellular with a fine collagenous stroma. The cells were fusiform with plump, deeply basophilic nuclei resembling those of the theca interna. Unlike the latter they did not contain lipid or P.A.S.-positive material. In other bundles, the connective tissue was relatively acellular and the collagen fibres were coarse and frequently hyalinised. In such bundles, the cells were small with scanty cytoplasm and elongated dense nuclei. There were no mitotic figures in any part of the tumour and smooth muscle cells were present only in the walls of vessels. Though normal ovarian structures were present in the hilar portion of the capsule, no epithelial elements were present within the tumour. The other ovary contained only primary follicles and remnants of corpora lutea. The endometrial histology was consistent with the terminal stages of metoestrus.
Secondary Tumours
Though 74 of the bitches submitted to autopsy had metastases from tumours arising in a variety of sites, only in 3 cases were secondary deposits found in the ovaries. Gross involvement of both ovaries was present at autopsy in 2 cases with widespread metastases from lymphoid tumours of the small intestine. A microscopic secondary was found in one ovary of an animal with a widely metastasising mammary carcinoma. Lymphosarcoma and mammary carcinoma were the commonest malignant tumours found at autopsy. Ovarian Cysts Follicular Cysts
Solitary cysts were found in 41 animals of 2 to 15 years of age and at various stages of the oestrus cycle. Twenty-nine of the bitches were nulliparous and only 7 were multiparous. The majority of the cysts measured I to 1·5 cm. in diameter but a few ranged up to 5 cm. On section they were thin-walled, monolocular and contained a clear watery fluid. Corpora lutea at various stages of growth and regression were present in the affected and normal ovaries of 3 I cases. There was cystic hyperplasia of the endometrium in I I cases. Multiple ovarian cysts were present in a further 22 animals of 5 years and over. Eighteen of these were maiden bitches. The cysts formed masses up to 10 cm. in diameter. The component cysts were commonly small and appeared to be monolocular. There was bilateral involvement in 7 cases. In 5 bitches, there was a concomitant cystic glandular hyperplasia of the endometrium. Histology. Evidence of follicular origin was readily apparent in the smaller solitary cysts. They were lined by several layers of granulosa cells within a capsule composed of regressing theca and compressed ovarian stroma. The nuclei of the granulosa cells were less regularly shaped than those of the normal follicle and showed complete loss of polarity. Degenerate remnants of the cumulus 06phorus could be seen in many of the cysts. The nuclei became compressed and orientated parallel to the capsule as the size of the cysts increased. The lining of the largest cysts consisted E
66
OVARIAN ABNORMALITIES IN THE BITCH
of a single layer of cuboidal or flattened cells. The capsule was dense, fibrous and often hyalinised. Where multiple cysts were present, the smaller ones were lined by a typical granulosa cell layer whilst the larger ones were lined by a low cuboidal epithelium. The epithelium in some of the larger cysts had completely disappeared and they were lined by dense hyalinised connective tissue. All the cysts presented a simple cavity with no evidence of intracystic growth or of communication between individual cysts. All stages of follicular aad luteal growth and regression were present in the cases with single cysts and in those with unilateral multicystic change but were absent in cases with bilateral involvement. There was no histological evidence of oestrogenic activity in the endometrium or vagina of any of these cases.
Lutein Cysts Solitary cysts lined by luteal tissue were found in the ovaries of 9 bitches of 2 to 13 years. Six of the animals were nulliparous. The cysts ranged from 1·5 to 3 cm. in diameter and had thicker and more opaque walls than those of the follicular type. The ovaries in all cases also contained normal corpora lutea. Histology. The normal corpus luteum in the dog shows central cavitation for a short time after ovulation but this rapidly disappears. None of the normal corpora lutea in these cases showed such cavitation. Six of the cysts were completely lined by several layers of granulosa lutein cells arranged on a capsule of compressed ovarian stroma. The lutein cells in the smaller cysts were similar to those of neighbouring normal corpora lutea and were supported by vascular connective tissue trabeculae extending in from the capsule. A thin layer of fibrin was deposited on the inner surface of the layer of lutein cells. The cells lining the larger cysts were less well preserved; the cells on the luminal surface were compressed and many contained pyknotic nuclei. A layer of fine connective tissue separated the lutein cells from the lumen in several of the larger cysts. Only a portion of the wall was lined by lutein cells in 3 cases, the remainder being lined by granulosa cells like those in follicular cysts (Fig. 6). Luteinisation of the granulosa cells was focal in 2 cases and a gradual transition from one cell type to another could be traced. Similar foci of luteinisation were present in the walls of the cyst in the third case, but the change appeared to be present in the theca interna since the granulosa cell layer continued over the lutein cells without interruption. All these lutein cysts contained a pale eosinophilic fluid which did not stain with sudan black B or with P.A.S. The morphology of the uterus and vagina in these cases was consistent with the progesterone phase of the cycle and this, in the presence of normal corpora lutea, gave no indication of the hormonal activity of the cysts.
Germinal Cysts These cysts were found on microscopic examination of the ovaries of 20 bitches of 6 to 13 years. Eleven of the animals were nulliparous. They were found in otherwise normal ovaries at all stages of the
C. DOW
oestrus cycle and also in association with other ovarian abnormalities. The cysts were microscopic in size, multiple and commonly bilateral. They were present as irregularly round or oval structures in the cortices of ovaries of the senile type in which there were numerous indentations of the germinal epithelium (Fig. 7). They were lined by a non-secretory cuboidal or low columnar epithelium of the germinal type. There was no evidence of cilia or of metaplastic change. The epithelium lay directly on the undifferentiated ovarian stroma with only slight condensation of argyrophil fibres without the formation of a definite basement membrane. The cysts contained a pale eosinophilic fluid which did not stain with sudan black B or P.A.S. Rete Cysts
Rete tubules were present as a small mass of irregular, anastomosing tubules in the hilar region of over 90 per cent of the ovaries examined. They were lined by a single layer of closely packed columnar cells with large, pale basal nuclei and ragged free borders. Single microscopic cysts were present near the hilus of thc ovary in close association with groups of rete tubules in 9 animals of 4 to I I years of age. The cysts were unilateral. The smaller cysts were irregular in outline and were lined by an epithelium similar to that of the normal rete tubules. The larger cysts were more rounded and were lined by an epithelium in which the cells showed loss of polarity. The cells were cuboidal in type and the free edge was more even in outline than in the normal tubules. Intra-cystic papillary processes were present in the smaller cysts but the larger ones were essentially monolocular. The fluid in the cysts was eosinophilic and stained pink with P.A.S. The stroma underlying the cysts was composed of dense fibrous tissue and did not contain muscle fibres. DISCUSSION
Twenty-five primary ovarian tumours were found in a histological study of the genital tract of 400 bitches submitted to routine post-mortem examination. Ovarian tumours represented 20 per cent of all tumours found in the animals under consideration. Both Mulligan (1949) and Cotchin (1951, 1954) observed that I per cent of all canine tumours examined by them were of ovarian origin. This disparity in incidence may possibly be explained by the fact that many of the tumours included in the present study were found only on histological examination. The affected animals ranged from 5 to 15 years of age and 18 out of the 25 were nulliparous which suggests an increased incidence of ovarian neoplasms with age and with absence of pregnancy. The survey suggests that there is a close similarity between the common ovarian tumours in the bitch and in women. Only in the
68
OVARIAN ABNORMALITIES IN THE BITCH
granulosa cell tumours are there obvious structural differences and, as Willis (1953) points out, the canine tumours show greater variations in morphology and some show distinct resemblances to the papillary adenomas. The granulosa cell tumours in the dog show no evidence of malignancy. Histological evidence of oestrogenic activity was found in the endometrium and vagina in only 3 of the 13 tumours of this type examined. Traut and Marchetti (1940) found that 39 out of 54 human granulosa cell tumours showed evidence of hormonal activity. Another major difference from a comparable series of human ovarian neoplasms is the absence of teratomata from the present series though both Storm (1947) and Cotchin (1956) have observed single specimens. It is noteworthy that tumours of the canine testis have not been recorded. It is interesting to note the low incidence of secondary tumours of the ovary in the bitch. Novak (1952) states that the commonest metastatic tumours of the human ovary arise from primary carcinomas of the pelvic organs and the intestines. Primary carcimoma of these regions is rare in the dog and though there were 18 cases of intestinal lymphosarcoma in the animals examined, only in 2 cases were there ovarian secondaries. Twenty-nine of the animals had carcinomata of the mammary glands with pulmonary metastases, yet ovarian involvement was present in only one case. Willis (1953) found a similarly low incidence of ovarian metastases from widely disseminated mammary carcinomas in women. Non-neoplastic cysts of the ovaries were found in 90 animals. Follicular cysts were the commonest and were found in 16 per cent of the ovaries examined. There was no histological evidence of oestrogen activity in any of the cases of follicular cysts. The incidence of all types of ovarian cysts was higher in nulliparous animals and in those over 5 years of age. CONCLUSIONS
A histological examination of the ovaries of 400 bitches, submitted to routine post-mortem, revealed primary tumours in 25 cases, secondary tumours in 3 cases and non-neoplastic cysts in a further 90 animals. A detailed histopathological description of the individual lesions is given in addition to an examination of their secretory activity with regard to mucin and lipid. Analysis of age incidence suggests that both tumours and cysts of the bitch are more common in animals over 5 years of age and in nulliparous animals. REFERENCES
Bloom, F. (1954). Pathology of the Dog and Cat. American Veterinary Publications; Evanston. Cotchin, E. (1951). Vet. Rec., 63, 67; (1954). Ibid., 66, 879; (1956). Neoplasms qf the Domesticated Animals. Commonwealth Agricultural Bureaux.
c.
DOW
[0 face /Jagc 68
C . DOW
69
Mulligan, R. M. (1949). Neoplasms oj the Dog. Williams and Wilkins Co.; Baltimore. Novak, E. (1952). Gynecologic and Obstetric Pathology. W. B. Saunders Co.; London. Storm, R. E. (1947). N . arner. Vet., 28,30. Traut, H. F ., and Marchetti, A. A. (1940). Surg. Gynec. & Obst., 70, 632. Willis, R. A. ( 1953) . Pathology of Tumours. Butterworth & Co.; London. [Received for publication, February 13th, 19591 LEGENDS
Fig.
G ranulosa cell tumour showing follicular structures of various sizes. Hand E x 65. Fig. 3· Serous cystadenoma showing thick branching intracystic papillary processes' Hand E x 2 0. Fig. 4· Pseudomucinous cystadenoma composed of numerous cysts of varying size. The cysts a re lined by a characteristic tall columnar epithelium with clear refractile cytoplasm. Hand E x 65. Fig. 5· Adeno-carcinoma showing long branching papillary processes extending into the lumen of the central cystic portion. Hand E x 65. Fig. 6. Simple ovaria n cyst lined partly by granulosa cells a nd partly by luteal cells. Hand E x 65 . Fig. 7. Group of germinal cysts in the cortex of the ovary. They are lined by a ow l cuboidal epithelium. Hand E x 2 0. 2.